METHODS OF DIAGNOSIS AND TREATMENT OF INFLAMMATORY BOWEL DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/728,452, filed on September 7, 2018, which is incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R35 CA197465 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The instant invention relates generally to the fields of inflammation, autoimmunity and autoimmune disease and methods for diagnosing and treating inflammatory bowel disease, ulcerative colitis, Crohn’s disease, and other autoimmune diseases.

BACKGROUND

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Inflammatory bowel diseases (IBDs) are disorders that involve chronic inflammation of the digestive tract. Ulcerative colitis and Crohn’s disease, the two main types of IBD, are

multifactorial immune -mediated disorders characterized by chronic relapsing inflammation of the gastrointestinal tract (Cohen and Sachar, BMJ 357, j2505 (2017); de Souza and Fiocchi, Nat Rev Gastroenterol Hepatol 13, 13-27 (2016)). While ulcerative colitis and Crohn’s disease differ in several respects, including the locations in the gastrointestinal tract that are affected and their symptoms, common to both is that they are incurable, often diagnosed at a young age, and are associated with significant morbidity (de Souza and Fiocchi, Nat Rev Gastroenterol Hepatol 13, 13-27 (2016)). The precise etiology of IBD remains unclear; however, genetic susceptibility, environmental factors, microbial flora, and dysregulation of the immune response are thought to be among the major factors involved (Halfvarson et al., 2017; Ni et al., Nat Microbiol 2, 17004 (2017) ; Peters et al., Nat Gene 49, 1437-1449 (2017)). In particular, the innate and adaptive immune systems are implicated as major contributors to the development and progression of IBD, predominantly through an imbalance between the pro-inflammatory responses of T-helper (Th)l, Th2, and Thl7 versus the anti inflammatory response of regulatory T cells (Canavan et al., Gut 65, 584-594 (2016); de Souza and Fiocchi, Nat Rev Gastroenterol Hepatol 13, 13-27 (2016)). Indeed, the use of immunosuppressive agents (such as azathioprine, 6-mercaptopurine, or methotrexate) and biologies (such as anti-tumor necrosis factor (TNF) agents) helped to reduce the use of corticosteroids and to augment response in IBD (Cohen and Sachar, BMJ 357, j2505 (2017); Neurath, Nat Rev Gastroenterol Hepatol 14, 573- 584 (2017)). Currently, various therapeutic approaches including integrin and cytokine inhibitors are exploited in IBD treatment (Yadav et al., Transl Res 176, 38-68 (2016)). Prevalent among them are inhibitors of proinflammatory cytokines, implicated in immune dysregulation which underlies intestinal inflammatory disorders, including inhibitors of TNF -a (Cohen and Sachar, BMJ 357, j 2505 (2017)), interleukin (IL)-6 (Allocca et al., Curr Drug Targets 14, 1508-1521 (2013)), IL-13 (Jovani et al., Curr Drug Targets 14, 1444-1452 (2013)), IL-17 (Yang et al, Trends Pharmacol Sci 35, 493-500 (2014), IL-18 (Harrison et al., Mucosal Immunol 8, 1226-1236 (2015); Nowarski et al., Cell 163, 1444-1456 (2015)), and IL-21 (MacDonald et al., Inflamm Bowel Dis 18, 2180-2189 (2012)).

Nonetheless, disappointing clinical trials with anti-IL-l7 in Crohn's diseases (Hueber et al., Gut 61, 1693-'700 (2012)) and anti-IL-l3 in ulcerative colitis (Danese et al., Gut 64, 243-249 (2015)), highlight the lack of thorough understanding of the complex biological processes underlying the pathogenesis of IBD. Therefore, there remains a need to determine genes, factors, and the pathogenic mechanisms of IBD and treatment for or protection against IBD, including but not limited to ulcerative colitis and Crohn’s disease.

SUMMARY OF THE INVENTION

[0006] The methods and compositions described herein provide advances to understanding of the role of S 100 family proteins, RNF5 and RNF5 regulation in autoimmune disorders including intestinal inflammatory disorders. The present disclosure identifies an untapped therapeutic approach to treat a variety of immune disorders, including inflammatory bowel disease.

[0007] In some aspects, provided herein is a method of treating or ameliorating symptoms of an inflammatory bowel disease (IBD) in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a S100A8 inhibitory agent that inhibits S100A8 expression or activity. In some embodiments, the inhibitory agent comprises a S100A8 neutralizing antibody. In some embodiments, the antibody neutralizes S100A8 and not S100A9. In some embodiments, the inhibitory agent decreases expression of S100A8. In some embodiments, the inhibitory agent comprises a small interfering RNA (siRNA) that targets a polynucleotide encoding S100A8. In some embodiments, the inhibitory agent comprises a guide sequence that targets a target sequence in a polynucleotide encoding S 100A8, and a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) enzyme comprising a nuclease domain.

In some embodiments, the CRISPR enzyme is a C2c2 enzyme or a fragment thereof. In some embodiments, the nuclease domain of the CRISPR enzyme comprises a mutation. In some embodiments, the CRISPR enzyme further comprises an effector domain. In some embodiments, the effector domain is a transcription repressor domain or a methyltrasferase domain. In some embodiments, the decrease in expression of S100A8 comprises a decrease of l.l-fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5 fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.l-fold, 2.2-fold, 2.3-fold, 2.4- fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.0-fold, 3.0-fold, 3. l-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5- fold, 4-fold, 5 -fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more in a first cell obtained from the subject post treatment with the method compared with a second cell obtained from the subject prior to treatment with the method, wherein the first cell and the second cell are of the same cell type. In some embodiments, the cell type of both the first cell and the second cell are neutrophils. In some embodiments, the cell type of both the first cell and the second cell are monocytes. In some embodiments, the cell type of both the first cell and the second cell are intestinal epithelial cells.

[0008] In some aspects, provided herein is a method of treating or ameliorating symptoms of an inflammatory bowel disease (IBD) in a subject in need thereof, the method comprising administering to the subject a composition comprising a therapeutically effective amount of a S100A8 inhibitory agent that inhibits S100A8 expression or activity. In some embodiments, the inhibitory agent increases expression of RNF5, wherein RNF5 modulates ubiquitination mediated degradation of S 100A8. In some embodiments, the inhibitory agent comprises a guide sequence that targets a target sequence in a polynucleotide encoding RNF5, and a CRISPR enzyme comprising a nuclease domain and an effector domain, wherein the nuclease domain of the CRISPR enzyme comprises a mutation.

In some embodiments, the effector domain is a transcription activator domain. In some embodiments, the effector domain is a scaffold domain that recruits one or more transcription activator domain. In some embodiments, wherein the inhibitory agent comprises a transcription activator like effector (TALE) or a fragment thereof that targets a target sequence in a polynucleotide encoding RNF5. In some embodiments, the increase in expression of RNF5 comprises an increase of l. l-fold, 1.2-fold,

1.3-fold, 1.4-fold, 1.5 fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2. l-fold, 2.2-fold, 2.3-fold,

2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.0-fold, 3.0-fold, 3.l-fold, 3.2-fold, 3.3-fold, 3.4-fold,

3.5-fold, 4-fold, 5-fold, lO-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more in a first cell obtained from the subject post treatment with the method compared with a second cell obtained from the subject prior to treatment with the method, wherein the first cell and the second cell are of the same cell type. In some embodiments, the cell type of both the first cell and the second cell are neutrophils. In some embodiments, the cell type of both the first cell and the second cell are monocytes. In some embodiments, the cell type of both the first cell and the second cell are intestinal epithelial cells.

[0009] In some embodiments, the IBD is ulcerative colitis or Crohn’s disease. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier or vehicle. In some embodiments, the composition further comprises a proinflammatory cytokine inhibitor. In some embodiments, the proinflammatory cytokine inhibitor is a TNF-a inhibitor, an IL-8 inhibitor, an IL-13 inhibitor, an IL-17 inhibitor, an IL-18 inhibitor, an IL-21 inhibitor, an I L I b inhibitor, an IL-6 inhibitor, an ILl2p35 inhibitor, or any combination thereof. In some embodiments, the composition further comprises an aminosalicylate, a corticosteroid, an immuno-suppressor, or any combination thereof. In some embodiments, the subject is identified as having poor responsiveness to treatment with anti-TNF treatment. In some embodiments, the composition further comprises a commensal bacterium. In some embodiments, the commensal bacterium is obtained from a microbiota of a healthy mammal. In some embodiments, the commensal bacterium is selected from the group consisting of bacteria species in genera Faecalibacterium, Clostridium, Bifidobacterium, Bacteroides, Helicobacter, Roseburia, and Eubacterium. In some embodiments, the composition further comprises a prebiotic. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered rectally. In some embodiments, the composition is administered with intraperitoneal injection. In some embodiments, the composition is released in the large intestine. In some embodiments, the composition is released in the colon. In some embodiments, the therapeutically effective amount is about lmg/kg to about lOmg/kg. In some embodiments, expression of MHC class I and/or MHC class II is decreased in a dendritic cell obtained from the subject post treatment with the method compared with a dendritic cell obtained from the subject prior to treatment with the method.

INCORPORATION BY REFERENCE

[0001] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 depicts that RNF5 is crucial for the maintenance of intestinal homeostasis (a) Representative immunoblot of RNF5 in intestinal epithelial cells (IECs) from different intestinal regions ofWT and Rnf5-/- mice: duodenum (D), jejunum (J), ileum (I), and colon (C). (b)

Representative IHC staining of RNF5 in small intestine and colon sections ofWT and Rnf5-/- mice. Scale bars, 50 pm. (c) Representative H&E staining ofWT and Rnf5-/- colon sections (The red triangles indicate infiltrating lymphocytes). Scale bars, 50 pm. (d) Representative images of immunofluorescence staining of CD45 in WT and Rnf5-/- colon sections, and quantification of CD45+ cells (n = 10, 2 fields each). Scale bars, 50 pm. (e) Flow cytometric analysis of the frequencies of CD4+, CD4+CD44+, CD4+CD25+, CD8+, and CD8+CD44+ T cells, colonic DCs (CD45+ CD1 lc+ CD103+ F4/80-), colonic macrophages (CD45+ CD1 lc+ CD103- F4/80+), inflammatory monocytes (CD45+ CD1 lc- Ly6C+), neutrophils (CD45+ CD1 lc- Ly6G+), and NK1.1+ NK cells among gated CD45+ cells isolated from the lamina propria of colons from WT and Rnf5-/-mice (n = 6). (f) Mean fluorescence intensity (MFI) of MHC class I and II on colonic DCs cells isolated from the lamina propria of colons from WT and Rnf5-/- mice (n = 6). (g) qRT-PCR analysis of elative mRNA expression of the indicated cytokines in WT and Rnf5-/-colon tissues (n = 6). (h) Heat map analysis of RNA-Seq data performed from 3 naive WT or Rnf5-/- colon tissues per group (left). 156 significantly dysregulated genes were identified in Rnf5-/- colon tissues compared with naive Rn£5-/- colon tissues (FDR adjusted P value<0.05 by Benjamini & Hochberg method). Right table shows top 10 upregulated genes were identified in Rnf5-/- colon tissues. All data are representative of three independent experiments. Graphs show the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by two-tailed t-test (d,e,f,g).

[0011] FIG. 2 depicts that DSS-induced colitis is exacerbated in Rn£5-/- mice (a) Initial weight (left) and percent body weight after DSS treatment (right) of WT and Rnf5-/- mice. 2.5% DSS was administered in drinking water for 7 days followed by a 48 h recovery period (n = 10). (b)

Representative photographs of colon and cecum (left) and quantification of colon length (right) from WT and Rnf5-/- mice on day 9 after initiation of DSS treatment (n = 10). (c,d) Disease activity index (c) and survival by Kaplan -Meier method (d) of DSS-treated WT and Rn£5-/- mice (n = 10). (e)

Representative H&E staining (left) and histological score (right) for colon sections from WT and Rn£5-/- mice on day 9 after DSS administration (n = 8, 2 fields each) (f) Representative images of Ki67, cleaved caspase-3, F4/80, Ly6G/C, CD1 lc, CD4, CD8, and periodic acid-Schiff/Alcian blue (PAS/AB) staining in colon sections of WT and Rnf5-/- mice on day 9 after DSS administration.

Scale bars, 50 pm. All data are representative of three independent experiments. Graphs show the means ± s.e.m. *P < 0.05 and **P < 0.01 by two-tailed t-test (a,b,c,e) or log-rank test (d).

[0012] FIG. 3 depicts that S100A8 is a novel substrate of RNF5. (a) Immunoprecipitation and immunoblot analysis of the RNF5-S100A8 interaction in HEK293T cells ectopically expressing Flag- tagged RNF5 or empty vector (EV) and V5 -tagged S100A8. (b) Immunoprecipitation and

immunoblot analysis of HEK293T cells co-expressing V5-S100A8, HA-ubiquitin (HA-Ub), plus Flag-RNF5. Cells were treated with MG132 (10 pM) for 4 h prior to lysis (c) Anti-Sl00A8 immunoprecipitation and anti-Ub immunoblot of MODEK cells transfected with EV or RNF5- targeting shRNA and treated with MG132 (10 pM) for 4 h before lysis (d) Immunoblot analysis of HEK293T cells expressing Flag-EV or Flag-RNF5 and treated with medium or 10 pM MG 132 for 4 h before lysis (e) Immunoblot analysis of MODEK cells expressing EV or shRNF5. Cells were treated with cycloheximide (CHX) for up to 24 h (n = 3). (f) Immunoprecipitation and immunoblot analysis of the interaction between endogenous RNF5 with S100A8 analyzed in MODE-K cells treated with MG132 (10 pM) for 4 h. (g) Immunoprecipitation and immunoblot analysis of cell lysates prepared from MODE-K cells treated with 10 ng/ml TNF-a for the indicated times followed by MG132 (10 pM) for 4 h before lysis. Data are representative of three independent experiments. Graphs show the means ± s.e.m.

[0013] FIG. 4 illustrates that increased level of S100A8 secreted by RNF5 -deficient intestinal epithelial cells (IECs) impacts host immune responses (a) Immunoblot analysis of endogenous proteins in MODE-K cells expressing empty vector (EV) or two RNF5 -targeting shRNAs. (b)

Representative images of IHC staining for S100A8 and S100A9 (left) and quantification of IHC scores (right) in colon sections from WT and Rn£5-/- mice (n = 6, 2 fields each). Scale bars, 50 pm.

(c) Immunoblot analysis of MODE-K cells expressing EV, shRNF5, or shRNF5 plus shSl00A8. (d) ELISA quantification of S 100A8 levels in the culture supernatants of MODE-K cells expressing EV, shRNF5, or shRNF5 plus shS l00A8 (n = 3). (e) ELISA quantification of IL-l2p70 and IL-l 1 b levels in the culture supernatants of bone marrow-derived dendritic cells (BMDCs) from WT mice incubated for 18 h with medium alone (no stimulation), recombinant S 100A8 (1 ng/ml), or conditioned medium (CM) from MODE-K cells expressing EV, shRNF5, or shRNF5 plus shS l00A8 (n = 3). (f) MFI of MHC class I and II on CD1 lc+ BMDCs incubated as in (e) (n = 3). (g) Relative luciferase activity of MODE-K-EV, MODE-K-shS 100A8, MODE-K-shRNF 5 , or MODE-K shRNF5/shS l00A8 cells transiently transfected with the NF-kB -dependent firefly luciferase reporter plasmid NF- KB -luc and Renilla luciferase plasmid pRL-TK (n = 3). All data are representative of three independent experiments. Graphs show the means ± s.e.m. n.s., not significant (P > 0.05), * * * * P < 0.0001 by two- tailed t-test (b). *P < 0.01, **P < 0.001, and ***P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (d,e,f,g).

[0014] FIG. 5 illustrates that RNF5 regulates the development of DSS-induced colitis through S l00A8-mediated CD4+ T cell activation (a) Representative immunoblots of S 100A8 and RNF5 in protein extracts of the distal colon from WT and Rnf5-/- mice on the indicated days after DSS administration (b) ELISA quantification of S 100A8 and TNF-a in the culture supernatants after 24 h incubation of distal colonic explants isolated from WT and Rnf5-/- mice on the indicated days after DSS administration (n = 3 mice/group) (c) ELISA quantification of S100A8 levels in the serum from WT and Rnf5-/- mice isolated on day 0 and day 9 after DSS administration (n = 4/group) (d) Flow cytometric analysis of the frequencies of CD4+, CD4+CD25+, CD4+CD44+, CD8+, and

CD8+CD44+ T cells, colonic DCs, colonic macrophages, neutrophils, monocytes, and NK cells in gated CD45+ cells isolated from the lamina propria of colons from WT and Rnf5-/- mice on the indicated days after DSS administration (n = 3-6 mice/group) (e) Intracellular IFN-g and TNF-a staining of the CD4+ T cells from LCMV -specific TCR transgenic SMARTA mice. BMDCs were generated from WT mice and incubated for 18 h with conditioned medium (CM) derived from MODE-K cells expressing EV, shSl00A8, shRNF5, or shRNF5 plus shS l00A8 treated with 0.5% DSS for 24 h. BMDCs were then incubated for 72 h with CFSE-labeled SMARTA CD4+ T cells (shown in Fig. l2h) in the presence of 2 pg/ml GP61-80 peptide. Right plot shows quantification of Intracellular IFN-y+ and TNF-a+ CD4+ T cells (n = 3 wells/condition) (f) Body weight of WT and Rnf5-/- mice administered 2.5% DSS and treated with control rat IgG2b or anti-CD4 neutralizing antibody (200 pg, i.p.) on days -1, 3, and 7, relative to the start of DSS treatment on day 0 (n = 8 mice/group), as shown by red arrows (on day 9: ****p < 0.0001; Rnf5-/— tisotype control vs Rnf5-/— tanti-CD4, ****p < 0.0001; WT+isotype control vs Rnf5-/— l-isotype control, n.s.; WT+anti- CD4 vs Rnf5-/— l-anti-CD4). (g) Representative images (top) of colon and cecum and quantification (bottom) of colon length from anti-CD4- or isotype control-treated WT or Rnf5-/- mice as described for (f) on day 9 after 2.5% DSS treatment (n = 8). (h) Disease activity index of anti-CD4- or isotype control-treated WT or Rnf5-/- mice after 2.5% DSS treatment as described for (f) (n = 8) (on day 9: ****P < 0.0001; Rnf5-/— l-isotype control vs Rn£5-/— tanti-CD4, ****p < 0.0001; WT+isotype control vs Rnf5-/-+isotype control, n.s.; WT+anti-CD4 vs Rnf5-/-+anti-CD4). All data are representative of two or three independent experiments. Graphs show the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by two-l tailed t-test (b,d).*P < 0.01, **P < 0.001, ***P < 0.0001 by one-way ANOVA test followed by Tukey's multiple comparison test (c,e). * * * * P < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test (f,g,h)

[0015] FIG. 6 illustrates that neutralizing antibodies to S 100A8 attenuate acute colitis in DSS- treated Rnf5-/- mice (a) Body weight of WT and Rnf5-/- mice administered 2.5% DSS and treated with control rabbit IgG or rabbit anti-Sl00A8 neutralizing antibody (200 pg, i.p.) on days -1, 1, and 3, relative to the start of DSS treatment on day 0 (n = 6 mice/group), as shown by red arrows (on day 9: * * * *p < 0.0001; Rnf5-/— tcontrol IgG vs Rnf5-/— l-anti-S l00A8, ****p < 0.0001; WT+control IgG vs Rnf5-/-+control IgG, n.s.; WT+anti-S l00A8 vs Rnf5-/-+anti-S l00A8) (b) Disease activity index of anti-Sl00A8- or control rabbit IgGl4treated WT or Rnf5-/- mice after 2.5% DSS treatment as described for (a) (n = 5) (on day 9: *P < 0.05; Rnf5-/-+control IgG vs Rnf5-/-+anti-Sl00A8, *P < 0.05; WT+control IgG vs Rnf5-/— tcontrol IgG, n.s.; WT+anti-Sl00A8 vs Rnf5-/— l-anti-S l00A8). (c) Survival of anti-Sl00A8- or control IgG-treated WT and Rnf5-/- mice after 2.5% DSS treatment (n = 6 mice/group) by Kaplan-Meier method (d) Representative images (left) of colon and cecum and quantification (right) of colon length from anti-Sl00A8- or control rabbit IgG-treated WT or Rnf5-/- mice as described for (a) on day 9 after 2.5% DSS treatment (n = 5). (e) Representative images of H&E-stained and anti-CD4-stained colon sections from WT and Rnf5-/- mice treated as described for (a). Sections are from mice sacrificed on day 9. Scale bars, 50 pm. (f) Quantification of histological score and CD4+ cells (per 20 field) from WT and Rnf5-/- mice treated with anti- S 100A8 antibody or control rabbit IgG after 2.5% DSS treatment for (e) (n = 3 mice/group, 3 field each) (g) Body weight of WT and Rnf5-/- mice administered 2.5% DSS for 5 days and treated with control rabbit IgG or rabbit anti-S l00A8 neutralizing antibody (200 pg, i.p.) on day 5, 7, and 9 (n = 6 mice/group), as shown by red arrows (on day 9: **P < 0.01; Rnf5-/-+control IgG vs Rnf5-/-+anti- S 100A8, **P < 0.01; WT+control IgG vs Rnf5-/— tcontrol IgG, n.s.; WT+anti-Sl00A8 vs

Rnf5-/-+anti-SlOOA8). All data are representative of two or three independent experiments. Graphs show the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test (a,b,d,f,g). Survival by log-rank test (c).

[0016] FIG. 7 illustrates the clinical relevance of S 100A8 by RNF5 in samples from IBD patients (a) Representative images of IHC staining for RNF5 and S 100A8 in colon sections from healthy control and IBD patients (un-inflamed and inflamed area). Scale bars, 50 pm. (b)

Quantification of RNF5 and S100A8 IHC scores of intestinal epithelial cells (IECs) in colon sections from healthy control (n =17) and IBD patients (un-inflamed and inflamed area) (n =19). (c) Scatter plots between the IHC score (IECs) of RNF5 and S100A8 in colitis from inflamed area of IBD patients (n=l9). (d) Number of RNF5- or Sl00A8-positive cells (40x) in stroma of colon sections from healthy control (n =17) and IBD patients (un -inflamed and inflamed area) (n =19). (e) Scatter plots between number of RNF5- and Sl00A8-positive cells in stroma of colitis from inflamed area of IBD patients (n =19). (f) RNF5 IHC score (IECs) and S100A8 IHC score (IECs) in the pathological severity (PS)_low (n =4) or high (n =12) colitis from inflamed area of IBD patients (g) Number of RNF5- or S100A8 -positive cells (stroma) in the pathological severity (PS)_low (n =4) or _high (n =12) colitis from inflamed area of IBD patients. Graphs show the means ± s.e.m. *P < 0.01, **P < 0.001, and ***P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (b,d).

Pearson's correlation (c,e). n.s., not significant (P > 0.05), *P < 0.05 and ***P < 0.001 by two-tailed t- test (f,g).

[0017] FIG. 8 depicts that RNF5 is crucial for the maintenance of intestinal homeostasis (a) qRT-PCR analysis of the indicated gene mRNA expression levels in WT and Rnf5-/- colon tissues for RNA-Seq validation (n = 3). (b,c) MFI of MHC class I and class II on CD1 lc+ splenic DCs (b) and BMDCs (c) from WT and Rnf5-/- mice treated with or without LPS (1 pg/ml for 24h) were determined by flow cytometry (n = 3). (d,e) Quantification of IL-l2p70 and IE-1b in the culture supernatants of splenic DCs (d) and BMDCs (e) from WT and Rnf5-/- mice treated with or without LPS (1 pg/ml for 24h) by ELISA (n = 3). All data are representative of three independent experiments. Graphs show the means ± s.e.m. n.s., not significant (P > 0.05), *P < 0.05, ***P < 0.001 by two-tailed t-test (a) not significant (P > 0.05) by oneway ANOVA test followed by Tukey's multiple comparison test (b,c,d,e).

[0018] FIG. 9 depicts that DSS-induced colitis is exacerbated in Rnf5-/- mice (a)

Quantification of Ki67, cleaved caspase-3, F4/80, Ly6G/C, CD1 lc, CD4, CD8, and PAS/Alcian blue (AB) staining in colon sections isolated from DSS-treated WT and Rnf5-/- mice on day 9 (n = 4, 2 fields each, 20 magnification) (b) Percent body weight after DSS treatment of WT and Rnf5-/- mice that were cohoused for 4 weeks prior to DSS treatment. 2.5% DSS was administered in drinking water for 7 days followed by a 48 h recovery period (WT alone and Rnf5-/- alone, n = 8; WT cohoused and Rnf5-/- cohoused, n = 8). (c) Disease activity index of DSS-treated WT and Rnf5-/- mice that were cohoused for 4 weeks prior to DSS treatment (WT alone and Rnf5-/- alone, n = 8;

WT cohoused and Rnf5-/- cohoused, n = 8). (d) Quantification of colon length (right) from WT and Rnf5-/- mice on day 9 after initiation of DSS treatment (WT alone and Rnf5-/- alone, n = 8; WT cohoused and Rnf5-/- cohoused, n = 8). (e) Representative H&E staining (left) and histological score (right) for colon sections from WT and Rnf5-/- mice on day 9 after DSS administration (WT alone and Rnf5-/- alone, n = 8; WT cohoused and Rnf5-/- cohoused, n = 8). All data are representative of three independent experiments. Graphs show the means ± s.e.m. n.s. not significant (P > 0.05), ***P < 0.001, ****p < 0.0001 by two-tailed t-test (a) n.s. not significant (P > 0.05) and ****P < 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test (b,c,d,e).

[0019] FIG. 10 depicts that S100A8 is a novel substrate of RNF5. (a,b) HEK293T cells were transfected with S100A8-V5 and Flag-EV or Flag-RNF5 and treated with cycloheximide (CHX) (20 mg/ml) for the indicated times. Representative immunoblot (a) and densitometric quantification (b) of S100A8 protein levels (n = 3). (c) Representative images (left) and quantification (right) of RNF5 staining in colon sections from WT mice on day 0 or day 9 after DSS administration. Scale bars, 50 pm. (n = 6, 2 fields each, 20 magnification). Data are representative of three independent experiments. Graphs show the means ± s.e.m. n.s. not significant (P > 0.05) by two-tailed t-test (c).

[0020] FIG. 11 depicts increased level of S100A8 secreted by RNF5 -deficient IECs impacts host immune responses (a) Densitometry measurements of S100A8, S100A9, and RNF5 expression in control MODE-K EV cells. Values are mean ± s.e.m. relative to HSP90 expression from three independent experiments (n = 3). (b) ELISA quantification of IL-l2p70 and IE-1b levels in the culture supernatants of BMDCs from WT mice incubated for 18 h with conditioned medium (CM) from MODE-K cells expressing EV or shRNF5 in the presence of IgG isotype control or anti-Sl00A8 blocking antibody (10 pg/ml) (n = 3). (c) MFI of MHC class I and II on CD1 lc+ BMDCs incubated as in (b) (n = 3). (d) qRT-PCR analysis of the indicated cytokines and chemokines in MODE-K-EV or MODE-K-shRNF5 cells (n = 3). (e,f) Concentration of cytokines (e) and chemokines (f) in the culture supernatants of MODE-K-EV and MODEK-shRNF5 cells analyzed by LEGENDplex kits (n = 3). All data are representative of three independent experiments. Graphs show the means ± s.e.m. n.s., not significant (P > 0.05), *P < 0.01, **P < 0.001, ***P < 0.0001 by one-way ANOVA test followed by Tukey's multiple comparison test (a,b,c). *P < 0.05, **P < 0.01 by two-tailed t-test (d,e,f).

[0021] FIG. 12 depicts elevated Thl cytokine production and CD4+ T cell activation in DSS- treated Rnf5 mice. (a,b) Concentration of cytokines (a) and chemokines (b) in 24 h culture supernatants of distal colonic explants from WT and Rnf5 ^; mice isolated on the indicated days after DSS administration (n = 3/group) using LEGENDplex kits (n.d., not detected) (c) qRT-PCR analysis of mRNA expression of the indicated cytokines and chemokines in WT and Rnf5 ^; colon tissues on day 0 and day 9 after DSS administration (n = 3). (d,e) Concentration of cytokines (d) and chemokines (e) in the serum from WT and Rnf5 ^; mice isolated on day 0 and day 9 after DSS administration (n = 4/group) using LEGENDplex kits (n.d., not detected) (f) ELISA quantification of S100A8 levels in the culture supernatants from MODEK-EV, MODE-K-shRNF5, or MODE-K- shRNF5/shSl00A8 cells treated with control medium or 0.5% DSS for 24 h (n = 3). (g) Flow cytometry of MHC class I and class II MFI on CD45+ CD1 lc+ BMDCs incubated with conditioned medium (CM) from MODE-K-EV, MODE-K-shRNF5, or MODE-K-shRNF5/shSl00A8 cells treated with control medium or 0.5% DSS for 24 h (n = 3). (h) Proliferation of CFSE-labeled CD4 ⁺ T cells from LCMV -specific TCR transgenic SMARTA mice. BMDCs were generated from WT mice and incubated for 18 h with CM derived from MODE-K cells expressing EV, shSl00A8, shRNF5, or shRNF5 plus shSl00A8 treated with 0.5% DSS for 24 h. BMDCs were then incubated for 72 h with CFSE-labeled SMARTA CD4 ⁺ T cells in the presence of 2 pg/ml GP _6I-8O peptide. Right plot shows quantification of the division index (n = 3 wells/condition) (i) Representative immunofluorescent images of anti-CD4 (red) and anti-Ki67 (green) stained colon sections from WT or Rnf5 ^; mice on day 9 after 2.5% DSS treatment. The white triangles indicate the single CD4 ⁺ Ki67 ⁺ lymphocyte in the field. Scale bars, 50 pm. All data are representative of three independent experiments. Graphs show the means ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001 by two-tailed t-test (a,b,i). *P < 0.01, **P < 0.001, ***P < 0.0001 by one-way ANOVA test followed by Tukey's multiple comparison test (c,d,e,f,g,h).

[0022] FIG. 13 depicts that neutralizing antibodies to S100A8 attenuate acute colitis in DSS- treated Rnf5 ^; mice (a) Representative images of anti-F4/80- or anti-Ly6G/C-stained colon sections from anti-Sl00A8- or control rabbit IgG-treated WT or Rnf5 mice as described for Fig. 6a on day 9 after 2.5% DSS treatment. Scale bars, 50 pm. (b) Quantification of histological score and F4/80 ⁺ or Ly6G/C ⁺ cells (per 20 field) from WT and Rnf5 ^; mice treated with anti-Sl00A8 antibody or control rabbit IgG after 2.5% DSS treatment for (a) (n = 3 mice/group, 3 field each) (c) Body weight of WT mice administered 4.5% DSS and treated with control rabbit IgG or rabbit anti-Sl00A8 neutralizing antibody (200 pg, i.p.) on days -1, 1, and 3, relative to the start of DSS treatment on day 0 (n = 6 mice/group), as shown by red arrows (d) Representative images (left) of colon and cecum and quantification (right) of colon length from anti-Sl00A8 or control rabbit IgG-treated WT mice as described for (c) on day 9 after 4.5% DSS treatment (n = 6 mice/group) (e) Disease activity index of anti-Sl00A8- or control rabbit IgG treated WT mice after 4.5% DSS treatment as described for (c) (n = 6 mice/group). All data are representative of three independent experiments. Graphs show the means ± s.e.m. ****p < 0.0001 by twoway ANOVA followed by Tukey’s multiple comparison test (b). *P < 0.05, ***P < 0.001, and ****P < 0.0001 by two-tailed t-test (c,d,e).

[0023] FIG. 14 depicts that RNF5 mRNA levels negatively correlated with S100A8 levels and TREM1-CCL7-CCR2 axis in IBD patients non-re sponsive to anti-TNF treatment at baseline and following first anti-TNF treatment (a) Heatmap of Spearman's rank correlation coefficients of targeted genes including RNF5 and S100A8 and TREM1-CCL7-CCR2 axis related genes prior to anti-TNF treatment in responding and non-responding patients, in three publicly available mucosal gene expression datasets. Correlation coefficient values are represented by color key. (b) Heatmap presenting correlation of Crohn's disease patients pre and post anti-TNF first treatment based on GSE16879 cohort. R:responsive, NR: nonre sponsive, Pre:Pre-treatment (baseline), Post: Post-treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Certain specific details of this description are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures and/or methods have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments. Unless the context requires otherwise, throughout the specification and claims which follow, the word“comprise” and variations thereof, such as,“comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as“including, but not limited to.” Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed disclosure.

[0025] As used in this specification and the appended claims, the singular forms“a,”“an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term“or” is generally employed in its sense including“and/or” unless the content clearly dictates otherwise.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All references cited herein are incorporated by reference in their entirety as though fully set forth.

Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

Definitions

[0027] When indicating the number of substituents, the term“one or more” refers to the range from one substituent to the highest possible number of substitution, e.g. replacement of one hydrogen up to replacement of all hydrogens by substituents.

[0028] The term“optional” or“optionally” denotes that a subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs and instances in which it does not.

[0029] The term“nucleic acid” as used herein generally refers to one or more nucleobases, nucleosides, or nucleotides, and the term includes polynucleobases, polynucleosides, and

polynucleotides.

[0030] The term“polynucleotide”, as used herein generally refers to a molecule comprising two or more linked nucleic acid subunits, e.g., nucleotides, and can be used interchangeably with “oligonucleotide”. For example, a polynucleotide may include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (P0 ₃ ) groups. A nucleotide can include a nucleobase, a five-carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides include nucleotides in which the sugar is ribose.

Deoxyribonucleotides include nucleotides in which the sugar is deoxyribose. A nucleotide can be a nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate or a nucleoside polyphosphate. For example, a nucleotide can be a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP), Exemplary dNTPs include deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP). dNTPs can also include detectable tags, such as luminescent tags or markers (e.g., fluorophores). For example, a nucleotide can be a purine (e.g., A or G, or variant thereof) or a pyrimidine (e.g., C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. Exemplary polynucleotides include, but are not limited to, short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA), and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single-stranded, double-stranded, triple-stranded, helical, hairpin, stem loop, bulge, etc. In some cases, a polynucleotide is circular. A polynucleotide can have various lengths. For example, a polynucleotide can have a length of at least about 7 bases, 8 bases, 9 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. For example, polynucleotide sequences may comprise isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules, and/or synthetic DNA/RNA analogs.

[0031] Polynucleotides may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s) and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, l-methylguanine, l-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5’- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5- oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, 5-methyl-2-thiouracil, 3-(3-amino- 3- N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine and the like. In some cases, nucleotides may include modifications in their phosphate moieties, including modifications to a triphosphate moiety. Non-limiting examples of such modifications include phosphate chains of greater length (e.g. , a phosphate chain having, 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., alpha-thiotriphosphate and beta-thiotriphosphates). Nucleic acid molecules may also be modified at the base moiety (e.g. , at one or more atoms that typically are available to form a hydrogen bond with a complementary nucleotide and/or at one or more atoms that are not typically capable of forming a hydrogen bond with a complementary nucleotide), sugar moiety or phosphate backbone. Nucleic acid molecules may also contain amine -modified groups, such as amino ally l-dUTP (aa-dUTP) and aminohexhylacrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine reactive moieties, such as N-hydroxysuccinimide esters (NHS). Alternatives to standard DNA base pairs or RNA base pairs in the oligonucleotides of the present disclosure can provide higher density in bits per cubic mm, higher safety (resistant to accidental or purposeful synthesis of natural toxins), easier discrimination in photo-programmed polymerases, or lower secondary structure. Such alternative base pairs compatible with natural and mutant polymerases for de novo and/or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012

Jul;8(7):6l2-4, which is herein incorporated by reference for all purposes.

[0032] As used herein, the terms“polypeptide”,“protein” and“peptide” are used

interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. The terms“polypeptide”,“protein” and “peptide” refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the L-optical isomer or the D-optical isomer. More specifically, the terms“polypeptide”,“protein” and“peptide” refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Proteins are essential for the structure, function, and regulation of the body’s cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant.

[0033] As used herein, the term“biological sample” means any biological material from which polynucleotides, polypeptides, biomarkers, and/or metabolites can be prepared and examined. Non limiting examples encompasses whole blood, plasma, saliva, cheek swab, fecal specimen, urine specimen, cell mass, or any other bodily fluid or tissue.

[0034] The terms“administer,”“administering”,“administration, ” and the like, as used herein, refer to the methods that may be used to enable delivery of compounds or compositions to the desired site of biological action. These methods include, but are not limited to oral routes (p.o.), intraduodenal routes (i.d.), parenteral injection (including intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), intravascular or infusion (inf.)), topical (top.) and rectal (p.r.) administration. Those of skill in the art are familiar with administration techniques that can be employed with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.

[0035] The terms“co-administration” or the like, as used herein, are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[0036] The terms“effective amount” or“therapeutically effective amount,” as used herein, refer to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated; for example a reduction and/or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an“effective amount” for therapeutic uses can be an amount of an agent that provides a clinically significant decrease in one or more disease symptoms.

An appropriate“effective” amount may be determined using techniques, such as a dose escalation study, in individual cases.

[0037] The terms“enhance” or“enhancing,” as used herein, means to increase or prolong either in amount, potency or duration a desired effect. For example, in regard to enhancing splicing of a target, the term“enhancing” can refer to the ability to increase or prolong splicing, either in amount, potency or duration, of a the target.

[0038] The term“subject” or“patient” encompasses mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human. The term“animal” as used herein comprises human beings and non-human animals. In one embodiment, a“non-human animal” is a mammal, for example a rodent such as rat or a mouse. In one embodiment, a non-human animal is a mouse.

[0039] The terms“treat,”“treating” or“treatment,” as used herein, include alleviating, abating or ameliorating at least one symptom of a disease or condition, preventing additional symptoms, inhibiting the disease or condition, e.g. , arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or stopping the symptoms of the disease or condition either prophylactically and/or therapeutically.

[0040] The term“preventing” or“prevention” of a disease state denotes causing the clinical symptoms of the disease state not to develop in a subject that can be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

[0041] The terms“pharmaceutical composition” and“pharmaceutical formulation” (or “formulation”) are used interchangeably and denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject, e.g., a human in need thereof.

[0042] The term“pharmaceutical combination” as used herein, means a product that results from mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term“fixed combination” means that the active ingredients, e.g., a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term“non-fixed combination” means that the active ingredients, e.g. a compound described herein and a co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., administration of three or more active ingredients.

[0043] The term“pharmaceutically acceptable” denotes an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use.

“Pharmaceutically acceptable” can refer a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, e.g., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0044] The terms“pharmaceutically acceptable excipient”,“pharmaceutically acceptable carrier”, “pharmaceutically acceptable vehicle” and“therapeutically inert excipient” can be used

interchangeably and denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives or lubricants used in formulating pharmaceutical products [0045] The term“pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. A “pharmaceutically acceptable salt” can refer to a formulation of a compound or agent that does not cause significant irritation to an organism to which it is administered and/or does not abrogate the biological activity and properties of the compound or agent.

[0046] Methods for detection and/or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0047] Methods for detection and/or measurement of RNA in biological material are well known in the art and include, but are not limited to, Northern-blotting, RNA protection assay, RT PCR.

Suitable methods are described in Molecular Cloning: A Laboratory Manual (fourth Edition) By Michael R. Green, Joseph Sambrook, Peter MacCallum 2012, 2,028 pp, ISBN 978-1-936113-42-2.

[0048] A ribonucleoprotein (RNP) refers to a nucleoprotein that contains RNA. A RNP can be a complex of a ribonucleic acid and an RNA-binding protein. Such a combination can also be referred to as a protein-RNA complex. These complexes can function in a number of biological functions that include, but are not limited to, DNA replication, gene expression, metabolism of RNA, and pre- mRNA splicing. Examples of RNPs include the ribosome, the enzyme telomerase, vault

ribonucleoproteins, RNase P, heterogeneous nuclear RNPs (hnRNPs) and small nuclear RNPs (snRNPs).

[0049] Nascent RNA transcripts from protein-coding genes and mRNA processing intermediates, collectively referred to as pre-mRNA, are generally bound by proteins in the nuclei of eukaryotic cells. From the time nascent transcripts first emerge from RNA polymerase (e.g. , RNA polymerase II) until mature mRNAs are transported into the cytoplasm, the RNA molecules are associated with an abundant set of splicing complex components (e.g., nuclear proteins and snRNAs). These proteins can be components of hnRNPs, which can contain heterogeneous nuclear RNA (hnRNA) (e.g.. p re in RNA and nuclear RNA complexes) of various sizes.

[0050] As used herein, the term“biomarker” or "marker" are used interchangeably to refer to any biochemical marker, serological marker, genetic marker, or other clinical or echographic

characteristic that can be used to classify a sample from a patient as being associated with an inflammatory condition, such as IBD. Non-limiting examples of markers used herein include anti- PMN antibodies (e.g., APMNA, pAPMNA, cAPMNA, ANSNA, ASAPPA, and the like),

antimicrobial antibodies (e.g., anti -Outer-Membrane Protein, anti-OmpC antibodies (ACA), anti- flagellin antibodies (AFA), and the like), lactoferrin, elastase, C-reactive protein (CRP), calprotectin, hemoglobin, S100A8, S100A9, other protein markers and the like and combinations thereof, as well as autoantibodies to endogenous inflammation-related proteins such as calprotectin, integrin, lactoferrin, and/or CRP. The recitation of specific examples of markers associated with inflammatory conditions is not intended to exclude other markers as known in the art and suitable for use in the present invention.

[0051] As used herein, the term "antibody" includes but is not limited to a population of immunoglobulin molecules, which can be polyclonal or monoclonal and of any class and isotype, or a fragment of an immunoglobulin molecule. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl (human), IgA2 (human), IgAa (canine), IgAb (canine), IgAc (canine), and IgAd (canine). Such fragment generally comprises the portion of the antibody molecule that specifically binds an antigen. For example, a fragment of an immunoglobulin molecule known in the art as Fab, Fab' or F(ab')2 is included within the meaning of the term antibody.

[0052] As used herein, the term“neutralizing antibody” includes antibodies which are capable of specifically binding to an epitope on a protein and neutralizing the protein. Neutralizing antibodies also include antibodies which are capable of binding to an epitope on a protein and rendering the protein inactive. Neutralizing antibodies also include antibodies which are capable of inhibiting binding of a protein to its receptor. In some embodiments, the neutralizing antibodies are capable of binding to and neutralizing S100A8. In some embodiments, the neutralizing antibodies include recombinant and chimeric antibodies. In some embodiments, the neutralizing antibodies include human antibodies. In some embodiments, the neutralizing antibodies include a human variable region. In some embodiments, the neutralizing antibodies include a human light chain constant region. In some embodiments, the neutralizing antibodies include a human heavy chain constant region.

[0053] As used herein, the term "endogenous antibodies" refers to antibodies made by or originating from a subject, which can be isolated from the patient's blood or tissue. Typically, endogenous antibodies are generated in response to a foreign antigen, for example in response to a bacterial antigen, as part of the body's natural defense against infection. In certain cases, however, the patient may generate endogenous antibodies against the body's own proteins, such endogenous antibodies being referred to herein as "autoantibodies".

[0054] As used herein, "Inflammation" or "inflammatory condition" refers to a immunovascular response to a stimuli, including but not limited to, an immune response to an antigen, a pathogen, or a damaged cell, which is mediated by white blood cells. In some embodiments, the inflammation may be chronic. In some embodiments, the inflammation may be an autoimmune condition, where the immune system causes damage to otherwise normal, non-foreign tissue, as is seen for example in rheumatoid arthritis, multiple sclerosis, and other autoimmune diseases.

[0055] The term "label," as used herein, refers to a detectable compound, composition, or solid support, which can be conjugated directly or indirectly (e.g., via covalent or non-covalent means, alone or encapsulated) to a monoclonal antibody or a protein. The label may be detectable by itself (e.g., radioisotope labels, chemiluminescent dye, electrochemical labels, metal chelates, latex particles, or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, and the like). The label employed in the current invention could be, but is not limited to alkaline phosphatase; glucose-6-phosphate dehydrogenase ("G6PDH"); horseradish peroxidase (HRP); chemiluminescers such as isoluminol, fluorescers such as fluorescein and rhodamine compounds; ribozymes; and dyes. The label may also be a specific binding molecule which itself may be detectable (e.g., biotin, avidin, streptavidin, digioxigenin, maltose, oligohistidine, e.g., hex-histidine, 2, 4 -dinitrobenzene, phenylarsenate, ssDNA, dsDNA, and the like). The utilization of a label produces a signal that may be detected by means such as detection of electromagnetic radiation or direct visualization, and that can optionally be measured.

[0056] A monoclonal antibody can be linked to a label using methods well known to those skilled in the art, e.g., Immunochemical Protocols; Methods in Molecular Biology, Vol. 295, edited by R. Bums (2005)). For example, a detectable monoclonal antibody conjugate may be used in any known diagnostic test format like ELISA or a competitive assay format to generate a signal that is related to the presence or amount of an IBD-associated antibody in a test sample.

[0057] Substantial binding" or "substantially binding" refer to an amount of specific binding or recognizing between molecules in an assay mixture under particular assay conditions. In its broadest aspect, substantial binding relates to the difference between a first molecule's incapability of binding or recognizing a second molecule, and the first molecules capability of binding or recognizing a third molecule, such that the difference is sufficient to allow a meaningful assay to be conducted to distinguish specific binding under a particular set of assay conditions, which includes the relative concentrations of the molecules, and the time and temperature of an incubation. In another aspect, one molecule is substantially incapable of binding or recognizing another molecule in a cross-reactivity sense where the first molecule exhibits a reactivity for a second molecule that is less than 25%, e.g. less than 10%, e.g., less than 5% of the reactivity exhibited toward a third molecule under a particular set of assay conditions, which includes the relative concentration and incubation of the molecules. Specific binding can be tested using a number of widely known methods, e.g, an

immunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or a western blot assay.

[0058] As used herein, the term "substantially the same amino acid sequence" includes an amino acid sequence that is similar, but not identical to, the naturally-occurring amino acid sequence. For example, an amino acid sequence, e.g., polypeptide, that has substantially the same amino acid sequence as a flagellin protein can have one or more modifications such as amino acid additions, deletions, or substitutions relative to the amino acid sequence of the naturally-occurring flagellin protein, provided that the modified polypeptide retains substantially at least one biological activity of flagellin such as immunoreactivity. The "percentage similarity" between two sequences is a function of the number of positions that contain matching residues or conservative residues shared by the two sequences divided by the number of compared positions times 100. In this regard, conservative residues in a sequence is a residue that is physically or functionally similar to the corresponding reference residue, e.g., that has a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like.

[0059] The term "heterologous" refers to any two or more nucleic acid or polypeptide sequences that are not normally found in the same relationship to each other in nature. For instance, a heterologous nucleic acid is typically recombinantly produced, having two or more sequences, e.g., from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous polypeptide will often refer to two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0060] As used herein, the term "fragment" includes a peptide, polypeptide or protein segment of amino acids of the full-length protein, provided that the fragment retains reactivity with at least one antibody in sera of disease patients.

[0061] An "epitope" is the antigenic determinant on a polypeptide that is recognized for binding by a paratope on antibodies specific to the polypeptide, for example, an IBD -associated antibody.

[0062] The term "clinical factor" includes a symptom in a patient that is associated with IBD. Examples of clinical factors include, without limitation, diarrhea, abdominal pain and/or discomfort, cramping, fever, anemia, hypoproteinemia, weight loss, anxiety, lethargy, and combinations thereof.

In some embodiments, a diagnosis of IBD is based upon a combination of analyzing the presence or level of one or more markers in a patient using statistical algorithms and determining whether the patient has one or more clinical factors.

[0063] The term "prognosis" includes a prediction of the probable course and outcome of IBD or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of IBD in a patient. For example, the prognosis can be surgery, development of a clinical subtype of IBD, development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.

Inflammatory disease

[0064] Inflammation is a crucial process in the normal defense mechanisms against various pathogens, and leukocytes are the principal cellular mediators of inflammation. Inflammation is characterized histologically by the accumulation of leukocytes in the affected tissue due to migration of circulating leukocytes out of the vasculature, a process which is actively mediated and precisely controlled by leukocytes, the cytokines they produce, and the vascular endothelium.

[0065] Inflammation is usually a normal, healthy response to injury or infection. However, excessive or uncontrolled inflammatory responses can lead to the pathologic inflammation seen in many rheumatologic and inflammatory disorders, where the inflammation, rather than promoting healing, seriously damages normal tissues, resulting in chronic pain, contributing to a wide variety of serious disorders, in some cases increasing the risk of cancer and heart disease, and in some cases even causing death. Inflammatory bowel disease (IBD), for example, is a debilitating and progressive disease involving inflammation of the gastrointestinal tract. Symptoms include abdominal pain, cramping, diarrhea and bleeding.

[0066] One indication of such inflammatory diseases is the presence of inflammatory cells such as neutrophils and macrophages at local sites of inflammation. Inflammation is a response of vascularized tissue to infection and/or injury and it is affected by adhesion of leukocytes to the endothelial cells of blood vessels and their infiltration into the surrounding tissues. Such local concentrations can be detected by invasive methods requiring biopsy procedures and pathology analysis. The inflammatory state can also he systemic, e.g. polypeptides secreted by inflammatory cells become detectable in the blood serum.

[0067] Non-limiting examples of autoimmune disease include inflammatory bowel disease, rheumatoid arthritis, diabetes mellitus, celiac disease, autoimmune thyroid disease, autoimmune liver disease, Addison’s Disease, Sjogren’s Syndrome, transplant rejection, graft vs. host disease, or host vs. graft disease.

[0068] Inflammatory bowel disease (IBD) encompasses a group of inflammatory disorders that affect areas of the gastrointestinal tract. IBD describes idiopathic gastrointestinal disorders

characterized by persistent or recurrent gastrointestinal (GI) signs and histological evidence of GI inflammation for which no underlying cause can be found.

[0069] Two main types of inflammatory bowel disease are Crohn’s disease and ulcerative colitis. Inflammatory bowel disease or "IBD" also refers to other chronic inflammation of all or part of the gastrointestinal tract, including, without limitation, the following sub-types: ulcerative colitis, Crohn's disease, lymphoplasmacytic enteritis (LPE), eosinophilic gastroenteritis (EGE) and granulomatous enteritis (GE).

[0070] Symptoms of Crohn’s disease and ulcerative colitis usually involve severe and chronic diarrhea, pain, fatigue and weight loss. Effective treatment of IBD requires differentiating the condition from other gastrointestinal disorders that do not necessarily involve chronic inflammation. Current methods of diagnosing IBD include using biomarkers associated with gastrointestinal infection, for example by detecting antibodies to anti-neutrophil cytoplasmic antibody (ANCA), anti- Saccharomyces cerevisiae immunoglobulin A (ASCA-IgA), anti-Saccharomyces cerevisiae immunoglobulin G (ASCA-IgG), an anti-outer membrane protein C (anti-OmpC) antibody, an anti- flagellin antibody, an anti -12 antibody, and a perinuclear anti -neutrophil cytoplasmic antibody

(pANCA), and other biomarkers.

[0071] Traditional treatments for IBD often require one or more surgical resections of the bowel, which are intrusive and can lead to a syndrome called“short gut syndrome,” where the remaining length of the bowel is insufficient to support life without lifetime intravenous feeding with total parenteral nutrition.

[0072] One focus in IBD research pertains to a group of immunogenic endogenous damage - associated molecular patterns (DAMPs). These include S100 proteins and high mobility group box-l (HMGB1), which are released during tissue damage and bind to pattern recognition receptors (PRRs) on innate immune cells, eliciting inflammatory responses (Boyapati et ah, Mucosal Immunol 9, 567- 582 (2016)). Notably, DAMPs are increasingly recognized to play a role in the etiology of IBD (Boyapati et ah, Mucosal Immunol 9, 567-582 (2016)). Several DAMPs, including the calprotectin complex of S100A8/S100A9 proteins, are present at high levels in the serum and corresponding intestinal tissues during inflammation, and some have been established as valuable biomarkers for a number of colonic inflammatory conditions (Sands, Gastroenterology 149, 1275-1285 el272 (2015)). Correspondingly, level of calprotectin in fecal samples is largely considered as a sensitive and specific biomarker, though inter- and intra-personal variation has been reported (Sands, Gastroenterology 149, 1275-1285 el272 (2015)). S100A8 and S100A9 are primarily expressed in neutrophils and monocytes, and induced under inflammatory conditions in keratinocytes and epithelial cells (Henke et ah, Exp Lung Res 32, 331-347 (2006); Mork et al.,Br J Dermatol 149, 484-491 (2003)). Disclosed herein are regulators and proteins associated with DAMPs and use as therapeutic targets in human inflammatory diseases.

[0073] The RING finger E3 ubiquitin ligase RNF5 was shown to be membrane anchored as part of the endoplasmic reticulum (ER) - associated degradation (ERAD) machinery (Matsuda and Nakano, 1998; Matsuda et ah, 2001). As part of ERAD, RNF5 is involved in the control of misfolded proteins implicated in cystic fibrosis (Y ounger et al, 2006) and inclusion body myocytis (Delaunay et ah, 2008). RNF5 plays a role in several pathological conditions. In tumor cells, RNF5 controls the stability of the glutamine carriers SLC1A5 and SLC38A2, which limits glutamine uptake and renders the tumor cells more sensitive to ER stress -inducing chemotherapy (Jeon et al., 2015). Under normal growth conditions, RNF5 was shown to control the level of the ATG4B protein, which is important for LC3 maturation and autophagosome formation and thus controls degree of group A streptococcus infection (Kuang et al., 2012). RNF5 was also implicated in the regulation of viral and bacterial infection, through control of immune sensing mechanism (Zhong et al., 2009), pointing to a possible role RNF5 may play in inflammatory diseases.

[0074] Disclosed herein are methods of diagnosing, predicting susceptibility, protection against, prognosing, treating, ameliorating symptoms of an immunodisorder in a subject in need thereof. In some embodiments, the immunodisorder is. In some embodiments, the immunodisorder is inflammatory bowel disease, rheumatoid arthritis, diabetes mellitus, celiac disease, autoimmune thyroid disease, autoimmune liver disease, Addison’s Disease, Sjogren’s Syndrome, transplant rejection, graft vs. host disease, or host vs. graft disease. In certain embodiments, the immunodisorder is IBD. In certain embodiments, the IBD is ulcerative colitis. In certain embodiments, the IBD is Crohn’s disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0075] In one aspect, the present invention provides inflammatory markers and methods for detecting the inflammatory markers useful in diagnosis and prevention of inflammatory disease, on a systematic and/or localized basis such as in the gastrointestinal tract. Non-limiting examples of inflammation related markers include anti-polymorphonuclear (PMN) leukocyte antibodies, calprotectin, beta -integrins, lactoferritin, and C-reactive protein, MHC class I and class II markers, H2-BI, Qpct, Lc6c2, Ang4, H2-T10, Padi2, Oxtr, Jchain, CcrlO, Gm8909, Ki67, Cdl lc, Ly6g, Ly6c, F4/80, TNF-a TNF-b, TNF-related ligand, INFy, S100 family proteins, S100A8, S100A9, and any combination thereof.

[0076] In one aspect, the present invention provides a method for monitoring the progression or regression of an inflammatory disease or condition. In some embodiments, the presence or level of at least one inflammation-associated marker can be determined, and the severity of the inflammatory disease or condition can be inferred based on the presence or level of the at least one inflammation- associated marker. In some embodiments, the inflammatory disease is IBD. Non -limiting examples of inflammation related markers include anti-polymorphonuclear (PMN) leukocyte antibodies, calprotectin, beta-integrins, lactoferritin, and C-reactive protein, MHC class I and class II markers, H2-BI, Qpct, Lc6c2, Ang4, H2-T10, Padi2, Oxtr, Jchain, CcrlO, Gm8909, Ki67, Cdl lc, Ly6g, Ly6c, F4/80, TNF-a TNF-b, TNF-related ligand, INFy, S100 family proteins, S100A8, S100A9, and any combination thereof.

[0077] In some embodiments, the frequency of at least one type of inflammation-associated cells can be determined, and the severity of the inflammatory disease or condition can be inferred based on the count or frequency of the at least one type of inflammation associated cells . In some

embodiments, the inflammation associated cells are dendritic cells. In some embodiments, the inflammation associated cells are T cells. In some embodiments, the inflammation associated cells are Th cells. In some embodiments, the inflammation associated cells are macrophages. In some embodiments, the inflammation associated cells are neutrophils. In some embodiments, the inflammation associated cells are inflammatory monocytes. In some embodiments, the inflammation associated cells are epithelial cells. In some embodiments, the inflammation associated cells are CD4+ cells, CD4+CD44+ cells, CD4+CD25+ cells, CD8+ cells, CD8+CD44+ cells, colonic DCs, colonic macrophages, or any combination thereof.

[0078] In another aspect, the present invention provides a method for monitoring, screening, and/or determining the efficiency of a drug candidate for treatment of an inflammatory disease or condition. For example, the presence or level of at least one inflammation related marker in a subject can be determined prior to treatment with a drug candidate and compared with the presence or level of the at least one inflammation related marker in the subject post treatment of the drug candidate. Non limiting examples of inflammation related markers include anti-polymorphonuclear (PMN) leukocyte antibodies, calprotectin, beta -integrins, lactoferritin, and C-reactive protein, MHC class I and class II markers, H2-BI, Qpct, Lc6c2, Ang4, H2-T10, Padi2, Oxtr, Jchain, CcrlO, Gm8909, Ki67, Cdl lc, Ly6g, Ly6c, F4/80, TNF-a TNF-b, TNF-related ligand, INFy, S100 family proteins, S100A8, S100A9, and any combination thereof.

[0079] In some embodiments, the presence or level of at least one inflammation associated cells in a subject can be determined prior to treatment with a drug candidate and compared with the presence or level of the at least one inflammation related marker in the subject post treatment of the drug candidate. In some embodiments, the inflammation associated cells are dendritic cells. In some embodiments, the inflammation associated cells are T cells. In some embodiments, the inflammation associated cells are Th cells. In some embodiments, the inflammation associated cells are

macrophages. In some embodiments, the inflammation associated cells are neutrophils. In some embodiments, the inflammation associated cells are inflammatory monocytes. In some embodiments, the inflammation associated cells are epithelial cells. In some embodiments, the inflammation associated cells are CD4+ cells, CD4+CD44+ cells, CD4+CD25+ cells, CD8+ cells, CD8+CD44+ cells, colonic DCs, colonic macrophages, or any combination thereof. [0080] In an aspect, the invention provides methods for treating or preventing an autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent inhibits the expression and/or activity of an

immunogenic endogenous damage associated protein. In some embodiments, the invention provides methods for treating or preventing an autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent inhibits the expression and/or activity of protein that binds to pattern recognition receptor (PRR) on innate immune cells. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a high mobility group box-l (HMGB1) protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a S 100 protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of S100A8. In some embodiments, the inhibitory agent inhibits the expression and/or activity of S100A8 and does not affect the expression and/or activity of S100A9.

[0081] In some embodiments, the invention provides methods for treating or preventing an autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent inhibits the expression and/or activity of a immunogenic endogenous damage associated protein, wherein the inhibitory agent binds to and neutralizes the immunogenic endogenous damage associated protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a high mobility group box-l (HMGB1), wherein the inhibitory agent binds to and/or neutralizes the HMGB1 protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a S 100 family protein, wherein the inhibitory agent binds to and/or neutralizes the S 100 family protein. In some embodiments, the inhibitory agent is an immunoglobulin. In some embodiments, the inhibitory agent is an anti-SlOO antibody. In some embodiments, the inhibitory agent is an anti-Sl00A8 antibody. In some embodiments, the inhibitory agent is an anti-Sl00A8 antibody that does not neutralize S100A9. In some embodiments, the inhibitory agent is an anti-Sl00A8 monoclonal antibody. In some embodiments, the inhibitory agent is an anti-Sl00A8 polyclonal antibody. In certain embodiments, the inhibitory agent binds to and/or neutralizes S100A8 in extracellular milieu of a subject. In some embodiments, the inhibitory agent binds to and/or neutralizes S 100A8 in extracellular milieu at inflammatory sites. In some embodiments, the inhibitory agent binds to and/or neutralizes S100A8 in extracellular milieu in the small intestine, in the large intestine, or in the colon. In some embodiments, the inhibitory agent binds to and/or neutralizes S100A8 secreted by neutrophils, monocytes, epithelial cells, or other cells of a subject.

[0082] In some embodiments, the invention provides methods for treating or preventing an immunodisorder comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent decreases the expression of an immunogenic endogenous damage associated protein. In some embodiments, the inhibitory agent decreases the expression and/or activity of a high mobility group box-l (HMGB1) protein in a cell. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes a HMGB1 protein in a cell. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a S100 family protein. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes the S100 family protein. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes S100A8.

[0083] In some embodiments, the inhibitory agent comprises a RNA molecule. In some embodiments, the inhibitory agent comprises a non-coding RNA molecule. In some embodiments, the non-coding RNA molecule comprises a microRNA, an siRNA, an anti-sense RNA, or any combination thereof. In some embodiments, the non-coding RNA molecule comprises a microRNA that targets mRNA that encodes an immunogenic endogenous damage associated protein. In some embodiments, the non-coding RNA comprises a siRNA that targets mRNA that encodes an immunogenic endogenous damage associated protein. In some embodiments, the non-coding RNA molecule comprises a microRNA that targets mRNA that encodes a HMGB1 protein. In some embodiments, the non -coding RNA comprises a siRNA that targets mRNA that encodes a HMGB1 protein. In some embodiments, the non-coding RNA molecule comprises a microRNA that targets mRNA that encodes a S100 family protein. In some embodiments, the non -coding RNA comprises a siRNA that targets mRNA that encodes a S 100 family protein. In some embodiments, the non-coding RNA molecule comprises a microRNA that targets mRNA that encodes a S 100A8 protein. In some embodiments, the non-coding RNA comprises a siRNA that targets mRNA that encodes S100A8 protein.

[0084] Therapeutic approaches based on siRNAs and microRNAs are available using methods well known to those skilled in the art. For example, a synthetic siRNA can be introduced into the target cells to elicit RNA interference (RNAi), thereby inhibiting the expression of a specific messenger RNA (mRNA) to produce a gene silencing effect. For example, single stranded RNAs acting as microRNA antagonists (also known as antagomirs or anti-miRs) can be introduced to inhibit the action of the endogenous miRNAs. In the replacement approach, synthetic miRNAs (also known as miRNA mimics) can be introduced to mimic the function of the endogenous miRNAs.

[0085] In some embodiments, the inhibitory agent comprises a protein. In some embodiments, the inhibitory agent comprises a nuclease that catalyzes cleavage of a polynucleotide that encodes an immunogenic endogenous damage associated protein in a cell. In some embodiments, the inhibitory agent comprises a nuclease that catalyzes cleavage of a polynucleotide that encodes a HMGB1 protein in a cell. In some embodiments, the inhibitory agent comprises a nuclease that catalyzes cleavage of a polynucleotide that encodes a S100 family protein in a cell. In some embodiments, the inhibitory agent comprises a nuclease that catalyzes cleavage of a polynucleotide that encodes S100A8 in a cell. In some embodiments, the inhibitory agent comprises a nuclease that catalyzes cleavage of a polynucleotide in a cell that does not involve insertion, deletion, substitution, frameshifting, or other genome editing events in the genome of the cell. Non-limiting examples of nucleases include zinc finger nuclease, fokl nuclease, meganuclease, Cas proteins. In some embodiments, the nuclease is a C2c2 nuclease.

[0086] In some embodiments, the inhibitory agent comprises a nucleic acid-guided protein complexed with a guide nucleic acid that recognize specific polynucleotide sequences in a cell. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, the inhibitory agent comprises a RNA-guided CRISPR/Cas protein. In some embodiments, the CRISPR/Cas protein is type II CRISPR/Cas protein, a type V CRISPR/Cas protein, a type VII CRISPR/Cas protein, Cas9, CasX, CasY, Cpfl, C2cl, C2c2, or C2c3, or other CRISPR/Cas proteins. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a polynucleotide that encodes a immunogenic endogenous damage associated protein in a cell. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a polynucleotide that encodes a HMGB1 protein in a cell. In some embodiments, the polynucleotide comprises a RNA sequence that is reverse complementary to a polynucleotide that encodes a S100 family protein in a cell. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a polynucleotide that encodes S 100A8 in a cell. In a preferred embodiment, the guide RNA comprises a RNA sequence that is reverse complementary to mRNA that encodes S100A8 in a cell. In a preferred embodiment, the CRISPR/Cas protein is C2c2.

[0087] In some embodiments, the inhibitory agent comprises a nucleic acid-guided protein complexed with a guide RNA that recognizes specific polynucleotide sequences in a cell. In some embodiments, the nucleic acid is a guide RNA. In some embodiments, the inhibitory agent comprises a RNA-guided CRISPR/Cas protein. In some embodiments, the CRISPR/Cas protein is type II CRISPR/Cas protein, a type V CRISPR/Cas protein, a type VII CRISPR/Cas protein, Cas9, CasX, CasY, Cpfl, C2cl, C2c2, or C2c3, or other CRISPR/Cas proteins. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a DNA that encodes a

immunogenic endogenous damage associated protein in a cell. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a DNA that encodes a HMGB1 protein in a cell. In some embodiments, the polynucleotide comprises a RNA sequence that is reverse complementary to a DNA that encodes a S100 family protein in a cell. In some embodiments, the guide RNA comprises a RNA sequence that is reverse complementary to a DNA that encodes S100A8 in a cell. In some embodiments, the In some embodiments, the CRISPR/Cas protein comprises a mutation in the nuclease domain. In some embodiments, the CRISPR/Cas protein comprises a mutation in the nuclease domain that reduces or abolishes the catalytic activity of the nuclease domain. In some embodiments, the CRISPR/Cas protein comprises a mutation in the nuclease domain that renders the nuclease domain a nickase domain. In some embodiments, the CRISPR/Cas protein is a Cas9 protein comprising mutations D10A and/or H840A. In some embodiments, the CRISPR/Cas protein lacks the HNH nuclease domain. In some embodiments, the CRISPR/Cas protein further comprises an effector domain. In certain embodiments, the effector domain is a transcriptional repressor domain, DNA methyl transferase domain, histone acetyltransferase domain, histone deacetylase domain, and combinations thereof.

[0088] A guide nucleic acid (e.g., guide RNA) can bind to a Cas protein and target the Cas protein to a specific location within a target polynucleotide. A guide nucleic acid can comprise a nucleic acid targeting segment and a Cas protein binding segment.

[0089] A guide nucleic acid can refer to a nucleic acid that can hybridize to another nucleic acid, for example, the target polynucleotide in the genome of a cell. A guide nucleic acid can be RNA, for example, a guide RNA. A guide nucleic acid can be DNA. A guide nucleic acid can comprise DNA and RNA. A guide nucleic acid can be single stranded. A guide nucleic acid can be double -stranded.

A guide nucleic acid can comprise a nucleotide analog. A guide nucleic acid can comprise a modified nucleotide. The guide nucleic acid can be programmed or designed to bind to a sequence of nucleic acid site-specifically.

[0090] A guide nucleic acid can comprise one or more modifications to provide the nucleic acid with a new or enhanced feature. A guide nucleic acid can comprise a nucleic acid affinity tag. A guide nucleic acid can comprise synthetic nucleotide, synthetic nucleotide analog, nucleotide derivatives, and/or modified nucleotides.

[0091] The guide nucleic acid can comprise a nucleic acid-targeting region (e.g., a spacer region), for example, at or near the 5’ end or 3’ end, that is complementary to a protospacer sequence in a target polynucleotide. The spacer of a guide nucleic acid can interact with a protospacer in a sequence-specific manner via hybridization (base pairing). The protospacer sequence can be located 5’ or 3’ of protospacer adjacent motif (PAM) in the target polynucleotide. The nucleotide sequence of a spacer region can vary and determines the location within the target nucleic acid with which the guide nucleic acid can interact. The spacer region of a guide nucleic acid can be designed or modified to hybridize to any desired sequence within a target nucleic acid.

[0092] A guide nucleic acid can comprise two separate nucleic acid molecules, which can be referred to as a double guide nucleic acid. A guide nucleic acid can comprise a single nucleic acid molecule, which can be referred to as a single guide nucleic acid (e.g., sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a fused CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid comprising a crRNA but lacking a tracRNA. In some embodiments, the guide nucleic acid is a double guide nucleic acid comprising non-fused crRNA and tracrRNA. An exemplary double guide nucleic acid can comprise a crRNA-like molecule and a tracrRNA- like molecule. An exemplary single guide nucleic acid can comprise a crRNA-like molecule. An exemplary single guide nucleic acid can comprise a fused crRNA-like and tracrRNA-like molecules. [0093] A crRNA can comprise the nucleic acid-targeting segment (e.g., spacer region) of the guide nucleic acid and a stretch of nucleotides that can form one half of a double -stranded duplex of the Cas protein- binding segment of the guide nucleic acid.

[0094] A tracrRNA can comprise a stretch of nucleotides that forms the other half of the double - stranded duplex of the Cas protein-binding segment of the gRNA. A stretch of nucleotides of a crRNA can be complementary to and hybridize with a stretch of nucleotides of a tracrRNA to form the double -stranded duplex of the Cas protein-binding domain of the guide nucleic acid.

[0095] The crRNA and tracrRNA can hybridize to form a guide nucleic acid. The crRNA can also provide a single- stranded nucleic acid targeting segment (e.g., a spacer region) that hybridizes to a target nucleic acid recognition sequence (e.g., protospacer). The sequence of a crRNA, including spacer region, or tracrRNA molecule can be designed to be specific to the species in which the guide nucleic acid is to be used.

[0096] Provided herein are methods and compositions for treatment and prevention of immunodisorders comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent. In some embodiments, the inhibitory agent comprises a protein comprising a nucleotide recognition domain, e.g. a DNA recognition domain. In some embodiments, the inhibitory agent comprises a protein comprising a nucleotide recognition domain, e.g. a DNA recognition domain, and an effector domain. In some embodiments, the effector domain is a transcriptional activator domain, transcriptional repressor domain, DNA methyl transferase domain, DNA demethylase domain, histone acetyltransferase domain, histone deacetylase domain, and combinations thereof. In some embodiments, the nucleotide recognition domain is derived from, or homologous to, a transcription activator like effector (TALE) DNA recognition domain. In some embodiments, the nucleotide recognition domain is derived from, or homologous to a zinc finger DNA recognition domain. In some embodiments, the nucleotide recognition domain is derived from, or homologous to a helix-tum-helix domain, a leucine zipper domain, a winged helix domain, a CRISPR/Cas protein DNA binding domain, a Wor3 domain, a HMG box, a OB fold domain, or any combination thereof. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes an immunogenic endogenous damage associated protein in a cell. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a high mobility group box-l (HMGB1) protein in a cell. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a S100 family protein in a cell. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes S100A8 in a cell. In some embodiments, the polynucleotide is a DNA.

[0097] In some embodiments, the decrease in expression of S100A8 comprises a decrease of 1.1- fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5 fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2. l-fold, 2.2- fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.0-fold, 3.0-fold, 3.l-fold, 3.2-fold, 3.3- fold, 3.4-fold, 3.5-fold, 4-fold, 5-fold, lO-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more in a treated with the method and composition described herein compared to expression of S100A8 in a control cell. In some embodiments, the decrease in expression of S100A8 comprises a decrease of about 20% to about 100%, about 50% to about 100%, about 20% to about 50%, at least about 20%, at least about 50%, compared to expression of S100A8 in a control cell.

[0098] In an aspect, the invention provides methods for treating or preventing an

immunodisorder comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent increases expression or activity of a protein associated with the immunodisorder. In some embodiments, the invention provides methods for treating or preventing an autoimmune disease comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent, wherein the inhibitory agent increases the expression and/or activity of a protein involved in protein expression control and targeted degradation. In some embodiments, the protein is involved in ubiquitination associated pathways. In some embodiments, the protein is involved in endoplasmic reticulum associated degradation (ERAD) machinery. In some embodiments, the protein is involved in control of immune sensing. In a preferred embodiment, the protein is RING finger E3 ubiquitin ligase RNF5.

[0099] In some embodiments, the inhibitory agent comprises a nucleic acid-guided protein complexed with a guide nucleic acid, e.g. a guide RNA, that recognize specific polynucleotide sequences in a cell. In some embodiments, the inhibitory agent comprises a RNA-guided CRISPR/Cas protein. In some embodiments, the CRISPR/Cas protein is type II CRISPR/Cas protein, a type V CRISPR/Cas protein, a type VII CRISPR/Cas protein, Cas9, CasX, CasY, Cpfl, C2cl, C2c2, or C2c3, or other CRISPR/Cas proteins. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is reverse complementary to a polynucleotide that encodes a protein that is involved in protein expression control and targeted degradation in a cell. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is reverse complementary to a polynucleotide that encodes a protein involved in endoplasmic reticulum associated degradation (ERAD) machinery in a cell. In some embodiments, the guide nucleic acid comprises a nucleic acid sequence that is reverse complementary to a polynucleotide that encodes a protein is involved in control of immune sensing in a cell. In a preferred embodiment, the guide nucleic acid comprises a nucleic acid sequence that is reverse complementary to a polynucleotide that encodes RING finger E3 ubiquitin ligase RNF5 in a cell. In some embodiments, the CRISPR/Cas protein comprises a mutation in the nuclease domain. In some embodiments, the CRISPR/Cas protein comprises a mutation in the nuclease domain that reduces or abolishes the catalytic activity of the nuclease domain. In some embodiments, the

CRISPR/Cas protein is a Cas9 protein comprising mutations D10A and/or H840A. In some embodiments, the CRISPR/Cas protein lacks the HNH nuclease domain. In some embodiments, the CRISPR/Cas protein further comprises an effector domain. In certain embodiments, the effector domain is a transcriptional activator domain, DNA methyl transferase domain, DNA demethylase domain, histone deacetylase domain, and combinations thereof.

[00100] Provided herein are methods and compositions for treatment and prevention of immunodisorders comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitory agent. In some embodiments, the inhibitory agent comprises a protein comprising a nucleotide recognition domain, e.g. a DNA recognition domain. In some embodiments, the inhibitory agent comprises a protein comprising a nucleotide recognition domain, e.g. a DNA recognition domain, and an effector domain. In some embodiments, the effector domain is a transcriptional activator domain, DNA demethylase domain, histone deacetylase domain, and combinations thereof. In some embodiments, the nucleotide recognition domain is derived from, or homologous to, a transcription activator like effector (TALE) DNA recognition domain. In some embodiments, the nucleotide recognition domain is derived from, or homologous to a zinc finger DNA recognition domain. In some embodiments, the nucleotide recognition domain is derived from, or homologous to a helix-tum-helix domain, a leucine zipper domain, a winged helix domain, a Wor3 domain, a HMG box, a OB fold domain, or any combination thereof. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a protein involved in protein expression control and targeted degradation. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a protein involved in ubiquitination associated pathways. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a protein involved in endoplasmic reticulum associated degradation (ERAD) machinery. In some embodiments, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes a protein involved in control of immune sensing. In a preferred embodiment, the nucleotide recognition domain recognizes and binds to a sequence in a polynucleotide that encodes RING finger E3 ubiquitin ligase RNF5.

[00101] In some embodiments, the increase in expression of RNF5 comprises an increase of 1.1- fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5 fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2. l-fold, 2.2- fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.0-fold, 3.0-fold, 3.l-fold, 3.2-fold, 3.3- fold, 3.4-fold, 3.5-fold, 4-fold, 5-fold, lO-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more in a treated with the method and composition described herein compared to expression of S100A8 in a control cell. In some embodiments, the increase in expression of RNF5 comprises a decrease of about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300%, compared to expression of RNF5 in a control cell. In some embodiments, the increase in expression of RNF5 comprises a decrease of about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 20% to about 50%, about 20% to about 100%, about 20% to about 150%, about 20% to about 200%, about 20% to about 250%, about 50% to about 100%, about 50% to about 150%, about 50% to about 200%, about 50% to about 250%, about 100% to about 150%, about 100% to about 200%, about 100% to about 250%, about 150% to about 200%, about 150% to about 250%, about 200% to about 250%, at least about 20%, at least about 50%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, or at least about 300% compared to the expression of Sl00A8 in a control cell.

[00102] In one aspect, provided herein is a pharmaceutical composition for treatment, prevention, or ameliorating symptoms of an immunodisorder, wherein the composition comprises an inhibitory agent. In some embodiments, the immnunodisorder is an inflammatory bowel disease (IBD), rheumatoid arthritis, diabetes mellitus, celiac disease, autoimmune thyroid disease, autoimmune liver disease, Addison’s Disease, Sjogren’s Syndrome, transplant rejection, graft vs. host disease, or host vs. graft disease. In some embodiments, the immunodisorder is Crohn’s disease. In some embodiments, the immunodisorder is ulcerative colitis. In some embodiments, the inhibitory agent inhibits the expression and/or activity of an immunogenic endogenous damage associated protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of protein that binds to pattern recognition receptor (PRR) on innate immune cells. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a high mobility group box-l (HMGB1) protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a S 100 protein. In some embodiments, the inhibitory agent inhibits the expression and/or activity of S 100A8. In some embodiments, the inhibitory agent inhibits the expression and/or activity of S100A8 and does not affect the expression and/or activity of S 100A9. In some embodiments, the inhibitory agent decreases the expression of an immunogenic endogenous damage associated protein. In some embodiments, the inhibitory agent decreases the expression and/or activity of a high mobility group box-l (HMGB1) protein in a cell. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes a HMGB1 protein in a cell. In some embodiments, the inhibitory agent inhibits the expression and/or activity of a S 100 family protein. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes the S 100 family protein. In some embodiments, the inhibitory agent decreases the amount of mRNA that encodes S100A8. In some embodiments, the inhibitory agent increases the expression and/or activity of a protein involved in protein expression control and targeted degradation. In some embodiments, the inhibitory agent increases the expression and/or activity of a protein involved in ubiquitination associated pathways. In some embodiments, the inhibitory agent increases the expression and/or activity of a protein involved in endoplasmic reticulum associated degradation (ERAD) machinery. In some embodiments, the inhibitory agent increases the expression and/or activity of a protein involved in control of immune sensing. In a preferred embodiment, the inhibitory agent increases expression and/or activity of RING finger E3 ubiquitin ligase RNF5.

[00103] In some embodiments, the composition described herein comprises an anti-inflammatory agent, e.g. glucocorticoids, progestins, mineralocorticoids, corticosteroids, ibuprofen, naproxen, aspirin, ketoprofen, nepafenac, diclofenac, indomethacin, piroxicam, meloxicam, sulindac, methotrexate, lefhmomide, hydroxychloroquine, sulfasalazine, prednisone, dexamethasone, cortisone, loteprendol, triamcinolone acetonide, fluocinolone acetonide, fluorometholone,

fluticasone, .aminosalicylate, or any combination thereof. In some embodiments, the composition described herein comprises an immuno-suppressor agent, e.g. cyclosporine, tacrolimus, rapamycin, or any combination thereof. In some embodiments, the composition described herein comprises a pro- inflammatory cytokine inhibitor, e.g., a TNF-a inhibitor, an IF-8 inhibitor, an IF-13 inhibitor, an IF- 17 inhibitor, an IF- 18 inhibitor, an IF-21 inhibitor, an IEIb inhibitor, an IF-6 inhibitor, an IFl2p35 inhibitor, or any combination thereof. In some embodiments, the composition described herein comprises a TFR antagonist. In some embodiments, the composition described herein further comprises an antibiotic, e.g. norfloxacin, fluconaxole, valacyclovir, ciprofloxacin, metronidazole, clarithromycin, levofloxacin, or any combination thereof. In some embodiments, the composition described herein further comprises a commensal bacterium. In some embodiments, the commensal bacterium is obtained from a microbiota of a healthy mammal. In some embodiments, the microbiota comprises bacteria belonging to In some embodiments, the microbiota comprises bacteria belonging to the genera Faecalibacterium, Clostridium, Bifidobacterium, Bacteroides, Helicobacter, Roseburia, Eubacterium, or any combination thereof. In some embodiments, the microbiota comprises bacteria species Oscillibacter valerici genes, Acetatifactor muris, Alistipes putredinis, Alistipes fimegoldii, Clostridium clostridioforme, Lactobacillus animalis, Lactobacillus murinus, Bacteroides massiliensis, Bacteroides sartorii, Muribaculum intestinale, Parasutterella excrementihominis, Bacteroides acidifaciens, Bacteroides xylanisolvens, Bacteroides chinchilla Clostridium methylpentosum, Bacteroides rodentium, Dysgonomonas wimpennyi, Parabacteroides merdae, Flavobacterium, Staphylococcus spp., Staphylococcus sciuri, Staphylococcus xylosus, Helicobacter ganmani,

Helicobacter hepaticus, Enterobacter hormaechei, Porphyromonas canis, Porphyromonas gingivicanis, Rickenella microfusus, Olivibacter spp, P. golds teinii, P. koreensis, Pedobacter spp., O. sinus, Blautia hansenii, Lachnospira pectinoschiza, or any combination thereof. In some

embodiments, the composition described herein further comprises a prebiotic. In some embodiments, the prebiotic comprises a mucin (e.g., porcine gastric mucin), inulin, N-acetyl- D-glucosamine, N- acetyl-D-mannosamine, glucose- 1 -phosphate, D-fructose, a galactomannan, N-acetyl mannosamine, N-acetylgalactosamine, N-acetylneuraminic acid, N- acetyl glucosamine, galactose, fucose, mannose, human milk oligosaccharides, guar gum, dextrin, a-cellulose, b-D glucan, pectin, com starch, potato starch, or any combination thereof.

[00104] In some embodiments, the compositions described herein are formulated into pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999), herein incorporated by reference for such disclosure.

[00105] A pharmaceutical composition can be a mixture of a composition or inhibitory agent described herein with one or more other chemical components (e.g. pharmaceutically acceptable ingredients), such as carriers, excipients, binders, filling agents, suspending agents, flavoring agents, sweetening agents, disintegrating agents, dispersing agents, surfactants, lubricants, colorants, diluents, solubilizers, moistening agents, plasticizers, stabilizers, penetration enhancers, wetting agents, anti foaming agents, antioxidants, preservatives, or one or more combination thereof. The pharmaceutical composition facilitates administration of the compound to an organism.

[00106] The compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally or intraperitoneally. In some embodiments, the small molecule splicing modulator or a pharmaceutically acceptable salt thereof is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the

pharmaceutical compositions can be administered parenterally, intravenously, intramuscularly or orally. The oral agents comprising a small molecule splicing modulator can be in any suitable form for oral administration, such as liquid, tablets, capsules, or the like. The oral formulations can be further coated or treated to prevent or reduce dissolution in stomach. The compositions of the present invention can be administered to a subject using any suitable methods known in the art. Suitable formulations for use in the present invention and methods of delivery are generally well known in the art. For example, the small molecule splicing modulators described herein can be formulated as pharmaceutical compositions with a pharmaceutically acceptable diluent, carrier or excipient. The compositions may contain pharmaceutically acceptable auxiliary substances as required to

approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[00107] Pharmaceutical formulations described herein can be administrable to a subject in a variety of ways by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release

formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[00108] In some embodiments, the pharmaceutical formulation is in the form of a tablet. In other embodiments, pharmaceutical formulations containing an composition or inhibitory agent described herein are in the form of a capsule. In one aspect, liquid formulation dosage forms for oral

administration are in the form of aqueous suspensions or solutions selected from the group including, but not limited to, aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[00109] For administration by inhalation, a composition or inhibitory agent described herein can be formulated for use as an aerosol, a mist or a powder. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner. In some embodiments, a composition or inhibitory agent described herein can be prepared as

transdermal dosage forms. In some embodiments, a composition or inhibitory agent described herein can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In some embodiments, a composition or inhibitory agent described herein can be administered topically and can be formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. In some embodiments, a composition or inhibitory agent described herein can be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly

suppositories, or retention enemas.

Biological samples

[00110] A sample, e.g., a biological sample can be taken from a subject and examined to determine whether the subject produces mRNA that is subject to alternative splicing. A biological sample can comprise a plurality of biological samples. The plurality of biological samples can contain two or more biological samples; for examples, about 2-1000, 2-500, 2-250, 2-100, 2-75, 2-50, 2-25, 2-10, 10-1000, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-500, 50-250, 50-100, 50-75, 60-70, 100-1000, 100-500, 100-250, 250- 1000, 250-500, 500-1000, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more biological samples. The biological samples can be obtained from a plurality of subjects, giving a plurality of sets of a plurality of samples. The biological samples can be obtained from about 2 to about 1000 subjects, or more; for example, about 2-1000, 2- 500, 2-250, 2-100, 2-50, 2-25, 2-20, 2-10, 10-1000, 10-500, 10-250, 10-100, 10-50, 10-25, 10-20, 15-20, 25-1000, 25-500, 25-250, 25-100, 25-50, 50-1000, 50-500, 50-250, 50-100, 100-1000,

100-500, 100-250, 250-1000, 250-500, 500-1000, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 68, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375,

400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, ot 1000 or more subjects.

[00111] The biological samples can be obtained from human subjects. The biological samples can be obtained from human subjects at different ages. The human subject can be prenatal (e.g., a fetus), a child (e.g. , a neonate, an infant, a toddler, a preadolescent), an adolescent, a pubescent, or an adult (e.g., an early adult, a middle aged adult, a senior citizen). The human subject can be between about 0 months and about 120 years old, or older. The human subject can be between about 0 and about 12 months old; for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months old. The human subject can be between about 0 and 12 years old; for example, between about 0 and 30 days old; between about 1 month and 12 months old; between about 1 year and 3 years old; between about 4 years and 5 years old; between about 4 years and 12 years old; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years old. The human subject can be between about 13 years and 19 years old; for example, about 13, 14, 15, 16, 17, 18, or 19 years old. The human subject can be between about 20 and about 39 year old; for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 years old. The human subject can be between about 40 to about 59 years old; for example, about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 years old. The human subject can be greater than 59 years old; for example, about 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,

75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 years old. The human subjects can include living subjects or deceased subjects. The human subjects can include male subjects and/or female subjects.

[00112] Biological samples can be obtained from any suitable source that allows determination of expression levels of genes, e.g., from cells, tissues, bodily fluids or secretions, or a gene expression product derived therefrom (e.g., nucleic acids, such as DNA or R A; polypeptides, such as protein or protein fragments). The nature of the biological sample can depend upon the nature of the subject. If a biological sample is from a subject that is a unicellular organism or a multicellular organism with undifferentiated tissue, the biological sample can comprise cells, such as a sample of a cell culture, an excision of the organism, or the entire organism. If a biological sample is from a multicellular organism, the biological sample can be a tissue sample, a fluid sample, or a secretion.

[00113] The biological samples can be obtained from different tissues. The term tissue is meant to include ensembles of cells that are of a common developmental origin and have similar or identical function. The term tissue is also meant to encompass organs, which can be a functional grouping and organization of cells that can have different origins. The biological sample can be obtained from any tissue.

[00114] The biological samples can be obtained from different tissue samples from one or more humans or non-human animals. Suitable tissues can include connective tissues, muscle tissues, nervous tissues, epithelial tissues or a portion or combination thereof. Suitable tissues can also include all or a portion of a lung, a heart, a blood vessel (e.g. , artery, vein, capillary), a salivary gland, a esophagus, a stomach, a liver, a gallbladder, a pancreas, a colon, a rectum, an anus, a hypothalamus, a pituitary gland, a pineal gland, a thyroid, a parathyroid, an adrenal gland, a kidney, a ureter, a bladder, a urethra, a lymph node, a tonsil, an adenoid, a thymus, a spleen, skin, muscle, a brain, a spinal cord, a nerve, an ovary, a fallopian tube, a uterus, vaginal tissue, a mammary gland, a testicle, a vas deferens, a seminal vesicle, a prostate, penile tissue, a pharynx, a larynx, a trachea, a bronchi, a diaphragm, bone marrow, a hair follicle, or a combination thereof. A biological sample from a human or non human animal can also include a bodily fluid, secretion, or excretion; for example, a biological sample can be a sample of aqueous humour, vitreous humour, bile, blood, blood serum, breast milk, cerebrospinal fluid, endolymph, perilymph, female ejaculate, amniotic fluid, gastric juice, menses, mucus, peritoneal fluid, pleural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, vomit, urine, feces, or a combination thereof. The biological sample can be from healthy tissue, diseased tissue, tissue suspected of being diseased, or a combination thereof.

[00115] In some embodiments, the biological sample is a fluid sample, for example a sample of blood, serum, sputum, urine, semen, or other biological fluid. In certain embodiments the sample is a blood sample. In some embodiments the biological sample is a tissue sample, such as a tissue sample taken to determine the presence or absence of disease in the tissue. In certain embodiments the sample is a sample of thyroid tissue.

[00116] The biological samples can be obtained from subjects in different stages of disease progression or different conditions. Different stages of disease progression or different conditions can include healthy, at the onset of primary symptom, at the onset of secondary symptom, at the onset of tertiary symptom, during the course of primary symptom, during the course of secondary symptom, during the course of tertiary symptom, at the end of the primary symptom, at the end of the secondary symptom, at the end of tertiary symptom, after the end of the primary symptom, after the end of the secondary symptom, after the end of the tertiary symptom, or a combination thereof. Different stages of disease progression can be a period of time after being diagnosed or suspected to have a disease; for example, at least about, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 years after being diagnosed or suspected to have a disease. Different stages of disease progression or different conditions can include before, during or after an action or state; for example, treatment with drugs, treatment with a surgery, treatment with a procedure, performance of a standard of care procedure, resting, sleeping, eating, fasting, walking, running, performing a cognitive task, sexual activity, thinking jumping, urinating, relaxing, being immobilized, being emotionally traumatized, being shock, and the like.

[00117] The methods of the present disclosure provide for analysis of a biological sample from a subject or a set of subjects. The subject(s) may be, e.g., any animal (e.g., a mammal), including but not limited to humans, non-human primates, rodents, dogs, cats, pigs, fish, and the like. The present methods and compositions can apply to biological samples from humans, as described herein.

[00118] A biological sample can be obtained by methods known in the art such as the biopsy methods provided herein, swabbing, scraping, phlebotomy, or any other suitable method. The biological sample can be obtained, stored, or transported using components of a kit of the present disclosure. In some cases, multiple biological samples, such as multiple thyroid samples, can be obtained for analysis, characterization, or diagnosis according to the methods of the present disclosure. In some cases, multiple biological samples, such as one or more samples from one tissue type (e.g., thyroid) and one or more samples from another tissue type (e.g. , buccal) can be obtained for diagnosis or characterization by the methods of the present disclosure. In some cases, multiple samples, such as one or more samples from one tissue type (e.g., thyroid) and one or more samples from another tissue (e.g., buccal) can be obtained at the same or different times. In some cases, the samples obtained at different times are stored and/or analyzed by different methods. For example, a sample can be obtained and analyzed by cytological analysis (e.g., using routine staining). In some cases, a further sample can be obtained from a subject based on the results of a cytological analysis. The diagnosis of an immnodisorder can include examination of a subject by a physician, nurse or other medical professional. The examination can be part of a routine examination, or the examination can be due to a specific complaint including, but not limited to, one of the following: pain, illness, anticipation of illness, presence of a suspicious lump or mass, a disease, or a condition. The subject may or may not be aware of the disease or condition. The medical professional can obtain a biological sample for testing. In some cases the medical professional can refer the subject to a testing center or laboratory for submission of the biological sample. The methods of obtaining provided herein include methods of biopsy including fine needle aspiration, core needle biopsy, vacuum assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy or skin biopsy. In some cases, the methods and compositions provided herein are applied to data only from biological samples obtained by FNA. In some cases, the methods and compositions provided herein are applied to data only from biological samples obtained by FNA or surgical biopsy. In some cases, the methods and compositions provided herein are applied to data only from biological samples obtained by surgical biopsy. A biological sample can be obtained by non-invasive methods, such methods including, but not limited to:

scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, feces collection, collection of menses, tears, or semen. The biological sample can be obtained by an invasive procedure, such procedures including, but not limited to: biopsy, alveolar or pulmonary lavage, needle aspiration, or phlebotomy. The method of biopsy can further include incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, or skin biopsy. The method of needle aspiration can further include fine needle aspiration, core needle biopsy, vacuum assisted biopsy, or large core biopsy. Multiple biological samples can be obtained by the methods herein to ensure a sufficient amount of biological material. Generic methods for obtaining biological samples are also known in the art and further described in for example Ramzy, Ibrahim Clinical Cytopathology and Aspiration Biopsy 2001 which is herein incorporated by reference in its entirety. The biological sample can be a fine needle aspirate of a thyroid nodule or a suspected thyroid tumor. The fine needle aspirate sampling procedure can be guided by the use of an ultrasound, X-ray, or other imaging device.

[00119] In some cases, the subject can be referred to a specialist such as an oncologist, surgeon, or endocrinologist for further diagnosis. The specialist can likewise obtain a biological sample fortesting or refer the individual to a testing center or laboratory for submission of the biological sample. In any case, the biological sample can be obtained by a physician, nurse, or other medical professional such as a medical technician, endocrinologist, cytologist, phlebotomist, radiologist, or a pulmonologist.

The medical professional can indicate the appropriate test or assay to perform on the sample, or the molecular profiling business of the present disclosure can consult on which assays or tests are most appropriately indicated. The molecular profiling business can bill the individual or medical or insurance provider thereof for consulting work, for sample acquisition and or storage, for materials, or for all products and services rendered.

[00120] A medical professional need not be involved in the initial diagnosis or sample acquisition. An individual can alternatively obtain a sample through the use of an over the counter kit. The kit can contain a means for obtaining said sample as described herein, a means for storing the sample for inspection, and instructions for proper use of the kit. In some cases, molecular profiling services are included in the price for purchase of the kit. In other cases, the molecular profiling services are billed separately.

[00121] A biological sample suitable for use by the molecular profiling business can be any material containing tissues, cells, nucleic acids, genes, gene fragments, expression products, gene expression products, and/or gene expression product fragments of an individual to be tested. Methods for determining sample suitability and/or adequacy are provided. The biological sample can include, but is not limited to, tissue, cells, and/or biological material from cells or derived from cells of an individual. The sample can be a heterogeneous or homogeneous population of cells or tissues. The biological sample can be obtained using any method known to the art that can provide a sample suitable for the analytical methods described herein.

[00122] Obtaining a biological sample can be aided by the use of a kit. A kit can be provided containing materials for obtaining, storing, and/or shipping biological samples. The kit can contain, for example, materials and/or instruments for the collection of the biological sample (e.g., sterile swabs, sterile cotton, disinfectant, needles, syringes, scalpels, anesthetic swabs, knives, curette blade, liquid nitrogen, etc.). The kit can contain, for example, materials and/or instruments for the storage and/or preservation of biological samples (e.g., containers; materials for temperature control such as ice, ice packs, cold packs, dry ice, liquid nitrogen; chemical preservatives or buffers such as formaldehyde, formalin, paraformaldehyde, glutaraldehyde, alcohols such as ethanol or methanol, acetone, acetic acid, HOPE fixative (Hepes-glutamic acid buffer-mediated organic solvent protection effect), heparin, saline, phosphate buffered saline, TAPS, bicine, Tris, tricine, TAPSO, HEPES, TES, MOPS, PIPES, cadodylate, SSC, MES, phosphate buffer; protease inhibitors such as aprotinin, bestatin, calpain inhibitor I and II, chymostatin, E-64, leupeptin, alpha-2-macroglobulin, pefabloc SC, pepstatin, phenylmethanesufonyl fluoride, trypsin inhibitors; DNAse inhibitors such as 2- mercaptoethanol, 2-nitro-5-thicyanobenzoic acid, calcium, EGTA, EDTA, sodium dodecyl sulfate, iodoacetate, etc. ; RNAse inhibitors such as ribonuclease inhibitor protein; double-distilled water; DEPC (diethyprocarbonate) treated water, etc.). The kit can contain instructions for use. The kit can be provided as, or contain, a suitable container for shipping. The shipping container can be an insulated container. The shipping container can be self-addressed to a collection agent (e.g., laboratory, medical center, genetic testing company, etc.). The kit can be provided to a subject for home use or use by a medical professional. Alternatively, the kit can be provided directly to a medical professional.

[00123] One or more biological samples can be obtained from a given subject. In some cases, between about 1 and about 50 biological samples are obtained from the given subject; for example, about 1-50, 1-40, 1-30, 1-25, 1-20, 1-15, 1-10, 1-7, 1-5, 5-50, 5-40, 5-30, 5-25, 5-15, 5-10, 10-

50, 10-40, 10-25, 10-20, 25-50, 25-40, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43

44, 45, 46, 47, 48, 49, or 50 biological samples can be obtained from the given subject. Multiple biological samples from the given subject can be obtained from the same source (e.g., the same tissue), e.g., multiple blood samples, or multiple tissue samples, or from multiple sources (e.g., multiple tissues). Multiple biological samples from the given subject can be obtained at the same time or at different times. Multiple biological samples from the given subject can be obtained at the same condition or different condition. Multiple biological samples from the given subject can be obtained at the same disease progression or different disease progression of the subject. If multiple biological samples are collected from the same source (e.g., the same tissue) from the particular subject, the samples can be combined into a single sample. Combining samples in this way can ensure that enough material is obtained for testing and/or analysis.

Methods of Administering

[00124] The compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally or intraperitoneally. In some embodiments, the small molecule splicing modulator or a pharmaceutically acceptable salt thereof is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, the

pharmaceutical compositions can be administered parenterally, intravenously, intramuscularly or orally. The oral agents comprising a small molecule splicing modulator can be in any suitable form for oral administration, such as liquid, tablets, capsules, or the like. The oral formulations can be further coated or treated to prevent or reduce dissolution in stomach. The compositions of the present invention can be administered to a subject using any suitable methods known in the art. Suitable formulations for use in the present invention and methods of delivery are generally well known in the art. For example, the small molecule splicing modulators described herein can be formulated as pharmaceutical compositions with a pharmaceutically acceptable diluent, carrier or excipient. The compositions may contain pharmaceutically acceptable auxiliary substances as required to

approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[00125] Pharmaceutical formulations described herein can be administrable to a subject in a variety of ways by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intralymphatic, intranasal injections), intranasal, buccal, topical or transdermal administration routes. The pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release

formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[00126] In some embodiments, the pharmaceutical compositions described herein are

administered orally. In some embodiments, the pharmaceutical compositions described herein are administered topically. In such embodiments, the pharmaceutical compositions described herein are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, shampoos, scrubs, rubs, smears, medicated sticks, medicated bandages, balms, creams or ointments. In some embodiments, the pharmaceutical compositions described herein are administered topically to the skin. In some embodiments, the pharmaceutical compositions described herein are administered by inhalation. In some embodiments, the pharmaceutical compositions described herein are formulated for intranasal administration. Such formulations include nasal sprays, nasal mists, and the like. In some embodiments, the pharmaceutical compositions described herein are formulated as eye drops. In some embodiments, the pharmaceutical compositions described herein are: (a) systemically administered to the mammal; and/or (b) administered orally to the mammal; and/or (c) intravenously administered to the mammal; and/or (d) administered by inhalation to the mammal; and/or (e) administered by nasal administration to the mammal; or and/or (f) administered by injection to the mammal; and/or (g) administered topically to the mammal; and/or (h) administered by ophthalmic administration; and/or (i) administered rectally to the mammal; and/or (j) administered non-systemically or locally to the mammal. In some embodiments, the pharmaceutical compositions described herein are administered orally to the mammal. In certain embodiments, a composition described herein is administered in a local rather than systemic manner. In some embodiments, a composition described herein is administered with intraperitoneal injection. In some embodiments, a composition described herein is administered topically. In some embodiments, a composition described herein is administered systemically.

[00127] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[00128] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[00129] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[00130] Injection can be conducted using sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline,

bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.

EXAMPLES

[00131] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.

Example 1. Experimental procedures

[00132] Animal studies. All animal experiments were approved by Sanford Burnham Prebys Medical Discovery Institute's Institutional Animal Care and Use Committee (AUF 17-001). Animal care was followed according to institutional guidelines. All mouse strains were generated in a C57BL/6 background. Rnf5 mice were generated as previously described (Delaunay et ah, PLoS One 3, el609 (2008)). SMARTA mice (Oxenius et ah, Eur J Immunol 28, 390-400 (1998)) were from Dr. Charles D. Surh (La Jolla Institute for Allergy and Immunology). C57BL/6 were used as WT and control littermates, respectively. For all experiments, 6-10-week-old mice were used. Mice were maintained under controlled temperature (22.5 °C) and illumination (12 h dark/light cycle) conditions. Experimental colitis was induced by the addition of 2.5% DSS (weight/volume) (molecular weight 36-50 kDa; MP Biomedicals) to the drinking water for 5 or 7 days, followed by regular drinking water through the end of the experiment. DSS-containing water was exchanged every other day. Body weight, water consumption, and the severity of diarrhea and rectal bleeding were monitored. Diarrhea was scored as: 0, normal; 2, loose stools; and 4, watery diarrhea. Blood in stools was scored as: 0, normal; 2, slight bleeding; and 4, gross bleeding. Weight loss was scored as: 0, none; 1, 1-5%; 2, 5- 10%; 3, 10-15%; 4, >15%. Disease activity index was the average of ([diarrhea + stool blood + weight loss scores] / 3) for each animal (Yoshihara et ah, 2006). Cecum and colon tissues were removed and cleaned, and sections were taken for tissue culture, flow cytometry, IHC, and histology.

[00133] Human samples. The protocol was approved by the Institutional Review Board at the Rambam Health Care Campus (RHCC), Gastroenterology Institute (IRB no. RMB-0405-17). Colonic mucosal biopsies were obtained with written informed consent and collected from healthy controls (n = 17) and patients with known IBD (ulcerative colitis (UC); n = 19) undergoing endoscopy. Formalin embedded human biopsies from colonoscopies of UC patients and healthy controls were retreated from the pathological archives at RHCC. Biopsies were obtained from UC patients who underwent colonoscopy at RHCC between 2015-2017 and had documented biopsies from both inflamed and un inflamed tissue were included. Clinical data (age, gender, concomitant UC related therapy) and endoscopic severity (severity defined according to the endoscopic Mayo score, range of 0-3, higher scores indicate higher mucosal inflammation) were recorded. Pathological severity of inflamed tissue was documented according to pathological report by normal, mild, moderate and severe inflammation (score 0-3, in increasing order, when 0 stands for normal tissue and 3 for severe inflammation). The clinical severity scores are categorized as 0-1 (low) and 2-3 (high). Biopsies from healthy persons undergoing screening colonoscopy at RHCC with random biopsies taken in this time period was retrieved to serve as healthy controls. Staining intensity of RNF5 and S100A8 (IECs) was scored using a four tier scale from 0 (no staining) to 3 (strongest staining). Intensity scores were multiplied by the percentage of intestinal epithelial cells (IECs) with positive staining to generate an IHC score (maximum score, 300) by Pannoramic viewer (3DHISTECH Ltd). Table 1 shows a summary of the patients and tissues used.

Table 1. Summary of sample tissues and characteristics of IBD patients

[00134] Antibodies and plasmids. The anti-RNF5 antibody was described previously (Delaunay et al., PLoS One 3, el609 (2008). Polyclonal rabbit S100A8 blocking antibody was produced as previously described (Vandal et al., J Immunol 171, 2602-2609 (2003)). Other antibodies were obtained from the following sources: b-actin (Santa Cruz Biotechnology, sc- 47778), HSP90 (Santa Cruz Biotechnology, SC-13119), CD45 (BD Biosciences, 550539), CDl lc (Abeam, abl l029), V5 (Invitrogen, 46-0705), FLAG (Sigma-Aldrich, F1804), HA(Santa Cruz Biotechnology, sc-805), ubiquitin (Cell Signaling Technology, 3936), S100A8 (Santa Cruz Biotechnology, sc-48352 and Abeam, ab9233 l), S100A9 (Santa Cruz Biotechnology, sc-53187), Ki-67 (Abeam, abl5580), cleaved caspase-3 (Cell Signaling Technology, 9661), F4/80 (Abeam, ab6640), Ly6G/C (R&D Systems, MAB1037), CD4 (Abeam, abl83685 and eBioscience, 14-9766-80), and CD8 (Invitrogen, 14-0808). Plasmids expressing FLAG-R F5 were described previously (Jeon et al., 2015). Plasmids expressing S100A8-V5 were purchased from Arizona State University Biodesign Institute. shRNAs were purchased from Sigma-Aldrich (MISSION shRNA Plasmid DNA). The RNAi Consortium lentiviral pLKO.l control vector (EV) served as the control for shRNA experiments.

[00135] Cell culture and transfections. MODE-K (established and kindly provided by Dr.

Dominique Kaisserlain) and HEK293T (from ATCC) cellswere cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (GE Healthcare Life Sciences) containing 10% fetal bovine serum (FBS) and penicillin-streptomycin. Bone marrow-derived dendritic cells (BMDCs) were differentiated [6 days in GM-CSF (20 ng/ml; eBioscience, #14-8331-80)]. BMDCs and splenic DCs were maintained in RPMI 1640 medium (Coming) containing 10% FBS and penicillin-streptomycin. Cells were incubated at 37 °C. Lipopolysaccharide (LPS) was purchased from Enzo Life Sciences. Recombinant mouse TNF-a and S100A8 proteins were purchased from BioLegend. Cells were transfected with plasmids or shRNAs using Jetprime (Poly Plus).

[00136] RNA extraction and analysis. Total RNA was extracted from cultured cells or colon tissues using QIAzol and RNeasy Mini Kits (Qiagen) according to the manufacturer's protocol. The purity and concentration of extracted RNA were checked and quantified by reading A260 nm and A280 nm in aNanoDrop 1000 spectrophotometer (Thermo Scientific). cDNA was synthesized using oligo(dT) and random primers (AB Bioscience), and qPCR analysis was performed with SYBR Green (Roche). Primer sequences are listed in Table 2. The results were normalized to Gapdh mRNA.

Table 2. Sequences of primers used

[00137] RNA-Seq.RNA was extracted from naive WT or Rnf5 ' colon tissues (3 per group). RNA was ribo depleted using the NEBNext rRNA Depletion Kit and barcoded libraries were made using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB). Libraries were pooled and single end sequenced (1X75) on the Illumina NextSeq 500 using the high output V2 kit (Illumina Inc). Read data was processed in BaseSpace (basespace.illumina.com). Reads were aligned to Mus musculus genome (mmlO) using STAR aligner (https://eode.google.eom/p/ma-star/) with default settings. Differential transcript expression was determined using the Cufflinks Cuffdiff package (https://github.com/cole-trapnell-lab/cufflinks). The RNA-Seq data were deposited in the Short Read Archive (SRA) of NCBI under BioProject accession number PRJNA422424. For the analysis of naive WT or Rnf5 ^; colon tissues, differentially expressed genes were selected based on the criteria of FDR-adjusted P value < 0.05 using Ingenuity Pathway Analysis (IP A, http://www.ingenuity.com).

[00138] Immunoprecipitation and immunoblotting. For immunoprecipitations, cell lysates were prepared using lysis buffer (1% Triton X-100 in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl) supplemented with protease and phosphatase inhibitors (Thermo Scientific). Lysates were incubated with the appropriate antibodies and protein A/G agarose beads (Santa Cruz Biotechnology) according to the manufacturer’s protocol. Beads were washed with lysis buffer, boiled in Laemmli buffer, and proteins were resolved by SDS-PAGE and transferred to membranes. To detect endogenous S100A8- RNF5 interactions, MODE-K cells were pretreated with 10 mM MG132 (Selleckchem) for 4 h before lysis. For immunoblotting without immunoprecipitation, cell or tissue lysates were prepared using M- PER buffer (Thermo Scientific) containing protease and phosphatase inhibitors. Equal amount of protein samples were fractionated using SDS-PAGE gels and transferred to PVDF membranes (Millipore, Sigma). After blocking with 5% BSA, the membranes were incubated with primary antibodies overnight at 4 °C, followed by 1 h incubation with HRP -conjugated secondary antibodies. Protein signals were visualized using the ECL detection system (Mortsel) or ChemiDoc imaging system (Bio -Rad) according to the manufacturer’s instructions.

[00139] Histology, immunohistochemistry, and immunofluorescence. Immediately after mouse sacrifice, the intestines were removed, cut open lengthwise, rinsed, and rolled up from the proximal to distal end to form a Swiss roll. Sections (5 pm) were cut in a Leica Microsystems cryostat and transferred onto Superfrost-Plus slides (Fisher Scientific), and stained with hematoxylin and eosin (H&E). Tissue damage in the colons was scored as follows: epithelium, 0 = normal morphology, 1 = loss of goblet cells, 2 = loss of goblet cells in large areas, 3 = loss of crypts, 4 = loss of crypts in large areas; and infiltration, 0 = no infiltrate, 1 = infiltrate around the crypt basis, 2 = infiltrate reaching the lamina muscularis mucosae, 3 = extensive infiltration reaching the lamina muscularis mucosae and thickening of the mucosa with abundant edema, 4 = infiltration of the submucosa. Total histological score was the sum of the epithelium and infiltration scores (Obermeier et ah, 2002). For

immunohistochemistry, the sections were deparaffmized and rehydrated, and antigen was retrieved using Dako target retrieval solution. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide for 30 min. Specimens were incubated with primary antibodies diluted in Dako antibody diluent overnight at 4 °C. Slides were then washed three times with PBS/0.03% Tween-20 and incubated with Dako Labeled Polymer-HRP for 1 h at room temperature. After three washes with PBS/0.03% Tween-20, the sections were incubated with DAB chromogen and counterstained with hematoxylin. Staining intensity of RNF5, S100A8, and S100A9 was scored using a four tier scale from 0 (no staining) to 3 (strongest staining). Intensity scores were multiplied by the percentage of intestinal cells with positive staining to generate an IHC score (maximum score, 300) by Aperio slide scanner. For immunofluorescence, sections were incubated with Alexa-conjugated secondary antibodies (Life Technologies), and the slides were covered with a drop of Vectashield with DAPI (H-1200, Vector Laboratories). Slides were visualized using a fluorescence microscope with a Slidebook or Aperio slide scanner. Periodic acid-Schiff (PAS) and alcian blue (AB) staining (Thermo Scientific) were performed according to standard protocols. [00140] Flow cytometry and cell isolation. The intestines were removed, carefully cleaned of the mesentery, opened longitudinally, and washed free of fecal content. The intestines were then cut into 0.5-cm pieces and transferred to 50-ml conical tubes. Lamina propria leukocytes were isolated using the Miltenyi Lamina Propria Dissociation Kit (Miltenyi Biotec, 130-097-410) according to the manufacturer’s instructions. For cell surface staining, 2 ^c 106 cells per sample were resuspended in staining buffer (PBS, 2% FBS, and 0.01% NaN3), pre-incubated in mouse Fc block (anti-CDl6/CD32; BioLegend) and then stained for 20 min at 4 °C with the following antibodies (BioLegend):CD45.2 (104), CD8a (53-6.7), CD4 (GK1.5), CD25 (3C7), CD44 (IM7), TNF-a (MP6-XT22), IFN-g

(XMG1.2), CDl lc (N418), Ly6G (1A8), Ly6C (HK1.4), CD103 (2E7), F4/80 (BM8), NK1.1

(PK136), MHC class I (AF6-88.5), and MHC class II (M5/114.15.2). Cells were washed twice with FACS staining buffer, fixed for 15 min with 1% formaldehyde in PBS, washed twice again, and resuspended in FACS staining buffer for analysis. All data were collected on an LSRFortessa (BD Biosciences) and analyzed using FlowJo software (Treestar). For splenic DC isolation, spleens from WT or Rnf5-/- mice were minced and digested with 1 mg/ml collagenase D (Roche) and 100 pg/ml DNase (Sigma) in 5% C02 incubator. Cells were passed through a 70-pm cell strainer and

erythrocytes were depleted using red cell lysis buffer (Sigma). Cells were then stained using anti mouse CD19 (1D3/CD19), Thyl.2 (53-2.1), NK1.1, CDl lc, and CD45.2 antibodies (Biolegend), and CD1 lc+ cells (DCs) were sorted using BD FACSAria II. The isolated cells were resuspended in RPMI 1640 medium, then cultured at 37°C in 5% C02 incubator for ELISA and FACS experiments.

[00141] BMDC activation and in vitro CD4+ T cell stimulation assay. To prepare conditioned media, MODE-K-EV, MODE-K-shRNF5, or MODE-K-shRNF5/shSl00A8 cells were incubated in DMEM supplemented with 10% FBS for 24 h at 37 °C to allow cell attachment. The cells were then treated with medium or 0.5 % DSS for 24 h, washed with PBS, and cultured for 48 h in Advanced DMEM (Thermo Fisher) containing 2 mM L-glutamine without serum. The medium was then collected and centrifuged at 2000 ^c g for 10 min at 4 °C to remove cellular debris. The resulting conditioned medium was used for treatment of BMDCs. BMDCs from WT mice were generated by incubating bone marrow cell suspensions for 6 days with GM-CSF. Cells were then washed and incubated for 18 h with MODE-K conditioned medium (20% v/v) prepared as described above. For S100A8 blockade, cells were incubated conditioned medium in the presence of IgG isotype control (SouthemBiotech) or anti-Sl00A8 blocking antibody (10 pg/ml). After 18 h, BMDC culture supernatants were removed and IL-l2p70 and P b levels were quantified by ELISA. CD4+ T cells were isolated from the spleens of LCMV -specific CD4 TCR transgenic SMARTA mice by negative bead enrichment (StemCell Technologies). Cells were incubated with 5 pM CFSE for 10 min at 37 °C and washed. Cells were then mixed 1 : 1 with the BMDCs and incubated for 72 h pulsed with 2 pg/ml of GP61-80 peptide (AnaSpec). T cell proliferation was monitored by CFSE dilution using flow cytometry and the division index (a measure of the average number of divisions which includes the undivided cells) was determined using FlowJo software (Treestar). For intracellular cytokine staining, the cells were fixed, permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with anti- TNF-a and anti-IFN-g.

[00142] ELISA. S100A8 (Abeam, ab2l3886), IL-l2p70 (R&D Systems, DY419), and P,-Ib (R&D Systems, DY401) levels in culture supernatants or in mouse serum were determined by ELISA according to the manufacturers’ protocols. Concentrations were determined by comparison with standard curves. Multiplexed analysis of cytokines and chemokines was performed using

LEGENDplex mouse cytokine and mouse proinflammatory chemokine kits (BioLegend). Data were collected on an LSRFortessa and analyzed using LEGENDplex software (BioLegend).

[00143] Luciferase reporter assay. MODE-K-EV, MODE-K-shSl00A8, MODE-K-shRNF5, or MODE-K-shRNF5/shSl00A8 cells were transiently transfected with the NF-kB -dependent firefly luciferase reporter plasmid NF-KB4UC and a Renilla luciferase plasmid pRL-TK (Kosco et ah, 2008) using Jetprime according to the manufacturer’s protocol. After 48 h, luciferase was assayed using a dual luciferase kit (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase activity as a control for transfection efficiency.

[00144] In vivo antibody treatment. For neutralizing S100A8, mice were injected intraperitoneally (i.p.) with 200 pg of rabbit anti-Sl00A8 or rabbit IgG isotype control 1 day before and 1 and 3 days after the start of 2.5% DSS treatment. For survival analysis, the mice were injected with antibodies 1 day before and 1, 3, 5, and 7 days after the start of DSS treatment. For the treatment after the colitis induction, mice were injected i.p. with the same amount of each antibody on day 5, 7, and 9 after 5 days of 2.5% DSS treatment. The efficiency of Sl00A8-depletion was tested by flow cytometry of blood samples. CD4+ T cells were depleted by i.p. injection of 200 pg anti-CD4 (clone GK1.5, GoInVivoTM grade, BioLegend) and rat IgG2b isotype control (GoInVivoTM grade, BioLegend) 1 day before and 3 and 7 days after the start of 2.5% DSS treatment. The efficacy of depletion was confirmed by FACS analysis of blood samples.

[00145] Public mucosal IBD gene expression data and targeted correlation analysis. For targeted co-expression analysis three IBD datasets of public colonic gene expression data were used at baseline, available in Gene Expression Omnibus (GEO) database: two cohorts of UC patients including GSE14580 (Arijs et ak, 2009) and GSE12251 (Arijs et ak, 2009) and additional cohort of CD patients from GSE16879 (Arijs et ak, 2010), for which expression 2 weeks post first treatment was also analyzed. Probe set annotation was performed using affycoretools and hgul33plus2cdf.db R packages. Response classification was used as reported, according to endoscopic and histologic findings at 4-8 weeks post first treatment. Pairwise Spearman's rank correlation coefficients were calculating between the genes of interest, separately for responders and non-responders groups using Hmisc R package and visualized by ComplexHeatmap R package.

[00146] Statistical analysis. Differences between two groups were assessed by two-tailed t-test, and differences between more than two groups were assessed by ANOVA followed by Tukey’s post hoc multiple comparisons test. Pearson's correlation was estimated between RNF5 and S100A8 expression. P < 0.05 was considered statistically significant. GraphPad Prism 7 software was used for all analyses.

Example 2. Rnf5 mice exhibit intestinal inflammation and infiltration of activated leukocytes

[00147] In wild-type (WT, C57BL/6) mice, RNF5 is widely expressed in the small intestine and colon, as demonstrated by immunoblot and immunohistochemical (IHC) analysis, and expression is particularly high in intestinal epithelial cells (IECs) (Fig. la,b). Given the importance of IECs in maintaining intestinal homeostasis (Peterson and Artis, Nat Revs Immunol 14, 141-153 (2014)), the inventors inquired whether loss of RNF5 affects the integrity and/or function of these cells.

Histological analysis of colonic sections from Rnf5 mice revealed mild inflammation with some prominent focal areas of immune cell infiltration compared with WT mice (Fig. lc).

Immunofluorescence staining of CD45+ cells in colon sections revealed that leukocytes were significantly more abundant in the colons of Rnf5 mice compared with WT mice (Fig. ld). Flow cytometric analysis of lamina propria CD4+ and CD8+ T cells, NK1.1+ natural killer cells, colonic macrophages (CD45+ CD1 lc+ F4/80+ CD103- ), neutrophils (CD45+ Ly6G+), and inflammatory monocytes (CD45+ Ly6C+), did not identify marked differences (Fig. le), except for a modest, albeit significant, increase in the frequency of total colonic DCs (CD45+ CD1 lc+ CD103+ F4/80-) in colonic lamina propria cells from Rnf5 mice compared with WT mice (Fig. le). In addition, the expression of MHC class I and II was higher on colonic DCs from Rnf5 ^; mice than from WT mice (Fig. lf), consistent with increased DC activation in the colon. Finally, qRT-PCR analysis of isolated colonic extracts demonstrated that expression of pro -inflammatory cytokines (II 1b, 116, 1112r35, and Tslp) was elevated in colons obtained from Rnf5 compared with WT genotype (Fig. lg). These data suggest that Rnf5 ^; mice exhibited a basal intestinal inflammation and infiltration of activated DCs compared with WT mice. To further examine the involvement of RNF5 in intestinal inflammation, changes in gene expression in the colon tissues of Rnf5 and WT mice were accessed. RNA-Seq analysis identified 156 significantly dysregulated (87 up- and 69 down-regulated) genes in colon tissues of Rnf5 ^; compared with WT mice (Fig. lh). The Ingenuity Pathway Analysis (IP A) pointed to the enrichment of genes involved in active antigen presentation in Rnf5 ^; colon tissues (Table 3).

Table 3. Top 10 genes upregulated in Rnf5-/- tissues

[00148] Increased expression of H2-B1, H2-Tl0,Gm8909, and Ly6c2 mRNA in Rnf5 ^; colon tissues implies that the infiltration of activated DCs may underlie colonic inflammation seen in Rnf5 ^; mice (Fig. 8a). To determine whether RNF5 plays a role in the activation of general DC population or specific intestinal DC subsets, splenic DCs (CD1 lc+) were sorted and bone marrow-derived dendritic cells (BMDCs) were generated from WT and Rnf5 ^; mice. No difference in the expression of MHC class I and II or in the production of IL-l2p70 and I L- 1 b after LPS stimulation was identified (Fig. 8b-e), suggesting that loss of RNF5 results in the selective activation of DCs in the intestinal microenvironment.

Example 3. DSS-induced acute colitis is exacerbated in Rnf5 mice

[00149] A dextran sulfate sodium (DSS) model of acute colitis was used to investigate the influence of RNF5 deficiency in the development of IBD. DSS induces inflammation throughout the gut, with particularly prominent damage in the colon, and is a commonly used model of human colitis. Rnf5 mice and WT littermates were administered to 2.5% DSS in drinking water for 7 days and were monitored for additional 48 h. Rnf5 ^; mice lost significantly more weight than control mice, with an estimated difference of 13% which coincided with more severe shortening of the colon on day 9 (Fig. 2a, b). Correspondingly, Rnf5 mice exhibited more severe colitis symptoms, including abundant bloody diarrhea and rectal bleeding, which resulted in a higher clinical activity index and earlier death, compared with WT mice (Fig. 2c, d). Histological analysis revealed larger areas of ulceration and crypt loss, more extensive inflammatory cell infiltration reaching the submucosa, and thickening of the mucosa with abundant edema in the colon of the Rnf5 ^; compared with that of WT mice, analyzed on day 9 after DSS administration (Fig.2e). Changes associated with enhanced inflammation in the colonic tissue include altered proliferation and cell death programs. Indeed, lower level of the proliferation marker Ki67 and higher level of the apoptotic marker cleaved caspase-3 were seen in colonic sections from Rnf5 compared with WT mice (Fig. 2f and Fig. 9a), suggesting that loss of RNF5 impaired the regenerative response. In addition, CD4+ T cells, F4/80+ macrophages, and Ly6G/C+ neutrophils, but not CD8+ T cells or CD1 lc+ DCs, were more abundant in colonic sections obtained 9 days following DSS-treatment of Rnf5 ^; . compared with WT mice (Fig. 2f and Fig. 9a). Lastly, colonic sections from DSS-treated Rnf5 ^; animals had markedly fewer goblet cells compared with WT mice, as indicated by periodic acid-Schiff/Alcian blue staining (PAS/AB) (Fig. 2f and Fig. 9a). Taken together, these data demonstrate that the absence of RNF5 not only exacerbates colonic damage during DSS-induced acute colitis leading to increased mouse mortality, but also impairs the subsequent regenerative process.

Example 4. Gut microbiota does not impact acute colitis in i¾i/5 ^“/_ mice.

[00150] Deregulation of intestinal homeostasis and susceptibility to inflammation are often associated with alterations in the commensal bacterial population (Zhang et ah, 2017). Therefore, it was tested whether co-housing of Rnf5 and WT mice, a condition in which the mice exchange their gut microbiota, prior to DSS treatment, would affect the susceptibility to develop acute colitis in either the WT and Rnf5 mice. Notably, while co-housing did not increase the degree of inflammation seen following DSS administration in the WT mice, it also did not diminish the degree of acute colitis (body weight loss, clinical activity index, colon length, and histological score), seen in the Rnf5 ^; mice (Fig. 9b-e). These data suggest that the acute colitis seen in DSS treated Rnf5 ^; mice, do not depend on altered gut microbiota composition, further pointing to altered inflammation as a key driver of this pathology.

Example 5. Identification of S100A8 1 as an RNF5 substrate

[00151] Liquid chromatography-tandem mass spectrometry was used to understand the mechanism by which RNF5 contributes to the maintenance of intestinal homeostasis. Proteins co- immunoprecipitated with RNF5 were identified. Focusing on proteins that may play a role in inflammation, RNF5 -interacting protein S100A8 was selected for further examination. S100A8 is a component of the calcium binding calprotectin complex, which has been shown to play a role as a gut mucosal DAMP involved in tissue inflammation (Bertheloot and Latz, Cell Mol Immunol 14, 43-64 (2017)). To confirm interaction of S100A8 with RNF5, ectopic expression of Flag-tagged RNF5 and V5-tagged S100A8 in HEK293T cells, followed by anti-Flag immunoprecipitation, confirmed that the two proteins interact (Fig.3a). Moreover, co-expression of hemagglutinin (HA)-tagged ubiquitin together with RNF5 and S100A8 proteins identified that S100A8 is ubiquitinated by RNF5 (Fig. 3b). To verify these findings, control or RNF5-targeted shRNA were expressed in MODE-K cells, a mouse IEC line. Analysis of the endogenous proteins in these cells revealed that the ubiquitination of S100A8 was lower upon RNF5 knockdown (KD) (Fig. 3c). Treatment of cells with the proteasome inhibitor MG132 increased the steady-state levels of S100A8, partially attenuating the effect of RNF5 co-expression (Fig. 3d). These observations suggest that RNF5-mediated ubiquitination of S100A8 targets it for proteasomal degradation. Indeed, treatment of shRNF5-expressing MODE-K cells with the translational inhibitor cycloheximide extended the half-life of S100A8 from 6.3 h to 11.9 h (Fig. 3e). Conversely, ectopic expression of Flag-RNF5 decreased the S100A8 half-life from 3.6 h to 1.3 h, compared with cells expressing the corresponding the control vectors (Fig. l0a,b). Notably, interaction of endogenous RNF5 and S100A8 proteins was confirmed in MODE-K cells (Fig. 3f). Collectively, these findings suggest that by its ubiquitination and proteasomal mediated degradation, RNF5 association with S100A8 determines its stability. To determine whether RNF5 regulation of S100A8 in IECs was affected by inflammation, RNF5-S100A8 interaction was examined in MODE-K cells exposed to the pro -inflammatory cytokine TNF. Interestingly, the interaction between S100A8 and RNF5 decreased within 6-12 h of TNF treatment and then increased over the next 24-48 h (Fig. 3g). Consistent with these observations, the RNF5 expression of WT colon tissues did not change following DSS treatment (Fig. lOc), supporting changes in the activity of this ubiquitin ligase following inflammatory stimuli. These results suggest that inflammatory stimuli determine RNF5 interaction and hence control of S 100A8 stability. Applicants thus set to test the possibility that increased level of S100A8 derived from RNF5 -deficient IECs may activate innate immune receptors and drive intestinal inflammatory responses.

Example 6. Increased level of S100A8 secreted by RNF5-deficient IECs impacts host immune responses in a paracrine and autocrine manner

[00152] S100A8 and S100A9 are highly expressed by neutrophils, monocytes, and epithelial cells and are found at high levels in the extracellular milieu at inflammatory sites. Consistent with this, S100A8 and S100A9 were both expressed in the MODE-K IEC line; however, expression of S100A8, but not S100A9, was increased upon RNF5 KD (Fig. 4a and Fig. 1 la). Similarly, S100A8 was markedly upregulated in colonic epithelial cells from Rnf5 ' mice compared with WT mice, whereas S100A9 expression was largely unaffected (Fig. 4b). Secretion of S100A8 by MODE-K cells was also elevated following RNF5 KD and reduced upon KD of both RNF5 and S100A8 (Fig 4c, d). To gain insight into the physiological significance of S100A8 secreted by IECs, the next inquiry was whether RNF5 -regulated S100A8 could activate DCs. BMDCs were incubated with conditioned medium (CM) collected from cultures of MODE-K, MODE-K-shRNF5, and MODE-K shRNF5/shSl00A8 cells. The effects of the MODE-K CM, medium alone, or recombinant S100A8 on BMDCs were then assessed by measuring the secretion of cytokines and chemokines and cell-surface expression of MHC class I and II. Indeed, production of IL-l2p70 and P b and MHC expression by BMDCs was strongly induced by CM from MODE-K cells and by recombinant S100A8. Notably, these effects were further enhanced in the presence of CM from cells expressing shRNF5 compared with empty vector (EV) or shRNF5/shSl00A8 (Fig. 4e,f), supporting the direct stimulatory component in CM as RNF5 -regulated S100A8. To further assess the contribution of RNF5 -regulated S100A8 to DC activation seen in these studies, BMDCs were cultured in the presence of CM that were supplemented with a specific

S100A8- blocking antibody or an immunoglobulin G (IgG) control antibody. The presence of S100A8 neutralizing antibodies, but not IgG control antibodies, diminished the effects of CM from the

MODE-K shRNF5 cells on BMDCs (Fig.l lb,c). These finding establish that RNF5 -regulated S100A8 is directly responsible for stimulation of BMDCs. In addition to S100A8, the CM of MODE-K cells contained a number of pro-inflammatory cytokines and chemokines. In particular, MODE-K-shRNF5 cells and their CM contained higher levels of CCL2 and CCL5 mRNA and protein, respectively, compared with control MODE-K cells and their CM (Fig. 1 l-f). As CCL2 and CCL5 have been implicated in the progression of IBD (Turner et ah, Biochim Biophys Acta 1843, 2563-2582 (2014)), the present disclosure points to a possible mechanism by which DCs may be recruited to the inflamed intestinal microenvironment in Rnf5 ' mice. Of note, CM from MODE-K-shRNF5 cells showed decreased levels of CCL11, which has been implicated in eosinophil recruitment, while the levels of major pro-inflammatory cytokines and chemokines were not significantly altered (Fig. 1 ld-f).

Collectively, these data indicate that increased level of S100A8, secreted by MODE-K IECs subjected to RNF5 KD, effectively activate DCs to produce pro -inflammatory cytokines and chemokines. S100A8 can activate receptor for advanced glycation end products (RAGE) and toll-like receptor (TLR)-4 that also result in the activation of NF-kB and the up-regulation of CCL2 and CCL5, involved in cellular inflammatory responses (Zackular et ah, J Biol Chem 290, 18991-998 (2015)). S100A8 proteins are secreted or released, impacting immune responses via activation of the corresponding receptors (Bresnick et ah, Nat Rev Cancer 15, 96-109 (2015)). Consistent with these, MODE-K-shRNF5 cells exhibited higher activation of the NF-kB pathway (Fig. 4g). Furthermore,

KD of both RNF5 and S100A8 abrogated NF-kB induction, seen upon RNF5 KD alone, suggesting that the chemokines induced upon RNF5 KD depend on Sl00A8-mediated NF-kB activation. Thus, the present disclosure shows that extracellular S100A8 which is secreted from RNF5 -deficient IECs can stimulate host innate immune responses in the intestinal microenvironment, in a paracrine andautocrine manner.

Example 7. Elevated Thl cytokine production and CD4+ T cell activation in DSS-treated Rnf5 mice

[00153] As discussed in previous examples, RNF5 KD elevates S100A8 production by IECs in vitro and secreted S100A8 can stimulate BMDCs. Contribution of RNF5 regulated S100A8 in exacerbation of DSS-induced colitis was then examined in Rnf5 ' mice compared with WT mice. For this, colon sections from mice treated with DSS for 3, 6, and 9 days were isolated and analyzed for S100A8 expression and the supernatants for secreted S100A8 and cytokines. In WT colons, S100A8 expression peaked at day 3 after initiation of DSS treatment and declined thereafter, and RNF5 expression did not change following DSS treatment (Fig. 5a), consistent with our finding of the temporal regulation of S100A8 by RNF5. In contrast, S100A8 levels were higher in colon tissue from Rnf5 ' mice on day 0, as noted (Fig 4b), but were further increased by DSS and remained high until the end of the analysis at day 9 (Fig. 5a) .Analysis of S 100A8, cytokine, and chemokine levels in the culture supernatants showed the Rnf5 ' colonic tissues produced significantly higher levels of S100A8 and TNF than did tissue from WT mice (Fig. 5b), as was also seen for other Thl cytokines and chemokines (IFN-g, IF-l2p70, CCF2, CCF3, CCF4, and CCF5) (Fig. l2a,b). In agreement, qRT- PCR analysis of colonic extracts isolated on day 9 following DSS administration confirmed elevated expression of pro -inflammatory cytokines and chemokines (Ill2p35, Tnf, Ifrry, Ccl2, Ccl3, Ccl4, and Ccl5) in colons obtained from Rnf5 ' compared with WT mice (Fig. l2c). Likewise, analysis of serum from DSS-treated mice identified higher levels of S 100A8, Thl cytokines, and chemokine (IFN-g, TNF, IL-l2p70, CCL2, CCL3, CCL4, and CCL5) in Rnf5 ' compared with WT mice (Figs. 5c, l2d,e). These observations suggest that the production of pro-inflammatory cytokines correlated strongly with the severity of disease and leukocyte infiltration seen in the colons of DSS-treated Rnf5 ' mice. Of note, the level of IL-6, IL-23, andIL-l7A, cytokines involved in the differentiation and function of Thl7 lymphocytes, was similar in both the colon tissues and serum of DSS-treated WT and Rnf5 ' mice (Fig. l2a,d). Taken together, these results suggest that DSS induces a Thl type inflammatory response in the colon that is exacerbated upon elevated levels of S 100A8, due to the absence of its E3 ubiquitin ligase RNF5. To further substantiate these findings, immune cells were extracted from the colonic lamina propria of DSS-treated mice after DSS treatment and showed increased number of total CD4+ and activated CD4+ CD44+ T cells, colonic DCs, colonic macrophages, monocytes, and neutrophils on days 3, 6, and 9 obtained from Rnf5 ' compared with WT mice (Fig. 5d). This observation suggests that lack of RNF5 expression in IECs increases the recruitment of antigen- presenting cells and CD4+ T cells to the inflamed intestines of DSS-treated mice, leading to increased production of Thl type inflammatory cytokines.

Example 8. RNF5 controls the severity of DSS-induced colitis through S100A8-1 mediated CD4+ T cell activation

[00154] Involvement of S100A8 in the exacerbation of DSS-induced colitis was investigated in the absence of RNF5. The effects of DSS on S 100A8 secretion by MODE5K cells expressing EV or shRNF5 was accessed, and the effects of MODE-K cell CM on BMDC activation were measured. Notably, DSS alone elevated S100A8 secretion by MODE-K cells, and KD of RNF5 further increased the amount of S 100A8 that was secreted (Fig. l2f). Moreover, the CM from DSS-treated MODE-K and MODE-K-shRNF5 further increased the expression of MHC class I and II on BMDCs compared with CM from untreated MODE-K cells (Fig. l2g), indicating that DSS treatment enhances IEC- mediated activation of DCs. This observation raised the possibility that DSS-treated MODE-K- shRNF5 cells may increase antigen presentation by DCs to T cells in the intestinal microenvironment. IECs can exert their influence through the priming of both cellular and humoral adaptive immune responses via a continuous dialogue with antigen-presenting cells. To test this, applicants treated BMDCs with CM from DSS-treated MODE-K and MODE-K-shRNF5 cells, and monitored the activation of CD4 ⁺ T cells purified from the GP _6i-80 -specific CD4+ TCR transgenic (SMARTA) mice. Proliferation (measured by CFSE dilution) and IFN-g and TNF -a production (intracellular staining) by CD4+ T cells were significantly higher after co-incubation with BMDCs stimulated by CM from DSS-treated MODE-K-shRNF5 cells compared with MODE-K or MODE-K-shRNF5/shS l00A8 cells (Fig. 5e and l2h). These data further support the notion that loss of RNF5 from IECs leads to enhanced secretion of S 100A8, which consequently activates DCs and enhances antigen specific CD4+ T cell proliferation and effector responses. Importantly, the reversal of these effects by simultaneous KD of both RNF5 and S100A8 in MODE-K cells confirms that these effects result from RNF5-mediated control of S100A8 ubiquitination and degradation in IECs. To substantiate the possible role of CD4+ T in the DSS-induced colitis, the level of CD4 cells were monitored to access possible contribution of CD4 cells to the severe colitis phenotype identified in the Rnf5 mice. A notable increase in the number of CD4+/Ki67+ T cells was identified in the colon of Rnf5 mice after DSS treatment (Fig. l2i), consistent with its expected role in promoting the intestinal inflammatory phenotype. Significantly, administration of neutralizing antibodies against CD4+ T cells abolished severe acute colitis phenotypes in Rnf5 mice including body weight loss, colon length, and disease activity index (Fig. 5f-h). These data establish the causative role of CD4+ T cell in the acute colitis phenotype driven by the RNF5-S100A8 axis.

Example 9. Neutralizing antibodies to S100A8 attenuate acute colitis in DSS-treated Rnf5 mice

[00155] To directly assess the significance of elevated S100A8 expression to the acute colitis phenotype identified in the Rnf5 ^; mice, the therapeutic potential of S100A8 blockade was evaluated in the DSS-induced colitis model. Intraperitoneal injection of rabbit anti-mouse S100A8 neutralizing antibodies (Raquil et al., J Immunol 180, 3366-3374 (2008); Vandal et al., J Immunol 171 2602-2609 (2003)) or a control rabbit IgG on days -1, +1, and +3 relative to the initiation of DSS treatment (day 0) ameliorated the body weight loss, colon shortening, clinical disease activity index (based on the severity of diarrhea, stool blood, weight loss), and elevated survival in DSS-treated Rnf5 mice, whereas it only modestly improved the disease phenotypes in WT mice (Fig. 6a-d). Histological analysis confirmed that administration of anti-Sl00A8 neutralizing antibody significantly reduced the DSS-induced colon damage, histological scores, and CD4+ T cell, F4/80+ cell, and Ly6G/C+ cell infiltration in Rnf5 mice (Fig. 6e,f and Fig. 13 a,b). These data suggest that high basal levels of S100A8 seen with absence of RNF5 in the colon underlie the severe acute colitis phenotype seen upon DSS stimuli. Furthermore, using neutralizing S100A8 antibodies was also effective in WT mice that developed severe colitis following treatment with higher concentration of DSS (4.5%) (Fig. l3c- e), pointing to the therapeutic potential of neutralizing S100A8 for IBD patients with severed colitis phenotypes associated with high S100A8 levels. Given the ability to attenuate the colitis phenotype upon administration of the neutralizing antibodies prior and during DSS treatment, the effectiveness of administration of S100A8 neutralizing antibodies after DSS administration was assessed.

Administration of the neutralizing antibody on day 5, 7, and 9 after 5 days of 2.5% DSS treatment effectively attenuated the progression of acute colitis in the Rnf5 ^; mice (Fig. 6g). These results highlight the role of S100A8 in the etiology of acute colitis and signify the importance of its regulation by RNF5, while pointing to the therapeutic potential of targeting the RNF5-S100A8 axis for IBD treatment. Example 10. Expression of S100A8 by RNF5 in samples from IBD patients

[00156] To further understand the potential biological significance of RNF5/S 100A8 protein expression in IBD pathogenesis, the IHC stains were performed on 17 healthy colon sections and 19 colon sections from un-inflamed area and from inflamed area of ulcerative colitis patients (Table 1). RNF5 was highly expressed in IECs and stroma of healthy controls and in colon sections from the un inflamed area of colitis patients (Fig. 7a). This suggests that RNF5 could be involved in maintaining human intestinal homeostasis. Notably, reduced level of RNF5 expression in IECs and stroma was observed in the damaged area of colitis patients (Fig. 7a,b,d). Conversely, S100A8 was highly detected in IECs and stromal infiltrating lymphocytes of colon sections from inflamed area of colitis patients (Fig. 7a,b,d). Quantification of RNF5 and S100A8 expression in IECs and stroma confirmed a significant degree of inverse correlation (Fig. 7c, e; P=0.0395, P=0.04l9). Noteworthy, pathological severity significantly correlated with low RNF5 and high S100A8 expression in the inflamed IECs, not stroma (Fig. 7f,g), suggesting that the control of S100A8 by RNF5 is primarily occurring in IECs and the importance of RNF5-S100A8 regulatory axis in IECs involved in IBD development and disease progression.

Example 11. Low RNF5 expression in IBD patients that are non-responders to anti-TNF therapy

[00157] Cell centered meta-analysis from mucosal gene -expression data has recently revealed that the triggering receptor expressed on myeloid cells (TREMl)-CCL7-CCR2 axis is upregulated in IBD patients that are non-responders to anti-TNF treatment, thus pointing to blood TREM-l expression as a predictor of non-response to anti-TNF treatment (Gaujoux et al., Gut 2018). Notably, comparing the expression of TREM1-CCL7-CCR2 axis with that of RNF5 revealed a negative correlation. Low level of RNF5 transcription coincided with high level of TREMl-axis related transcripts in non-responders (Fig. l4a) both prior and two weeks following anti-TNF treatment (Fig. l4b). While at the transcript level, this observation points to the importance of RNF5 in an orthogonal inflammatory axis for IBD patients receiving anti-TNF therapy.

[00158] The examples and embodiments described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.