INDUCTION OF LAMINA PROPRIA REGULATORY T CELLS

RELATED APPLICATIONS

This application claims the benefit of priorirty to U.S. Provisional Patent

Application serial number 62/183,019, filed June 22, 2015, and U.S. Provisional Patent Application serial number 62/183,021, filed June 22, 2015, each of which are hereby incorporated by reference in their entirety.

GOVERNMENT INTEREST

This invention was made with Government support under National Institutes of Health Grant R01-AI51530 and R56 All 10630. The Government has certain rights in the invention.

BACKGROUND

Regulatory T cells (Tregs) are a subset of T cells that help keep the immune system in check by suppressing immune responses, thereby reducing immune-mediated pathology, such as autoimmune disease, inflammatory disease and allergies. Tregs regulate the immune response through a number of mechanisms, such as the secretion of inhibitory cytokines, such as IL-10 and TGF-β, the expression of inhibitory receptor CTLA-4 and granzyme-mediated cytolysis.

One of the key regulators of Treg development and function is the transcription factor FoxP3. Reduced numbers of FoxP3 expressing Tregs are associated with

autoimmune diseases such as systemic lupus erythematosus (SLE). Mutation of FoxP3 in humans results IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome, which is characterized by a dysfunction of regulatory T cells and subsequent autoimmunity.

Beyond their role in regulating immune activity, Tregs located in parenchymal tissues, referred to as "tissue Tregs," are also involved in certain other, non-immunological, processes. For example, tissue Tregs that reside in visceral adipose tissue regulate metabolic parameters, while those located in muscle tissue channel tissue inflammation and regeneration following muscle injury.

An important population of tissue Tregs resides in the lamina propria (LP) of the digestive tract, and in particular in the colon. In fact, under normal conditions, compared with all other tissues, the intestinal lamina propria has the greatest population of Tregs. Such LP Tregs modulate responses to commensal microbes, thereby reducing the risk of intestinal immunopathologies. LP Tregs are an unusual population, which has provoked some contradictory observations. T cell receptors expressed by LP Tregs show a marked reactivity against microbial antigens, which are important drivers of LP Treg differentiation and/or expansion (Lathrop et al, Nature 478:250 (2011); Cebula et al, Nature 497:258 (2013)), and some LP Tregs may arise by conversion from FoxP3 ^" conventional T cells (Tconv) ( Lathrop et al, Nature 478:250 (2011)), although arguments for a thymic origin have also been made ( Cebula et al, Nature 497:258 (2013)). Many LP Tregs express marker profiles (Nrpl ^" , Helios ^" ) that differ from Tregs found in peripheral lymphoid tissue (Bilate and Lafaille, Annu. Rev. Immunol. 30:733 (2012), though the significance of the different marker expression patterns is unclear. Generally, germ-free (GF) mice exhibit a reduced abundance of LP Tregs (Ai et al, Immunol. Rev. 259:60 (2014)), and colonization of GF mice by pools of microbes (particularly Schadler's flora and Clostridia

combinations) elicited the differentiation or expansion of Helios ^" Nrpl ^" LP Tregs (Geuking et al, Immunity 34:794 (2011); Atarashi et al, Nature 500:232 (2013); Atarashi et al, Science 331 :337 (2011)). The ability of single microbes to induce colonic Tregs has been more controversial, and the need for complex combinations has been suggested Atarashi et al, Nature 500:232 (2013); Atarashi et al, Science 331 :337 (2011); Faith et al, Sci. Transl. Med. 6:220ral 1 (2014). Accordingly, induction of LP Tregs is a promising treatment for intestinal immunopathologies, including inflammatory bowel diseases such as ulcerative colitis and Crohn's disease.

SUMMARY

In certain aspects, provided herein are methods and compositions for inducing the production of Rory ⁺ Helios ^" lamina propria regulatory T cells (LP Tregs) and/or for treating or preventing a disease associated with a pathological immune response {e.g., an

inflammatory bowel disease) in a subject.

In certain aspects provided herein is a method for inducing the production of Rory ⁺ Helios ^" LP Tregs in a subject comprising administering to the subject a composition comprising a bacteria or a combination of bacteria that induce the production of

Rory ⁺ Helios ^" LP Tregs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is a food product supplemented with the bacteria. In some embodiments, the food product is or comprises a dairy product {e.g., yogurt, frozen yogurt, ice cream, milk or cheese). In some embodiments, the food product is a non-dairy food product. In some embodiments, the food product is a beverage. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered rectally.

In certain aspects provided herein is a method of treating or preventing an inflammatory bowel disease in a subject comprising administering to the subject a composition comprising a bacteria or a combination of bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs. In some embodiments, the inflammatory bowel disease is Crohn' s disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis, indeterminate colitis, infectious colitis, pseudomembranous colitis, ischemic inflammatory bowel disease or Behcet' s disease. In some embodiments, the disease is Crohn' s disease or ulcerative colitis. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is a food product supplemented with the bacteria. In some embodiments, the food product is or comprises a dairy product (e.g., yogurt, frozen yogurt, ice cream, milk or cheese). In some embodiments, the food product is a non-dairy food product. In some embodiments, the food product is a beverage. In some embodiments, the composition is administered orally. In some embodiments, the composition is administered rectally.

In some embodiments of the methods described herein, the subject has or is predisposed to a disease associated with a pathological immune response. In some embodiments, the disease is an inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis, indeterminate colitis, infectious colitis, pseudomembranous colitis, ischemic inflammatory bowel disease or Behcet's disease). In some embodiments, the disease is Crohn' s disease or ulcerative colitis.

In some embodiments of the methods described herein, the subject has reduced levels of bacteria present in their gut. In some embodiments, the subject had been administered an antibiotic prior to administration of the composition. In some

embodiments, the antibiotic was administered less than a month, less than 30, 28, 21, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 days prior to administration of the composition.

In certain aspects, provided herein is a composition for inducing the production of Rory ⁺ Helios ^" LP Tregs in a subject comprising a bacteria or a combination of bacteria that induce the production of Rory^elios ^" LP Tregs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition is a food product supplemented with the bacteria. In some embodiments, the food product is or comprises a dairy product (e.g., yogurt, frozen yogurt, ice cream, milk or cheese). In some embodiments, the food product is a non-dairy food product. In some embodiments, the food product is a beverage. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated for rectal administration.

In certain aspects, provided herein is a method of making a composition for inducing the production of Rory ⁺ Helios ^" LP Tregs in a subject comprising combining a bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs with a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for oral administration. In some embodiments, the composition is formulated for rectal

administration.

In certain aspects, provided herein is a method of making a composition for inducing the production of Rory ⁺ Helios ^" LP Tregs in a subject comprising combining a bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs with a food product. In some embodiments, the food product is or comprises a dairy product (e.g., yogurt, frozen yogurt, ice cream, milk or cheese). In some embodiments, the food product is a non-dairy food product. In some embodiments, the food product is a beverage.

In some embodiments of the compositions and methods described herein, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is selected from a species of bacteria listed in Figure 5. In some embodiments, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs does not belong to the Clostridia class. In some embodiments, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and Bifidobacterium breve. In some embodiments, the bacteria is selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum and Parabacteroides johnsonii. In some embodiments, the bacteria is selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron and Bacteroides vulgatus. In some embodiments, the bacteria is selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus and Enterococcus faecalis. In some embodiments, the bacteria is Bacteroides ovatus. In some embodiments, the bacteria is Campylobacter jejuni. In some embodiments, the bacteria is Staphylococcus saprophyticus. In some embodiments, the bacteria is Enterococcus faecalis. In some embodiments, the bacteria are live, replication competent bacteria.

In some embodiments of the compositions and methods described herein, at least

10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and Bifidobacterium breve. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the

composition are selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum and Parabacteroides johnsonii. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the

composition are selected from the group consisting of Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron and Bacteroides vulgatus. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) of the bacteria in the composition are selected from the group consisting of

Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus and Enterococcus faecalis. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria is Bacteroides ovatus. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Campylobacter jejuni. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is

Staphylococcus saprophyticus. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Enterococcus faecalis.

In some embodiments of the compositions and methods described herein, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is selected from a strain of bacteria listed in Figure 5. In some embodiments, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs does not belong to the Clostridia class. In some embodiments, the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79,

Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides

uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium

nucleatum_F0419, Enterococcus faecalis OGlRF, Bacteroides

thetaiotaomicron_ATCC29148, Parabacteroides johnsonii_CL02T12C29, Bacteroides oleiciplenus_DSM22535, Lactobacillus rhamnosus_LMS2-l, Bacteroides

massiliensis D SMI 7679, Parabacteroides merdae_CL03T12C32, Fusobacterium

mortiferum_A016, Bifidobacterium breve_SK134, Bacteroides finegoldii_DSM17565 and Bacteroides fragilis_3_l_12. In some embodiments, the bacteria is selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79,

Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides

uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium

nucleatum_F0419, Enterococcus faecalis OGlRF, Bacteroides

thetaiotaomicron_ATCC29148 and Parabacteroides johnsonii_CL02T12C29. In some embodiments, the bacteria is selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides

thetaiotaomicron_ATCC29741 and Bacteroides vulgatus_ ATCC8482. In some

embodiments, the bacteria is selected from the group consisting of Bacteroides

ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus

saprophyticus DLKl and Enterococcus faecalis_HH22. In some embodiments, the bacteria is Bacteroides ovatus_CL02T12C04. In some embodiments, the bacteria is Campylobacter jejuni_AS-84-79. In some embodiments, the bacteria is Staphylococcus

saprophyticus DLKl. In some embodiments, the bacteria is Enterococcus faecalis_HH22. In some embodiments, the bacteria are live, replication competent bacteria.

In some embodiments of the compositions and methods described herein, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni AS- 84-79, Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium

nucleatum_F0419, Enterococcus faecalis OGlRF, Bacteroides

thetaiotaomicron_ATCC29148, Parabacteroides johnsonii_CL02T12C29, Bacteroides oleiciplenus_DSM22535, Lactobacillus rhamnosus_LMS2-l, Bacteroides

massiliensis D SMI 7679, Parabacteroides merdae_CL03T12C32, Fusobacterium mortiferum_A016, Bifidobacterium breve_SK134, Bacteroides finegoldii_DSM17565 and Bacteroides fragilis_3_l_12. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides

thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium

nucleatum_F0419, Enterococcus faecalis OGIRE, Bacteroides

thetaiotaomicron_ATCC29148 and Parabacteroides johnsonii_CL02T12C29. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides thetaiotaomicron_ATCC29741 and Bacteroides vulgatus_ ATCC8482. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from the group consisting of Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus

saprophyticus DLKl and Enterococcus faecalis_HH22. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Bacteroides ovatus_CL02T12C04. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Campylobacter jejuni_AS-84-79. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Staphylococcus saprophyticus DLKl. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition is Enterococcus faecalis_HH22.

In certain aspects, provided herein are methods and compositions for inducing the production or activity of regulatory T cells in a subject. In some embodiments, the methods comprise administering to the subject an agent that activates Rory (i.e., a Rory agonist). In some embodiments, the regulatory T cells are lamina propria regulatory T cells.

In certain aspects, provided herein are methods and compositions for treating or preventing a disease associated with a pathological immune response. In some

embodiments, the methods comprise administering to the subject an agent that activates Rory (i.e., a Rory agonist). In some embodiments, the agent is administered in an amount sufficient to induce the production or activity of regulatory T cells (e.g., lamina propria regulatory T cells) in the subject. In some embodiments, the disease associated with a pathological immune response is an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease, ulcerative colitis, irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis, indeterminate colitis, infectious colitis, pseudomembranous colitis, ischemic inflammatory bowel disease or Behcet's disease. In some embodiments, the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

In some embodiments of the methods described herein, a Rory agonist is

administered to a subject (e.g., an effective dose of a Rory agonist). In some embodiments, the Rory agonist is a small molecule. In some embodiments, the Rory agonist is a sterol. In some embodiments, the sterol Rory agonist is selected from the group consisting of cholesterol sulfate, 25-OHC, 25-OHC sulfate, desmosterol, desmosterol sulfate, 5α,6α- epoxycholestanol sulfate, 7a,27-diOHC, 5a,6a-epoxycholestanol, 24S,25-epoxycholesterol, 7a-OHC, 20a-OHC, 22R-OHC, 24S-OHC, 27-OHC, 7p,27-OHC, 7-keto,27-OHC, 7a,27- OHC, 4AC4MA°, A7-daf, zymosterol, 4C, 7DHC, zymosterone, 4C220H, 4,7-cholesten, 250H, OR-12872 and OR-942

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows Rory, encoded by Rorc, is preferentially expressed in colonic Tregs. (A) Heatmap of gene clusters that are enriched in tissue Tregs and colonic Tregs. Top: Genes that are overrepresented or underrepresented in tissue Tregs vs. splenic Tregs (at a Fold Change>2) were clustered using hierarchical clustering (Pearson correlation). Bottom: Heatmap of colon preferential gene clusters that are biased in colonic Tregs (at a Fold Change>1.5) compared with other tissue Tregs. (B) Heatmap of transcription factors that are overrepresented in colonic Tregs compared with Tregs from other lymphoid and non- lymphoid tissues. Rorc was the most differential based on hierarchical clustering analysis. (C) Representative dot plots (left) and respective frequencies (right) of Rory^elios ^" Tregs within the Foxp3 ⁺ CD4 ⁺ TCRp ⁺ population. (n=26) ***P<0.0001, paired t test. (D)

Representative dot plots of Rory vs Helios, Nrpl or 1133R expression within the colonic Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population. Quantification of these plots is shown in FigS2 (n>5). (E) depicts representative dot plots (top) and frequency (bottom) of Rory^elios ^" Tregs within the Foxp3 ⁺ CD4 ⁺ TCRp ⁺ population across different tissues- colon, small intestinal lamina propria (SI), Peyer's patches (PP), mesenteric lymph nodes (MLN), peripheral lymph nodes (LN), spleen (Spl), injured muscle, thymus and MC138 tumor. (F) Lymphocytes were treated with PMA and ionomycin and stained for II 17- A, I117-F and FoxP3. Representative dot plots of I117-A and Foxp3 expression (top), and frequencies of I117-A or I117-F producing cells (bottom) within Foxp3 ⁺ Tregs or Foxp3 ^" Tconvs from the colon (n=16), SI (n=4) and spleen (n=10) are shown.

Figure 2 shows a comparison of gene expression profiles of Tregs from colon and spleen uncovered 933 differential transcripts. Colonic and splenic Tregs were sorted from Foxp3igfp mice. Fold change difference and corresponding p values between gene expression of colonic and splenic Tregs are shown in volcano plot. Colonic signature is highlighted at a FoldChange>2 and p value <0.05.

Figure 3 shows Rory ⁺ Tregs express low levels of Nrpl, Helios and IL33R. (A) Colonic lymphocytes were stained with Foxp3, Helios, Nrpl and Rory. Frequencies of Helios ⁺ or Nrp ⁺ cells are reported within the Rory ⁺ Tregs or Rory ^" Tregs. (B) Representative dot plots of IL33R and Foxp3 expression within the CD4 ⁺ TCRP ⁺ population and IL33R and Helios expression within the Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population.

Figure 4 shows Rory ⁺ Helios ^" Tregs are induced by gut microbacteria. (A) Graphical representation of colonic Rory ⁺ Helios ^" Tregs within the Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population from adult SPF (n=14) and germ free mice (n=26). ***P<0.0001, unpaired t test. (B)

Induction of Rory in colonic Tregs during normal post-natal development in SPF mice. Left: Representative dot plots of Rory ⁺ Helios ^" and RoryTTelios ^" Tregs within the

Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population before (dl4) during (dl9) and after (d24) Rory induction. Right top: Frequency of Foxp3 ⁺ Tregs within CD4 ⁺ TCRP ⁺ cells. Right bottom: Frequency of Rory ⁺ Helios ^" Tregs (red) and RoryTTelios ^" Tregs (black) within the Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population. (C) Mice were treated with single (Neomycin, Vancomycin, Ampicillin, Metronidazole) or full cocktail (VMNA) of antibiotics for 4 weeks. Frequency of colonic Rory ⁺ Helios ^" Tregs within the Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population is shown. ***P<0.0001, unpaired t test. (D) Germ free mice were colonized with single bacterial species (mono- colonization) for 2 weeks. Representative dot plots (top) and frequency of Rory^elios ^" Tregs (bottom) within the colonic Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population following mono- colonization are shown. Coloring corresponds to different phyla. (E) Mice were mono- colonized with a focused sequenced set of Bacteroides. The frequency of Rory^elios ^" Tregs within the colonic Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population is shown (E). (F) Colonic

Rory ⁺ Helios ^" Tregs and RoryTTelios ^" Tregs were compared at different points of

colonization in mice colonized with Bacteroides thetaiotaomicron ATCC29741.

Representative dot plots (top) and respective frequencies within the Foxp3 ⁺ CD4 ⁺ TCRp ⁺ population (bottom) are shown. Figure 5 is a table showing the induction of colonic Rory Helios ^" Tregs by the indicated bacterial species. FDR refers to false discovery rate.

Figure 6 shows induction of Rory ^' Helios ^" Tregs is not accompanied by

inflammation and is independent of bacterial load. (A) Haematoxylin and eosin staining of the distal colon from mono-colonized mice and the corresponding frequency of colonic Rory ⁺ Helios ^" Tregs. (B) Correlation between bacterial load (measured as CFU) and frequency of colonic Rory ⁺ Helios ^" Tregs in mono-colonized mice. Correlation coefficient = 0.046.

Figure 7 shows characterization of Treg populations in Foxp3-Cre. Ror ¹ ^ mice. Colonic and splenic lymphocytes were isolated from WT or Foxp3-Cre. Ror f ¹ ^ mice, which lack Rory expression specifically in Tregs. Representative dot plots (left) and frequencies of Foxp3 ⁺ Tregs (middle) within the CD4 ⁺ TCRP ⁺ population and Helios ^" Tregs (right) within the Foxp3 ⁺ CD4 ⁺ TCRP ⁺ population are shown. Foxp3, **P=0.004; Helios ^" Tregs, *P=0.03, unpaired t test.

Figure 8 shows Rory ⁺ Tregs control colitis. (A) Colonic and splenic lymphocytes from WT (n=7) mice and FoxpS-Cre.Rorc ¹ ^ (n=8) littermates were treated with PMA and ionomycin. Frequencies of I117-A (left) and IFNy (right) producing cells within the CD4 ⁺ TCRp ⁺ population are shown. I117-A,***P=0.0002; IFNy, **P=0.004, unpaired t test. (B) T BS-colitis score of WT (n=9) and Foxp3-Cre.Ron ^ (n=8) littermates challenged with TNSB is shown. Colitis score is calculated based on weight loss, histologic score and other physical parameters (detailed in FigS5). **P=0.001, paired t test. (C) Haematoxylin and eosin staining of the distal colon. (D) Correlation of TNBS-colitis score (x-axis) with Rory ⁺ Helios ^" Treg frequency (y-axis) in mice monocolonized for 2 weeks with bacteria that elicit high, medium or low Rory ⁺ Helios ^" Treg phenotypes prior to the induction of TNBS - colitis. Correlation coefficient= 0.82, ***P>0.0001.

Figure 9 shows Rory Tregs protect from colitis. TNBS- colitis was induced in WT (n=9) and Foxp3-Cre.Ror f ^{/fi '} (n=8) littermates. Percent weight loss (left) and colon thickness (right) are shown. Data is representative of three independent experiments.

Figure 10 shows Rory contributes to colonic Treg homeostasis and determines a part of the colonic Treg signature. (A) Colonic Rory ⁺ or Rory ^" Tregs were sorted from an intercross of Foxp3 ^{thyl 1} and Rorc^ reporter male mice. Mean gene expression values are shown for Rory ⁺ or Rory ^" Tregs and colonic Treg signature, shown in FigSl, is highlighted in red (induced) or blue (repressed) (n=3). (B) Some of the genes (Havcr2, Cxcr3), over- represented in Rory Tregs, were validated at protein level. Representative dot plots of Cxcr3 and Tim3 encoded by Havcr2 are shown. (C) Fold change differences in gene expression between CD4 T cells and Treg cells that are enriched for or lack Rory expression are shown. CD4 T cells were sorted from SI of germ free mice or mice monocolonized with SFB. Gene changes that are specific to Rory ⁺ Tregs (red), shared by both Rory ⁺ Tregs and TH17 cells (green) and specific to TH17 cells (blue) are

highlighted.(n=3).

DETAILED DESCRIPTION

General

In certain aspects, provided herein are methods and compositions related to the use of bacteria that induce the production of Rory ⁺ Helios ^" lamina propria regulatory T cells (LP Tregs) for the production of such LP Tregs in a subject and/or the treatment or prevention an inflammatory bowel disease in a subject.

As described herein, the present inventors discovered that Rory is expressed in a FoxP3 ⁺ Helios ^" population of LP Tregs. Indeed, Rory ⁺ Tregs are a major subset of those Tregs elicited in response to antigens of commensal microbes in the gut. The present inventors have further identified a number of human commensal bacterial species and strains that are capable of inducing production of Rory ⁺ Helios ^" LP Tregs when administered to a subject. Examples of such bacteria are provided in Figure 5 and include Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis,

Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and Bifidobacterium breve. Compositions comprising such bacteria are therefore useful for the induction of Rory ⁺ Helios ^" LP Tregs in a subject and/or for the treatment or prevention of diseases associated with a pathological immune response, such as an inflammatory bowel disease.

In certain aspects, provided herein are methods and compositions for the induction of lamina propria regulatory T cells (LP Tregs) and/or for the treatment of a disease associated with a pathological immune response (e.g., an inflammatory bowel disease) through the induction of Rory expression or activity. Thus, in certain aspects, provided herein are compositions and methods for the treatment and/or prevention of inflammatory bowel disease in a subject comprising administering to the subject an agent that activates

Rory (i.e., an Rory agonist).

Rory is a transcription factor encoded by the Rote gene that has previously been reported to be antagonistic to FoxP3 (Korn t a/., A mm. Rev. Immunol. 27:485 (2009)). As FoxP3 is a key regulator of Treg development, inhibition of Rory has been proposed as a mechanism for the treatment of autoimmune diseases (e.g.. U.S. Pat. No. 8,912,219, U.S.

Pat. Pub. 2014/0163110, each of which is hereby incorporated by reference). Mice lacking

FoxP3 ⁺ Treg expression of Rory have increased disease severity in a colitis model. This role for Rory contrasts strongly with the previously accepted dichotomy between FoxP3 and Rory, a notion that stems mainly from their antagonism in the outcome of TGFP

supplemented in vitro cultures (Zou et al, Nature 453 :236 (2008)).

As described herein, in LP Tregs, Rory controls a specific transcriptional signature, overlapping but mainly distinct from its transcriptional signature in conventional T cells.

Notably missing are most transcripts encoding the pro-inflammatory IL-17 cytokine family, but the shared aspects of the transcriptional signature include 1123r. Notably, human IL23R genetic variants are strongly associated with inflammatory bowel disease (Abraham and

Cho, Annu. Rev. Med. 60:97 (2009)). Rory ⁺ Tregs do not respond to the alarmin IL-33, and are phenotypically distinct from I133R ⁺ Helios ⁺ cells that congregate or expand in response to IL-33 in response to tissue damage. Mutually exclusive expression of IL-33 receptor and Rory in LP Tregs indicates that these molecules distinguish responses to commensal versus aggressive microbes.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The articles "a" and "a«" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

As used herein, the term "administering" means providing an agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering.

The term agenf is used herein to denote a chemical compound, a small molecule, a mixture of chemical compounds and/or a biological macromolecule (such as a nucleic acid, an antibody, an antibody fragment, a protein or a peptide). Agents may be identified as having a particular activity by screening assays described herein below. The activity of such agents may render them suitable as a "therapeutic agent" which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

As used herein, an "effective amounf is an amount effective in treating or preventing a disease associated with a pathological immune response, including, for example, inflammatory bowel disease.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.

"Small molecule ^' " as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules. Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures, often fungal, bacterial, or algal extracts, which can be screened with any of the assays described herein.

As used herein, the term "subject" means a human or non-human animal selected for treatment or therapy. In certain embodiments, of the methods and compositions described herein the subject is a human subject.

The phrases "therapeutically-effective amount" and "effective amounf as used herein means the amount of an agent which is effective for producing the desired therapeutic effect in at least a sub-population of cells in a subject at a reasonable

benefit/risk ratio applicable to any medical treatment.

"Treating' a disease in a subject or "treating' a subject having a disease refers to subjecting the subject to a pharmaceutical treatment, e.g., the administration of a drug, such that at least one symptom of the disease is decreased or prevented from worsening.

Bacteria that Induce LP Tregs

In certain aspects, provided herein are compositions and methods related the use of bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs. Examples of such bacteria are provided in Figure 5. In some embodiments, the bacteria does not belong to the Clostridia class. In some embodiments, the species of the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the strain of the bacteria that induces the production of Rory ⁺ Helios ^" LP Tregs is selected from the group consisting of Bacteroides

ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus

saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides

thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides

uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium

nucleatum_F0419, Enterococcus faecalis OGlRF, Bacteroides

thetaiotaomicron_ATCC29148, Parabacteroides johnsonii_CL02T12C29, Bacteroides oleiciplenus_DSM22535, Lactobacillus rhamnosus_LMS2-l, Bacteroides

massiliensis D SMI 7679, Parabacteroides merdae_CL03T12C32, Fusobacterium

mortiferum_A016, Bifidobacterium breve_SK134, Bacteroides finegoldii_DSM17565 and/or Bacteroides fragilis_3_l_12.

In some embodiments, the bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs do not express a heterologous gene (i.e., are not recombinant). In some embodiments, the bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs are recombinant . In some embodiments, the bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs are attenuated.

In some embodiments, the bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs has a genomic sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or 100%) homology the genomic sequence of a bacteria strain described herein (e.g., Bacteroides ovatus_CL02T12C04, Campylobacter jejuni_AS-84-79, Staphylococcus saprophyticus DLKl, Enterococcus faecalis_HH22, Bacteroides

thetaiotaomicron_ATCC29741, Bacteroides vulgatus_ ATCC8482, Bacteroides

uniformis_ATCC8492, Enterococcus faecalis_TX0104, Lactobacillus casei_A047, Bacteroides fragilis_CL03T00C08, Acinetobacter lwoffii_F78, Fusobacterium nucleatum_F0419, Enterococcus faecalis OGlRF, Bacteroides

thetaiotaomicron_ATCC29148, Parabacteroides johnsonii_CL02T12C29, Bacteroides oleiciplenus_DSM22535, Lactobacillus rhamnosus_LMS2-l, Bacteroides

massiliensis D SMI 7679, Parabacteroides merdae_CL03T12C32, Fusobacterium mortiferum_A016, Bifidobacterium breve_SK134, Bacteroides finegoldii_DSM17565 and/or Bacteroides fragilis_3_l_12).

The bacteria described herein can be grown in culture using methods known in the art. For example, Bacteroides Clostridia, Bifidobacteria, Lactobacilli, Enterococci, Fusobacteria, Propionibacteria and Peptostreptococcus can be grown in supplemented Yeast extract-peptone-glycerol (YPG) medium, Blood Brucella Agar or Blood TSA Agar plates. Acinetobacter can be grown in SB medium and LB Agar plates. Lachnospiraceae, Veillonella, Coprobacillus can be grown in chopped meat broth. Anaerobic bacteria can be cultured under strictly anaerobic conditions (80% N ₂ , 10% H ₂ , 10% C0 ₂ ) at 37 °C in an anaerobic chamber. Staphylococcus can be grown aerobically, at 37 °C in L-broth and on LB Agar plates.

In some embodiments, combinations of species or strains of bacteria induce the production of Rory ⁺ Helios ^" LP Tregs {e.g., the strains and species of bacteria listed in Figure 5) are used in the methods and/or compositions provided herein. In certain embodiments, a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 of species or strains of bacteria induce the production of Rory ⁺ Helios ^" LP Tregs are used in the methods and/or compositions described herein.

In some embodiments, the combination of bacteria used in the compositions and/or methods described herein includes Bacteroides ovatus and one or more bacterial strains selected from the group consisting of Campylobacter jejuni, Clostridia ramosum,

Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum,

Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni and one or more bacterial strains selected from the group consisting of Clostridia ramosum, Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum and one or more bacterial strains selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum, Bacteroides

thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus and one or more bacterial strains selected from the group consisting of Enterococcus faecalis, Clostridium histolyticum, Bacteroides thetaiotaomicron,

Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis and one or more bacterial strains selected from the group consisting of Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of

Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of

Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides merdae and one or more bacterial strains selected from the group consisting of

Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of

Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides finegoldii and Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides ovatus, Campylobacter jejuni and one or more bacterial strains selected from the group consisting of Clostridia ramosum, Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Clostridia ramosum and one or more bacterial strains selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum,

Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Staphylococcus saprophyticus and one or more bacterial strains selected from the group consisting of Enterococcus faecalis, Clostridium histolyticum, Bacteroides

thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei,

Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus,

Enterococcus faecalis and one or more bacterial strains selected from the group consisting of Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus,

Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus,

Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of

Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus,

Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides ovatus, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides ovatus, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Clostridia ramosum and one or more bacterial strains selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Clostridium histolyticum,

Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Staphylococcus saprophyticus and one or more bacterial strains selected from the group consisting of Enterococcus faecalis, Clostridium histolyticum, Bacteroides

thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Enterococcus faecalis and one or more bacterial strains selected from the group consisting of Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus,

Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Clostridium histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni,

Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Campylobacter jejuni, Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni,

Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Campylobacter jejuni, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Campylobacter jejuni, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Staphylococcus saprophyticus and one or more bacterial strains selected from the group consisting of Enterococcus faecalis, Clostridium histolyticum, Bacteroides

thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Enterococcus faecalis and one or more bacterial strains selected from the group consisting of Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus,

Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Clostridium histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum,

Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum,

Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridia ramosum, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis and one or more bacterial strains selected from the group consisting of Clostridium histolyticum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Clostridium histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum,

Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Acinetobacter woffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides fmegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Staphylococcus saprophyticus, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Clostridium histolyticum and one or more bacterial strains selected from the group consisting of Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis,

Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis,

Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Enterococcus faecalis, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides thetaiotaomicron and one or more bacterial strains selected from the group consisting of Bacteroides vulgatus, Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis,

Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Clostridium

histolyticum, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides thetaiotaomicron, Bacteroides vulgatus and one or more bacterial strains selected from the group consisting of Bacteroides uniformis, Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides thetaiotaomicron, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Acinetobacter woffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus,

Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis,

Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides

thetaiotaomicron, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Bacteroides uniformis and one or more bacterial strains selected from the group consisting of Lactobacillus casei, Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus,

Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus,

Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides vulgatus, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Lactobacillus casei and one or more bacterial strains selected from the group consisting of Bacteroides fragilis, Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Acinetobacter Iwoffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides fmegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis,

Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides uniformis, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei,

Bacteroides fragilis and one or more bacterial strains selected from the group consisting of Acinetobacter Iwoffii, Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei,

Acinetobacter woffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei,

Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei,

Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus casei, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Acinetobacter woffii and one or more bacterial strains selected from the group consisting of Fusobacterium nucleatum, Parabacteroides johnsonii, Bacteroides oleiciplenus,

Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae,

Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Bacteroides fragilis, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis,

Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium

mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides fragilis,

Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Fusobacterium nucleatum and one or more bacterial strains selected from the group consisting of Parabacteroides johnsonii, Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of

Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii,

Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of ^' Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Acinetobacter Iwoffii, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Parabacteroides johnsonii and one or more bacterial strains selected from the group consisting of Bacteroides oleiciplenus, Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium nucleatum, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Bacteroides oleiciplenus and one or more bacterial strains selected from the group consisting of Lactobacillus rhamnosus, Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides johnsonii, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides oleiciplenus, Lactobacillus rhamnosus and one or more bacterial strains selected from the group consisting of Bacteroides massiliensis, Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides oleiciplenus, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides oleiciplenus, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides finegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides oleiciplenus, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides finegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides oleiciplenus, Bacteroides finegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus, Bacteroides massiliensis and one or more bacterial strains selected from the group consisting of Parabacteroides merdae, Fusobacterium mortiferum, Bacteroides finegoldii and/ 'or Bifidobacterium breve. In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides fmegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Lactobacillus rhamnosus, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides massiliensis, Parabacteroides merdae and one or more bacterial strains selected from the group consisting of Fusobacterium mortiferum, Bacteroides fmegoldii and/or

Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides massiliensis, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Bacteroides massiliensis, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides merdae, Fusobacterium mortiferum and one or more bacterial strains selected from the group consisting of Bacteroides fmegoldii and/ 'or Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Parabacteroides merdae, Bacteroides fmegoldii and Bifidobacterium breve.

In some embodiments, the combination of bacteria includes Fusobacterium mortiferum, Bacteroides fmegoldii and Bifidobacterium breve.

Bacterial Compositions

In certain embodiments, provided herein is a composition {e.g., a pharmaceutical composition, a dietary supplement or a food product) containing bacteria or combinations of bacteria that induce the production of Rory ⁺ Helios ^" LP Tregs. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from among the bacterial species described herein. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are selected from among the bacterial strains described herein.

In some embodiments, the compositions described herein may include only one species of bacteria described herein or may include two or more species of the bacteria described herein. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the species described herein, in any combination, can be included in the compositions provided herein.

In some embodiments, the compositions described herein may include only one strain of the bacteria described herein or may include two or more strains of the bacteria described herein. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the strains described herein, in any combination, can be included in the

compositions provided herein.

In some embodiments, the composition described herein may be a pharmaceutical composition, a dietary supplement, or a food product (e.g., a food or beverage). In some embodiments, the food product is an animal feed.

As described in detail below, the pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for oral or rectal administration.

In certain embodiments, the pharmaceutical composition for oral administration described herein comprises an additional component that enables efficient delivery of the bacteria to the colon, in order to more efficiently induce the production of LP Tregs in the colon. In some embodiments, pharmaceutical preparation that enables the delivery of the bacteria to the colon can be used. Examples of such formulations include pH sensitive compositions, such as buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach. When a pH sensitive composition is used for formulating the pharmaceutical preparation, the pH sensitive composition can be a polymer whose pH threshold of the decomposition of the composition is between about 6.8 and about 7.5.

Another embodiment of a pharmaceutical composition useful for delivery of the bacteria to the colon is one that ensures the delivery to the colon by delaying the release of the bacteria by approximately 3 to 5 hours, which corresponds to the small intestinal transit time. In some embodiments, the pharmaceutical composition for delayed release includes a hydrogel shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, with the result that the contents are effectively released (released predominantly in the colon). Delayed release dosage units include bacteria-containing compositions having a material which coats or selectively coats the bacteria. Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and/or enzyme degradable polymers. A wide variety of coating materials for efficiently delaying the release is available and includes, for example, cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

Examples of composition enabling the delivery to the colon further include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of U.S. Pat. No. 6,368,586, hereby incorporated by reference) and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.

An example of a system enabling the delivery to the colon is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach. Such a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).

Another example of the system enabling the delivery to the colon is a system of delivering a composition to the colon, the system being specifically decomposed by an enzyme (for example, a carbohydrate hydrolase or a carbohydrate reductase) present in the colon. Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

In some embodiments, the compositions described herein further comprise an immunosuppressive agent. Examples of immunosuppressive agents include, but are not limited to, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast- cell stabilizers, monoclonal anti-IgE antibodies, vaccines (e.g., vaccines used for vaccination where the amount of an allergen is gradually increased), cytokine inhibitors, such as anti-IL-6 antibodies, TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept, and combinations thereof.

In some embodiments, the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.

In some embodiments the composition is a food product for animals, including humans. The animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.

Rory

In certain embodiments, provided herein are methods inducing LP Tregs and/or treating a disease or disorder associated with a pathological immune response, such as an inflammatory bowel disease, by inducing Rory. Activation of Rory can, for example, be via an increase in Rory protein activity or Rory protein amount. For example, agents that induce Rory include agents that increase Rory protein activity, agents that decrease Rory protein degradation, agents that increase Rory mRNA stability, and agents that increase

transcription and/or translation of nucleic acids encoding Rory protein.

Rory is a DNA-binding transcription factor and is a member of the R1 subfamily of nuclear hormone receptors. Rory is encoded by the Rorc gene. While Rory has previously been reported to be antagonistic to Treg inducing transcription factor FoxP3, as disclosed herein, Rory is expressed in a major population of LP Tregs. The amino acid sequence human Rory is available at NCBI accession number XP 006711547.2, which is incorporated by reference herein. The nucleic acid sequence of the human Rory isoform mRNA is available at NCBI accession numbers XM_006711484.2, which is incorporated by reference herein.

Rory Agonists

In certain embodiments, provided herein are compositions and methods for inducing Tregs and/or for treating inflammatory bowel disease. These methods include administering an agent that activates Rory {i.e., a Rory agonist). Such agents include those disclosed below, those known in the art and those identified using the screening assays described herein. In some embodiments, any agent that activates Rory can be used to practice the methods disclosed herein. Rory agonists may be small molecules, proteins, peptides, nucleic acids, carbohydrates or antibodies. In some embodiments, the Rory agonist is a sterol. Exemplary Rory agonists are described in Hu et al, Nature Chemical Biology

11 : 141-147 (2015), Soroosh et al, Proc. Natl. Acad. Sci. USA 111 : 12163-12168 (2014) and Santori et al, Cell Metabolism 21 :286-297 (2015) and U.S. Pat. 8,389,739, each of which is hereby incorporated by reference.

In some embodiments, the Rory agonist is a sterol selected from the group consisting of cholesterol sulfate, 25-OHC, 25-OHC sulfate, desmosterol, desmosterol sulfate, 5a,6a-epoxycholestanol sulfate, 7a,27-diOHC, 5a,6a-epoxycholestanol, 24S,25- epoxycholesterol, 7a-OHC, 20a-OHC, 22R-OHC, 24S-OHC, 27-OHC, 7p,27-OHC, 7- keto,27-OHC, 7a,27-OHC, 4AC4MA°, A7-daf, zymosterol, 4C, 7DHC, zymosterone, 4C220H, 4,7-cholesten, 250H, OR-12872, OR-942, or a prodrug, active derivative or pharmaceutically acceptable salt thereof.

In some embodiments, the Rory agonist is 25-OHC, a 25-OHC prodrug, an active derivative of 25-OHC or a pharmaceutically acceptable salt thereof. 25-OHC has the following chemical structure:

In some embodiments, the Rory agonist is hyodeoxycholic acid methyl ester (OR- 942), a hyodeoxycholic acid methyl ester prodrug, an active derivative of hyodeoxycholic acid methyl ester or a pharmaceutically acceptable salt thereof, hyodeoxycholic acid methyl ester has the following chemical structure:

In some embodiments, the Rory agonist is OR-12872 (4R-[3R,6R-Bis-(tert-butyl- dimethly-silanyloxy)- 1 OR, 13R-dimethyl-5R-8 S-9S- 14S- hexadecahydrocyclopenta[a]phenanthren-17R-yl]-pentanoic acid methyl ester), an OR- 12872 prodrug, an active derivative of OR-12872 or a pharmaceutically acceptable salt thereof. Synthesis of OR-12872 is described in U.S. Pat. 8,389,739, which is hereby incorporated by reference in its entirety. OR-12872 has the following chemical structure:

In some embodiments, the Rory agonist is desmosterol, a desmosterol prodrug, an active derivative of desmosterol or a pharmaceutically acceptable salt thereof. Desmosterol has the following chemical

In some embodiments, the Rory agonist is 7p,27-OHC, a 7p,27-OHC prodrug, an active derivative of 7p,27-OHC or a pharmaceutically acceptable salt thereof. 7p,27-OHC has the following chemical structure:

In some embodiments, the Rory agonist is 7keto,27-OHC, a 7keto,27-OHC prodrug, an active derivative of 7keto,27-OHC or a pharmaceutically acceptable salt thereof.

7keto,27-OHC has the following chemical structure:

In some embodiments, the Rory agonist is 4a-carboxy,4P-methyl-zymosterol (4ACD8), a 4ACD8 prodrug, an active derivative of 4ACD8 or a pharmaceutically acceptable salt thereof. 4ACD8 has the followin chemical structure:

In some embodiments, agents useful in the methods described herein can be identified by screening compound libraries (including sterol libraries) to identify compounds that induce Rory activity (e.g., in a reporter assay, such as those described in Hu et al, Nature Chemical Biology 11 : 141-147 (2015), Soroosh et al, Proc. Natl. Acad. Sci. USA 111 : 12163-12168 (2014) and Santori et al, Cell Metabolism 21 :286-297 (2015) and U.S. Pat. 8,389,739, each of which is hereby incorporated by reference).

Agents useful in the methods disclosed herein may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Agents may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al, 1994, J. Med. Chem. 37:2678-85); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12: 145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al.

(1994) Proc. Natl. Acad. Sci. USA 91 : 11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261 : 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33 :2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33 :2061; and in Gallop et al. (1994) J. Med. Chem. 37: 1233.

Libraries of agents may be presented in solution (e.g., Houghten, 1992,

Biotechniques 13 :412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, USP 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89: 1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.). Pharmaceutical Compositions

In certain embodiments, provided herein is a composition, e.g., a pharmaceutical composition, containing at least one agent described herein together with a

pharmaceutically acceptable carrier. In one embodiment, the composition includes a combination of multiple {e.g., two or more) agents described herein.

As described in detail below, the pharmaceutical compositions disclosed herein may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; or (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation.

Methods of preparing these formulations or compositions include the step of bringing into association an agent described herein with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association an agent described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product. Pharmaceutical compositions suitable for parenteral administration comprise one or more agents described herein in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Regardless of the route of administration selected, the agents provided herein, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions disclosed herein, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

Methods

In certain aspects, provided herein are methods for inducing production of

Rory ⁺ Helios ^" LP Tregs and/or for treating or preventing a disease or disorder associated a pathological immune response, such as an autoimmune disease, an allergic reaction and/or an inflammatory disease. In some embodiments, the disease or disorder is an inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis).

The methods described herein can be used to treat any subject in need thereof. As used herein, a "subject in need thereof includes any subject that has a disease or disorder associated with a pathological immune response {e.g., an inflammatory bowel disease), as well as any subject with an increased likelihood of acquiring a such a disease or disorder.

The compositions described herein can be used, for example, as a composition for preventing or treating (reducing, partially or completely, the adverse effects of) an autoimmune disease, such as chronic inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, or Hashimoto's disease; an allergic disease, such as a food allergy, pollenosis, or asthma; an infectious disease, such as an infection with Clostridium difficile; an inflammatory disease such as a TNF-mediated inflammatory disease (e.g., an inflammatory disease of the gastrointestinal tract, such as pouchitis, a cardiovascular inflammatory condition, such as atherosclerosis, or an inflammatory lung disease, such as chronic obstructive pulmonary disease); a

pharmaceutical composition for suppressing rejection in organ transplantation or other situations in which tissue rejection might occur; a supplement, food, or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of effector T-cells.

In some embodiments, provided herein are methods of treating an inflammatory bowel disease. Inflammatory bowel diseases include, for example, certain art-recognized forms of a group of related conditions. Several major forms of inflammatory bowel diseases are known, with Crohn's disease (regional bowel disease, e.g., inactive and active forms) and ulcerative colitis (e.g., inactive and active forms) the most common of these disorders. In addition, the inflammatory bowel disease encompasses irritable bowel syndrome, microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common forms of IBD include indeterminate colitis, pseudomembranous colitis (necrotizing colitis), ischemic

inflammatory bowel disease, Behcet' s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, and primary sclerosing cholangitis.

In some embodiments, administration is in combination with administration of at least one prebiotic substance (e.g., a prebiotic substance that favors the growth of the bacterial species in the composition over the growth of other human commensal bacterial species). In some embodiments, the prebiotic substance is a nondigestible oligosaccharide. In some embodiments, the prebiotic substance is almond skin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose, amylopectin, acetyl-Co A, biotin, beet molasses, yeast extracts, and resistant starch.

In some embodiments, the compositions described herein are administered in combination with an immunosuppressive agent. Examples of immunosuppressive agents include corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast- cell stabilizers, monoclonal anti-IgE antibodies, vaccines, anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept, and combinations thereof. Also described herein is a composition that comprises the bacterial composition and at least one substance selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines,

glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anticholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines, anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab, or etanercept, and combinations thereof.

In some embodiments, the methods provided herein include the step of

administering at least one antibiotic before or in combination with, the administration of a composition described herein.

In some embodiments, the methods provided herein include the step of determining the subject's microbiome prior to the administration of a composition described herein. In some embodiments, the selection of the bacteria or combination of bacteria administered to the subject is determined based upon the make-up of the subject's microbiome.

Actual dosage levels of the bacteria in the compositions described herein may be varied so as to obtain an amount of the bacteria which is effective to achieve the desired therapeutic response for a particular patient. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.

The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally and parenterally. In certain embodiments the pharmaceutical compositions are delivered generally ( e.g., via oral or parenteral administration).

EXEMPLIFICATION

Experimental Procedures

Mice

Mice were bred in a pathogen-free facility or in germ-free isolators. For IL-33 administration, recombinant mouse IL-33 (BioLegend) was administered i.p. at 2 μg/ injection every other day for 7 days.

Preparation of lymphocytes and flow cytometry Intestinal tissues were treated with RPMI containing 1 mM dithiothreitol, 20 mM EDTA and 2% FBS at 37C for 15 min to remove epithelial cells. The tissues were then minced and dissociated in collagenase solution containing 1.5 mg/ml collagenase II (Gibco) 0.5 mg/ml, dispase and 1% FBS containing RPMI solution constantly stirring at 37 °C for 45 min. Single cell suspensions were then filtered and washed with 4% RPMI solution. The Peyer's Patches were treated in a similar fashion except for the first step of removal of epithelial cells. Lymph nodes and spleens were mechanically disrupted.

Single-cell suspensions were stained with antibodies against CD4, CD8, TCR-b, CD45, IL-17A, ΓΡΝγ, Helios (Biolegend), Rory, Foxp3, ST2( (eBioscience), Nrpl(R&D Systems), anti-ST2 conjugated to biotin (mdBioproducts). For cytokine analysis, cells were treated with RMPI containing 10% FBS, 10 ng/ml phorbol 12-myristate 13-acetate (Sigma), 1 uM Ionomycin (Sigma) in presence of GolgiStop (BD Biosciences) for 3.5 hours. For intracellular staining of cytokines and transcription factors, cells were stained for surface markers and fixed in eBioscience Fix/Perm buffer overnight, followed by permeabilization in eBioscience permeabilization buffer for 45 min in the presence of antibodies. Cells were acquired with a BD LSRII and analysis was performed with FlowJo (Tree Star) software. Gene expression profiling

Cells were double-sorted into TRIzol (Invitrogen) using a MoFlo sorter. All samples were generated in duplicate or triplicate. Sample processing and data analysis were performed on GeneChip Mouse Genome M1.0 ST chip arrays (Affymetrix) as described in D. Cipolletta et al., Nature 486, 549 (2012), which is hereby incorporated by reference. TNBS Colitis

Mice were desensitized via administration of 1% TNBS (prepared in 4: 1 acetone: olive oil solution) on shaven skin between the shoulders. A week later, colitis was induced by intrarectal administration of 150-200 μg TNBS per gram of mouse (Sigma) in 50% ethanol into anaesthetized mice via a thin round-tip needle. The TNBS concentration was optimized for each batch of TNBS used in both germ free and SPF mice. The tip of the needle was inserted 4 cm proximal to the anal verge, and mice were held in a vertical position for at least 1 min after the injection. All the mice were observed and weighed daily and were sacrificed on day 4 after intrarectal TNBS administration at the peak of the disease. A combined colitis score was calculated based on weight loss, histology, diameter of the colon, and the appearance of the stool. For TNBS induction in gnobiotic mice, mice were colonized with single bacterial species for 2 weeks, as described earlier, prior to TNBS administration. Weight loss was scored as follows: 0, 0-4% weight loss or weight gain; 1, 4-10% weight loss; 2, 10-15%) weight loss; 3, 15-20%) weight loss; 4, more than 20%) weight loss. The appearance of the stool was assessed as follows: 0, hard; 1, softened stool; 2, diarrhea; 3, bloody diarrhea. The thickness of the colon was assessed based on differential in diameter (Adiameter=diameter of TNBS treated colon- diameter of untreated colon): 0,no change; 1, Adiameter <lmm;2, Adiameter= l-2mm; 3, Adiameter= 2-3mm; 4, Adi ameter>3 mm .

Histology

Colons were fixed with Bouin's fixative, sectioned, and stained with haematoxylin and eosin. The degree of inflammation in the distal part of colon was graded from 0 to 4 as follows: 0, normal intact structure; 1, mild inflammation with intact structure; 2, infiltration of leukocytes and some damage to structure; 3, severe inflammation accompanied by complete loss of structure; 4, necrosis of the tissue.

Bioinformatic and statistical analysis

Significance was assessed by the Student's t test. When necessary paired t test was used and a p value of < 0.05 was deemed statistically significant.

Microarray data were background-corrected and normalized using the robust multi- array average (RMA) algorithm implemented in the GenePattern software package and replicates were averaged. Genes that have a mean expression value of at least 120 in any of the samples with a coefficient of variance set for >0.25 were analyzed. Genes that over- or under-represented in colonic Tregs compared with splenic Tregs or in Rory ⁺ Tregs compared with Rory ^" Tregs were determined by at least 2-fold or 1.5-fold differential expression respectively. Colon-preferential and tissue-specific gene clusters were determined by clustering genes that are over or under-expressed by 2-fold or higher in any of the tissues compared with spleen. Hierarchical clustering analysis used Pearson correlation in all comparisons. Genes that were over- or under-represented by 1.5 fold or higher in colonic Tregs compared with all non-lymphoid tissues were considered colon- preferential. Genes that have p values>0.05 at and FDR of 0.1 were deemed significant. Example 1: Expression of Rory by colonic regulatory T cells

Comparison of gene expression profiles of highly purified CD4 ⁺ FoxP3 ⁺ Tregs (from

Foxp3 ^ig f ^p reporter mice, described Bettelli et al., Nature 441, 235 (2006), which is hereby incorporated by reference) from colon or spleen uncovered 933 differential transcripts (at a FoldChange > 2 and FDR < 0.1; Fig. 1A (top), Fig. 2). Among these differential transcripts, several important signaling and effector pathways (including Icos, Gzmb, Lag3, Areg, ILlrll; Fig. 1 A (top)), were shared in patchwork manner by other tissue Tregs, but -39% (at a colon specific bias > 1.5 fold compared to other tissues) had preferential expression in colonic Tregs (including 1110, Ctla4, Havcr2, Ccl20, Jak2, Fosl2; Fig. 1A (bottom)).

Correspondingly, several transcription factors were over-expressed in colon, including Ahr, Epasl, Heyl, Bcl6, Npas2, Nrldl, and Maf. The most differential of these transcription factors was Rorc (gene that encodes Rory; Fig. IB, 1C). Rory controls many aspects of innate and adaptive immunocyte differentiation, and is known as the key regulator of IL17 producing CD4 ⁺ T cells (Thl7), and as a reciprocal antagonist of FoxP3 during in vitro differentiation in which iTreg and Thl7 represent alternative cell fates (reviewed in Korn et al., Annu. Rev. Immunol 27, 485 (2009), which is hereby incorporated by reference).

Flow cytometry confirmed that a large proportion of colonic CD4 ⁺ FoxP3 ⁺ Tregs indeed expressed Rory (40-60% in adult SPF mice), a phenotype largely absent in spleen or lymph node (LN) (Fig. 1C). Helios and Nrpl, which are considered markers of thymus- derived Tregs (reviewed in Bilate and Lafaille, Annu. Rev. Immunol. 30, 733 (2012), which is hereby incorporated by reference), were absent on colonic Rory ⁺ Tregs (Fig. ID, Fig. 3 A). The combination of Rory and Helios demarcated three distinct subsets of colonic Tregs, Rory ⁺ representing the majority of Helios ^" cells (Fig. ID, 3 A left). This pattern was also seen for Treg cells in other tissues, and consistent with the RNA data. Rory ⁺ Tregs were also detected in the small intestine and in the regenerating muscle, albeit at lower frequencies (Fig. IE). Rory ⁺ Tregs were distinct from those expressing the IL-33 receptor, most of which were Helios ⁺ (Fig. ID, Fig. 3B). Rare Tregs expressing IL-17 and Rory have been observed during chronic inflammation or cancer, usually with high levels of Helios suggestive of a thymic origin (reviewed in Du et al, J. Leukoc. Biol. 96, 39 (2014), hereby incorporated by reference). IL-17 production in colonic Rory ⁺ Tregs was tested. In contrast to the clear subset of IL-17 expressing Tregs in the small intestine LP, colonic Rory+ Tregs did not secrete IL-17A or F (Fig. IF).

Example 2: Certain Species of Gut Microbacteria Induce Rory ⁺ Regulatory T cells

The properties of colonic Rory ⁺ Treg population suggested a link to the gut microbiota. Germ free (GF) mice had a far lower proportion of Rory ⁺ Tregs than their conventionally-raised specific-pathogen-free (SPF) counterparts (Fig. 4A). During normal maturation of SPF mice, Rory ⁺ Tregs appeared between 15 and 25 days of age (Fig. 4B), coincident with the radical changes in the gut microbiota that accompany the transition to solid food. Interestingly, Rory Tregs appeared a few days after cells with a RoryTTelios ^" phenotype. Oral antibiotic treatment of adult mice strongly affected Rory ⁺ Tregs (Fig. 4C), a large reduction following a broad-spectrum combination (vancomycin, neomycin, metronidazole, ampicillin "VMNA") while individual antibiotics have less or no effect suggested the contribution of several microbes.

A panel of bacterial species selected from the microbiota of the human

gastrointestinal tract were tested for their ability to induce the production of Rory ⁺ Tregs. Bacteria were obtained from the ATCC, BEI Resources, or the German Collection of Microorganisms and Cell Cultures (DSMZ), or from the laboratory collections at Harvard Medical School. Anaerobic bacteria were cultured under strictly anaerobic conditions (80% N ₂ , 10% H ₂ , 10% C0 ₂ ) at 37°C in an anaerobic chamber.

Germfree C57BL/6J were maintained in sterile isolators, and were inoculated by gavage with single bacterial species at 4 weeks of age, then housed in gnobiotic isolators. After 2 weeks, mice were sacrificed, colon tissue was harvested, cleaned of luminal content and treated with RPMI containing 1 mM DTT, 20 mM EDT A and 2% FB S at 37°C for 15 min to remove epithelial cells. The colon tissue was then minced and dissociated in collagenase solution (1.5mg/ml collagenase II (Gibco), 0.5mg/ml dispase and 1%FBS in RPMI) with constantly stirring at 37°C for 45min. Single cell suspensions were then filtered and washed in RPMI buffer, and stained with antibodies against CD4, CD8, TCR-β, CD45, then fixed in eBioscience Fix/Perm buffer overnight, followed by permeabilization in eBioscience permeabilization buffer for 45 min in the presence of conjugated anti-Foxp3 and anti-Rory antibodies. Cells were analyzed with a BD LSRII flow cytometer, and data analysis was performed with Flow Jo (Tree Star) software. The proportion of Rory ⁺ Tregs was determined within the total population of CD4 ⁺ FoxP3 ⁺ Treg cells, and the significance of the difference relative to unmanipulated germfree mice determined using a two-tailed Student's T-test. False-discovery rates were estimated using the Benjamini-Hochberg procedure.

A number of microbes elicited colonic Rory ⁺ Tregs, with a reproducible gradient of responses, and for some at frequencies comparable with those of SPF mice (Fig. 4D and 5). This restoration of Rory ⁺ Tregs was independent of bacterial load and not accompanied by inflammation (Fig. 6). Bacteria able to induce Rory ⁺ Treg (and FoxP3 ⁺ Tregs more generally) belonged to several phyla and genera. The ability to promote colonic Treg production within the Bacteroides genus varied (high proportions with B. thetaiotaomicron, low with B. fragilis). A wider Bacteroides panel was assessed (Fig. 4E and 5). Here again, a range of abilities to induce colonic Rory ⁺ Tregs was observed. Colonic Rory ⁺ Tregs did not appear immediately after GF colonization, but only after a few days. Here again,

Rory ⁺ Helios ^" Tregs appeared to follow RoryTTelios ^" cells (Fig. 4F), suggesting that the Rory ^" Helios ^" cells are a first differentiation intermediate, prior to the induction of Rory.

Example 3: Reduced gut regulatory T cells in Rory de ficient mice

Foxp3-cre Ror f^ mice were generated. Such mice have a Treg-selective deficiency in Rorc. These mice do not show systemic Treg deficiency or scurfy-like pathology, but the amount of total colonic Tregs, and more specifically that of Helios ^" Tregs, was reduced (Fig. 7). The loss of Rory Tregs in Foxp3-cre Ror^fl mice was accompanied by an increase of IL-17 and IFNy production among FoxP3 ^" conventional CD4 ⁺ T cells (Fig. 8A), suggesting a decreased regulatory activity of colonic Tregs in the absence of Rory. To verify this point, Foxp3-cre Ror^ mice were assessed in the Trinitrobenzenesulfonic acid- (TNBS) induced colitis model. Foxp3 ^' cre. mice showed significant exacerbation of disease severity, reflected in the overall colitis score and the histopathology (Figs. 8B and 8C, Fig. 9). In GF mice monocolonized with microbes that induce different levels of Rory ⁺ Tregs, and challenged in the TNBS sensitization model, a significant correlation between the frequency of Rory ⁺ Tregs and the colitis score was observed (Fig. 8D). These results demonstrate a non-redundant role for Rory ⁺ Tregs in colonic homeostasis.

Example 4: Rory function in Rory ⁺ regulatory T cells

What transcripts Rory controls in Rory ⁺ Tregs, and whether it is necessary to specify this particular Treg lineage was also examined. Gene expression profiles of Rory ⁺ and Rory ^" colonic Tregs (from an intercross of Foxp3 ^{thyl 1} and Rorc^ reporter mice) were compared. Rory ⁺ Tregs were enriched relative to Rory ^" Tregs in some, but not all, transcripts of the colonic Treg signature (Fig. 10A), notably 1123r, Cxcr3, Tbx21 and Havcr2. Preferential expression of some of the corresponding proteins, including Cxcr3, was validated (Fig. 10B). Illrll (encodes IL-33R), Nrpl and Ikzf2 were underrepresented in Rory ⁺ Tregs.

It was next examined how the transcripts associated with Rory in colonic Tregs relate to the Rory-dependent signature in conventional Thl7 cells, as defined from a comparison of CD4 ⁺ T cells from the small intestine of mice colonized, or not, with

Segmented Filamentous Bacteria, classic inducers of Rory ^" dependent Thl7 cells (Fig. IOC). Much of the classic Thl7 signature did not correlate with Rory in colonic Tregs (Fig. IOC), and there was only a minority of shared transcripts (e.g. Rorc itself, 1123r, Ccl20, as well as 1117a). In contrast, Rory in colonic Tregs was specifically associated with Treg preferential transcripts (Fig. IOC, e.g. Havrc2, Lag3, Tbx21).

Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.