CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/146,321, filed February 5, 2021, and U.S. Provisional Application Serial No. 63/278,740, filed November 12, 2021, which applications are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to treatments to prevent the risk of sepsis.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grants R01-GM132149 and R01- HL 160811, awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0004] Despite recent advances in antibiotic therapy and supportive critical care, sepsis remains a leading cause of death in intensive care units. Currently, the increased vascular permeability is well-recognized to be responsible for sepsis-triggered organ failure and patient mortality. This is particularly the case in the lungs where capillary leak induces lung edema, leading to acute respiratory distress syndrome (ARDS). However, whether vascular permeability increases in the heart that contributes to cardiac dysfunction during sepsis is not well-studied. Recently, we and others showed a positive connection between vascular leakage, cardiac depression, and mortality during sepsis. Nonetheless, the mechanisms underlying sepsis-induced cardiac capillary leak remains obscure. In general, vascular barrier integrity is maintained by junctional complexes including tight junctions and adherens junctions which are anchored to the actin cytoskeleton between endothelial cells (ECs). Upon sepsis conditions, proinflammatory factors can disrupt EC barrier integrity by either altering protein levels of junctional molecules or disturbing actin dynamics, leading to paracellular gap formation. At present, a variety of mediators (/.< ., Racl/RhoA) have been identified to play a critical role in regulating vascular permeability. However, to date, no specific treatment targeting the vascular leak in sepsis is yet available. Thus, a deeper understanding of mediators and their associated mechanisms in sepsis-elicited vascular leak is of great importance for the development of future therapeutic strategies.

SUMMARY OF THE INVENTION

[0005] One embodiment of the present invention addresses this need by providing a method of reducing the risk of a sepsis-induced vascular leak, tissue edema or organ dysfunction in a subject. The method involves administering an effective amount of a composition selected from the group consisting of Lipocalin 10 (SEQ ID NO: 1), a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2, LcnlO-expressing vectors for full length/truncated LcnlO, or combinations thereof to the subject. In one embodiment, the method is used to reduce the risk of a sepsis-induced vascular leak. In another embodiment, the method is used to reduce the risk of tissue edema. In one embodiment, the method is used to reduce the risk of organ dysfunction. In one embodiment, the subject is administered Lipocalin 10 (SEQ ID NO: 1). In another embodiment, the subject is administered a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2. In one embodiment, the subject is administered with Lipocalin 10 (SEQ ID NO: 1) or a truncated Lipocalin 10 (SEQ ID NO: 2) at a dosage of 50-200 ng/g body weight. In another embodiment, the subject is administered with Lipocalin 10 (SEQ ID NO: 1) or a truncated Lipocalin 10 (SEQ ID NO: 2) via vein injection.

[0006] In another embodiment of the present invention, a method of reducing the risk of a heart attack-induced cardiac dysfunction, atherosclerosis, inflammatory bowel disease or diabetes- induced cardiomyopathy in a subject is provided. The method involves administering an effective amount of a composition selected from the group consisting of Lipocalin 10 (SEQ ID NO: 1), a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2, LcnlO- expressing vectors for full length/truncated LcnlO, or combinations thereof to the subject. In one embodiment, the method is used to reduce the risk of a heart attack-induced cardiac dysfunction. In another embodiment, the method is used to reduce the risk of atherosclerosis. In one embodiment, the method is used to reduce the risk of inflammatory bowel disease. In another embodiment, the method is used to reduce the risk of diabetes-induced cardiomyopathy. In one embodiment, the subject is administered Lipocalin 10 (SEQ ID NO: 1). In another embodiment, the subject is administered a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2.

[0007] In another embodiment of the present invention, a pharmaceutical composition comprising a truncated Lipocalin 10 protein having the amino acid sequence SEQ ID NO: 2 is provided. In one embodiment, the composition is for use in a method of reducing the risk of a sepsis-induced vascular leak, tissue edema or organ dysfunction in a subject. In another embodiment, the composition is for use in a method of reducing the risk of a heart attack-induced cardiac dysfunction, atherosclerosis, inflammatory bowel disease or diabetes-induced cardiomyopathy in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The foregoing summary, as well as the following detailed description of preferred embodiments of the application, will be better understood when read in conjunction with the appended drawings.

[0009] FIG. 1 A is a schematic showing cofilin-mediated actin dynamics in endothelial cells upon resting and sepsis conditions. TJ = tight junction; AJ = adherence junction.

[0010] FIG. IB is a graph showing expression levels of LIMK2 and Sshl in blood samples collected from 6 cohorts of sepsis patients and healthy donors.

[0011] FIG. 2 is a blot showing genotyping of a global LcnlO-KO mouse model. +/+: wild-type (WT); +/-: heterozygous; -/-: homozygous (KO).

[0012] FIGs. 3A and 3B are an image and a graph showing the generation of endothelial cellspecific LcnlO-transgenic mice (EC-LcnlO-Tg). FIG. 3A is a diagram of a transgenic vector; and FIG. 3B is RT-qPCR results showing that the mRNA levels of LcnlO were only increased in cardiac ECs (C-ECs) but not in cardiac fibroblasts or myocytes. (n=4, *, p<0.01 vs. WTs).

[0013] FIGs. 4A-4D are a series of graphs showing dynamic alterations of LcnlO expression in mouse hearts following LPS injection (FIG. 4A) occurs only in cardiac endothelial cells but not in either fibroblasts or cardiomyocytes (FIG. 4B). Similar results were observed in mouse hearts (FIG. 4C) following CLP surgery and in cardiac ECs (FIG. 4D) (n=4-9, *, p<0.05 vs. Oh).

[0014] FIGs. 5A-5D are a series of images and graphs showing: (FIG. 5A) Representative echocardiography images and their analysis reveal the significant reduction of EF% (FIG. 5B) and FS% (FIG. 5C) in LPS-treated LcnlO-KO mouse hearts, compared to LPS-WTs (n=5-7, *, p<0.05 vs. LPS-WTs). (FIG. 5D) The survival rate is greatly lower in LcnlO-KO mice than WT-mice following LPS injection. (n=10, *, p < 0.05 vs. WTs).

[0015] FIG. 6 is a volcano plot graph summarizing the RNA-seq. results of gene expression profiles in Ad.LcnlO- vs. Ad.GFP-infected ECs.

[0016] FIGs 7A-7C are a series of graphs showing (FIG. 7A) Exogenous addition of rLcnlO and (FIG. 7B) endogenous elevation of LcnlO by adenoviral vectors could upregulate both LRP2 and Sshl expression. (FIG. 7B, FIG. 7C) Knockdown of LRP2 could block LcnlO-induced elevation of Sshl in either (FIG. 7B) Ad.LcnlO-ECs or (FIG. 7C) rLcnlO-treated ECs, compared to respective controls. *, p < 0.05, n=4-6; ns: not significant.

[0017] FIGs 8A and 8B are a pair of graphs showing that adenovirus vector-mediated overexpression of LcnlO protects against LPS-caused EC leakage, which is dependent on the Sshl signaling, as measured by (FIG. 8A) FITC-dextran and (FIG. 8B) EB-albumin flux. (n=5, *p < 0.05; ns, not significant).

[0018] FIG. 9 is a scheme showing the LcnlO-induced reduction of vascular leakage via the LRP2- Sshl-Cofilin signaling during sepsis.

[0019] FIGs 10A-10C are a series of images and graphs showing that treatment of ECs with rLcnlO greatly protects against endothelial leakage, as evidenced by increases TEER (FIG. 10 A) and decreases leakage of FITC-dextran (FIG. 10B) and EB-albumin (FIG. 10C), compared to BSA-controls (n=4; *, p < 0.05).

[0020] FIGs 11A-11C are a series of graphs showing that the knockdown of Sshl by siRNAs greatly offset rLcnlO-induced protection against vessel leakage, as evidenced by the increases of TEER (FIG. 11 A), decreases of FITC (FIG. 1 IB) and EB (FIG. 11C) flux, (n=4-6; *, p < 0.05; ns, not significant).

[0021] FIGs 12A-12E are a series of graphs showing the expression levels of LcnlO in tissues of wild-type mice as well as in the blood and spleen of septic mice.

[0022] FIGs 13A-13H are a series of graphs showing that LcnlO deficiency increases inflammatory response locally and systemically in sepsis. Serum and peritoneal lavage fluid were harvested at 16 h after CLP-operation. The pro-inflammatory cytokines TNF-a (FIG. 13A, FIG. 13E), IL-6 (FIG. 13B, FIG. 13F) and MCP-1 (FIG. 13C, FIG. 13G) and anti-inflammatory cytokine IL-10 (FIG. 13D, FIG. 13H) were measured using ELISA kits. #, P<0.05; n=6-8 mice per group. [0023] FIGs 14A-14E are a series of images and graphs showing that LcnlO deficiency attenuates bacterial clearance and suppressed phagocytic capability of peritoneal macrophages (PMs) from septic mice.

[0024] FIGs 15A-15F are a series of graphs showing the dynamic expression of LcnlO in septic hearts and septic endothelial cells. CLP: cecal ligation and puncture.

[0025] FIGs 16A-16H are a series of graphs showing that knockdown of LcnlO augments the permeability in mouse cardiac endothelial cells (MCECs).

[0026] FIGs 17A-17G are a series of images and graphs showing that overexpression of full-length LcnlO reduces LPS- or TNF alpha-caused endothelial cell leakage.

[0027] FIGs 18A and 18B are a series of graphs showing expression levels of LcnlO in different tissues of wild type (WT) mouse and efferocytic macrophages.

[0028] FIGs 19A-19E are a series of graphs showing that the absence of LcnlO exacerbates I/R- induced cardiac dysfunction and cardiac damage.

[0029] FIGs 20A-20D are a series of graphs showing that addition of recombinant LcnlO protein increases macrophage efferocytosis in vitro through the upregulation of MerTK.

[0030] FIGs 21A-21E are a series of graphs showing that the overexpression of LcnlO in macrophgges upregulates anti-inflammatory genes.

[0031] FIG. 22 is a series of graphs showing the reduced LcnlO expression in macrophages upon different metabolic stress conditions.

[0032] FIGs 23A and 23B are a pair of graphs showing that the overexpression of LcnlO in Raw264.7 cells reduced inflammatory response to LPS, evidenced by reduced levels of IL-6 (FIG 23 A) and TNF-alpha (FIG 23B).

DETAILED DESCRIPTION OF THE INVENTION

[0033] The details of one or more embodiments of the disclosed subject matter are set forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided herein. [0034] The present disclosure may be understood more readily by reference to the following detailed description of the embodiments taken in connection with the accompanying drawing figures, which form a part of this disclosure. It is to be understood that this application is not limited to the specific devices, methods, conditions or parameters described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting. Also, in some embodiments, as used in the specification and including the appended claims, the singular forms “a,” “an,” and “the” include the plural, and reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” or “approximately” one particular value and/or to “about” or “approximately” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0035] While the following terms are believed to be well understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of the disclosed subject matter. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter belongs.

[0036] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0037] The present invention has found the therapeutic potential of LcnlO protein as a novel regulator of vascular barrier integrity and as a new protector against sepsis-induced organ failure. This invention provides new strategies to inhibit sepsis-induced vascular hyperpermeability and as a consequence, minimize fluid resuscitation and tissue edema. This novel approach can improve the survival of septic patients.

[0038] Lipocalin 10 (LcnlO) is a secreted protein that is a member of lipocalin family. Of specific interest, LcnlO is highly expressed in the heart, lymph node, spleen and thyroid. Data has shown that LcnlO was significantly down-regulated in the hearts of both endotoxin LPS- and cecal ligation-puncture (CLP)-treated mice, compared to their controls. Interestingly, further analysis of LcnlO expression in different cell types isolated from LPS- and CLP -treated hearts showed that reduction of LcnlO occurred only in cardiac ECs rather than in fibroblasts or cardiomyocytes. These compelling data indicate that LcnlO is involved in sepsis-induced cardiac vascular leak.

[0039] Using a global LcnlO-knockout (KO) mouse model, the present invention found that deficiency of LcnlO significantly increased vascular permeability, which correlated with more severe cardiac depression and higher mortality following LPS challenge, compared to LPS-treated wild-type (WT) mice. By contrast, in vitro overexpression of LcnlO in ECs showed a greater resistance to LPS-caused monolayer leak compared to control cells. An initial mechanistic analysis by RNA-sequencing and RT-qPCR showed that both endogenous and exogenous elevation of LcnlO in ECs could significantly upregulate slingshot homolog 1 (Sshl) expression. Sshl is a phosphatase known to dephosphorylate and activate Cofilin, a key actin-binding protein that plays an essential role in controlling actin filament re-arrangement. Importantly, knockdown of Sshl in ECs by siRNA greatly offset LcnlO-induced reduction of monolayer permeability upon LPS insult. Thus, based on these data, the inventors have found that LcnlO is critical for protecting against sepsis-induced cardiovascular leak via the activation of the Sshl-Cofilin pathway.

[0040] To define the precise role of LcnlO in vascular permeability during polymicrobial sepsis, a global LcnlO-KO mouse model can be utilized to test whether LcnlO-deficient mice are sensitive to sepsis-induced vascular leak, cardiac dysfunction, and death. By contrast, EC-specific LcnlO- transgenic (Tg) mice can be used to test if EC-specific elevation of LcnlO protects against sepsis- triggered vascular leak and heart injury, leading to improved myocardial function and survival rate. Polymicrobial sepsis can be induced by cecal ligation and puncture (CLP) surgery, and animal survival can be monitored over time. Cardiovascular leakage, edema, leukocyte infiltration and cardiac function can then be measured. This helps to evaluate LcnlO as a novel anti-sepsis mediator, which enhances vascular barrier integrity during sepsis.

[0041] LcnlO may have autocrine effects on EC function during sepsis. Importantly, our data shows that the addition of recombinant LcnlO protein (rLcnlO) to cultured ECs resulted in a reduced monolayer leakage upon LPS insult. Injection of the rLcnlO protein into septic mice can suppress vascular leak and cardiac edema, improve myocardial function as well as animal survival outcomes.

[0042] Further, the present invention can help identify the mechanism by which LcnlO-elicited inhibition of cardiovascular leak is dependent on Sshl -mediated actin dynamics. We have found that LcnlO can upregulate Sshl expression in ECs, and knockdown of Sshl greatly reduced LcnlO- elicited protection against EC monolayer leak upon LPS exposure. Given that Sshl is a key regulator of actin dynamics in ECs, this indicates that LcnlO-mediated inhibition of sepsis-induced vascular leak is dependent on Sshl -mediated actin filament reorganization.

LcnlO as novel regulator of sepsis-caused microvascular endothelial leakage

[0043] Sepsis is initiated by an uncontrolled immune response to a local severe infection. Prompt antibiotic therapy and adequate intravenous fluid therapy are essential for the treatment of septic patients to reduce mortality. However, fluid overload can be harmful, because vascular leakage is increased during sepsis, which in turn causes hypoperfusion, tissue edema and finally, loss of organ function (e.g. heart failure). Therefore, therapeutic interventions aimed at reducing vascular leak could be an effective treatment option against sepsis. Unfortunately, no therapies targeting the increased vascular permeability in sepsis have been successful thus far, indicating the need to understand better the mediators and mechanisms involved in sepsis-triggered endothelial dysfunction. At present, most prior work has focused on the sepsis-induced pulmonary capillary hyperpermeability that results in severe lung edema and acute respiratory distress syndrome. Few studies have investigated coronary vascular leakage, which is a major cause of heart failure and death in human patients with septic shock.

[0044] Of the known lipocalin (Lcn) family members, only Lcn2 (also called neutrophil -gelatinase associated lipocalin, NGAL) has been well characterized for its role in cancer, infectious disease and metabolic disorders. Interestingly, recent RNA-sequencing analysis of peripheral blood collected from sepsis patients showed that Lcn2 expression was significantly elevated in sepsis non-survivals versus survivals. By contrast, LcnlO expression was greatly reduced in sepsis nonsurvivals, compared to survivals.

[0045] Our data shows that expression levels of LcnlO were greatly reduced in mouse hearts following either LPS treatment or cecal ligation and puncture (CLP) surgery. Of interest, such reduction of cardiac LcnlO occurred only in endothelial cells (ECs), but not in either fibroblasts or cardiomyocytes. Furthermore, LcnlO-knockout (LcnlO-KO) mice exhibited higher mortality rate than control wild-type (WT) mice following endotoxin LPS challenge (n=10, p<0.05). Remarkably, LPS-treated LcnlO-KO mice revealed greater cardiac dysfunction, which was accompanied by the increased vascular leakage, compared to LPS-WTs. Importantly, in vitro overexpression of LcnlO in mouse cardiac ECs significantly reduced monolayer permeability, compared to control cells, following LPS insult. Together, these data prompted us to test whether ablation of LcnlO is sensitive, whereas elevation of LcnlO in ECs is resistant, to vascular leak and cardiac dysfunction during polymicrobial sepsis induced by CLP surgery.

[0046] Cofilin-mediated actin filament dynamics in endothelial permeability and the mechanism underlying LcnlO-controlled vascular permeability. The current paradigm states that the increase of paracellular endothelial leakage is driven, on the one hand, by the generation of the centripetal contractile forces and, on the other hand, by the loss of junctional integrity, provided mostly by the tight and adherence junctions. Both endothelial contraction and the maintenance of junctional integrity depend on actin filament reorganization (FIG. 1 A). Notably, there are two forms of actin known to exist in ECs including monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin). Under resting conditions, a subset of G-actin monomers assemble along the cell periphery as cortical actin filaments, which connect to transmembrane junction proteins (e.g., Occludin, VE-cadherin) for maintaining barrier integrity (FIG.1A, left). During sepsis, inflammatory mediators disturb normal actin dynamics, leading to increased actin fiber bundling and stress fiber formation that generate pulling forces and compromise EC contact stability (FIG. 1A, right). Hence, the modification of actin filament dynamics in ECs would be a valuable approach to the development of treatment options for sepsis. Over the past decade, most studies have focused on two major pathways, i.e., those associated with small GTPases and those associated with myosin light chain (MLC) kinases and phosphatases that control actin dynamics and cell contraction. However, another key signaling pathway involving LIM kinases (LIMK) and their downstream target, Cofilin, has been less well studied in sepsis-induced vascular leak.

[0047] At present, there is evidence that Cofilin depolymerizes F-actin to provide new G-actin monomers for polymerization. Cofilin activity is tightly controlled by LIMK1/2, which phosphorylate Cofilin at serine 3, whereby its activity is blocked. In contrast, dephosphorylation by the sling-shot homolog 1 (Sshl) can reactivate Cofilin, which stimulates the severance and depolymerization of F-actin filaments (FIG.1A). Interestingly, recent meta-analysis of the genome-wide mRNA expression profile of whole blood collected from 6 cohorts of 450 individuals (n=323 sepsis; n=127 healthy donors) identified both LIMK2 and Sshl.

[0048] Currently, understanding LcnlO functionality is limited to its potential role as a biomarker for heart failure. However, it has never been investigated whether LcnlO plays any major roles in cardiovascular leakage during sepsis. Our data shows that LPS-caused vascular leak is associated with loss of LcnlO, contributing to myocardial dysfunction and animal mortality. Importantly, we discovered that LPS- and CLP-induced reduction of cardiac LcnlO occurred only in cardiac endothelial cells (ECs) but not in two other cardiac cell types (myocytes and fibroblasts). Accordingly, in vitro overexpression of LcnlO in mouse cardiac ECs greatly decreased monolayer leakage upon LPS challenge.

[0049] Regarding the putative LcnlO-Sshl signaling connection, our RNA sequencing data showed that Sshl was the most significantly upregulated gene in LcnlO-overexpressing ECs. Consistently, treatment of ECs with recombinant LcnlO protein (rLcnlO) also resulted in a significant upregulation of Sshl. Accordingly, the actin-binding protein, Cofilin, was activated in both LcnlO-overexpressing and rLcnlO-treated ECs, as evidenced by a great reduction of its phosphorylation levels, leading to less stress fiber generation and more cortical actin formation, compared to respective controls.

[0050] Further, our data shows that pre-addition of recombinant LcnlO protein (rLcnlO) to cultured cardiac ECs suppresses stress fiber formation and meanwhile, promote cortical actin generation, resulting in decreased monolayer permeability, compared to control cells upon LPS challenge. rLcnlO can be injected into septic mice to reduce vascular permeability, attenuate cardiac edema and dysfunction, and thus lead to enhanced animal survival. Given that increased vascular leakage is a hallmark of many pathological conditions, such as heart failure, atherosclerosis, and a variety of inflammatory diseases including sepsis, the present invention is likely to have a profound impact on improving human health.

LcnlO as a novel Cofilin activator

[0051] Over the past decade, in order to restore vascular barrier integrity in sepsis, tremendous effort has been spent on how to stabilize endothelial junctions and glycocalyx by controlling the activation of small GTPase signaling, metalloproteases (e.g., ADAMs, MMPs) and transmembrane receptors (e.g., Robo4, Tie2). Far fewer studies have focused on exploring the Sshl-Cofilin signaling pathway as a means to enhance actin re-organization against vascular leak. Our data has identified LcnlO as a novel activator of Cofilin in ECs though the upregulation of Sshl expression. [0052] FIG. IB suggests that the net activity of Cofilin might be reduced in blood cells during sepsis. Moreover, Gorovoy et al. recently showed that LIMK deficiency in mice greatly suppressed endotoxin-induced vascular leak and improved animal survival. Nonetheless, whether elevation or activation of Sshl provides protection against sepsis-triggered vascular leak remains unclear. The present invention has found that both addition of recombinant LcnlO protein (rLcnlO) and forced overexpression of LcnlO in ECs significantly upregulated Sshl expression (see data below). By contrast, siRNA-mediated knockdown of Sshl remarkably diminished LcnlO-induced reduction of endothelial permeability upon LPS insult.

[0053] The present invention concerns the role of LcnlO in vascular permeability during polymicrobial sepsis. Based on available data, we know that: 1) increased vascular leakage is responsible for sepsis-caused hypoperfusion, heart failure and mortality; 2) LcnlO expression is significantly down-regulated in the blood of sepsis non-survivals, compared to survival patients; and 3) the expression level of LcnlO is greatly reduced in human failing hearts and septic mouse hearts (data below). Of interest, our data showed that sepsis-induced reduction of cardiac LcnlO occurred only in ECs, but not in either fibroblasts or cardiomyocytes. Taken together, these results indicate that LcnlO plays a critical role in the regulation of vascular permeability during sepsis. Relevant to this indication, our data revealed that LcnlO-KO mice had a higher vascular leakage, a deteriorated cardiac function, and a lower survival rate, compared to WT mice following LPS insult. By contrast, in vitro overexpression of LcnlO in ECs showed a greater resistance to LPS- caused monolayer leak than control ECs.

[0054] The present invention also involves investigating the therapeutic potential of recombinant LcnlO protein in treating sepsis. Pre-clinical studies conducted for the present invention found that addition of recombinant LcnlO protein (rLcnlO, 200ng/ml) to cardiac ECs greatly upregulated Sshl expression (FIG. 7A), resulting in enhanced formation of cortical actin and reduced permeability upon LPS exposure (data shown below). These findings indicate that administration of rLcnlO to septic mice will decrease cardiovascular leakage and cardiac dysfunction, and as a consequence, improve animal survival.

Treatment

[0055] In one embodiment, the present invention is a method of reducing the risk of a sepsis- induced vascular leak, tissue edema or organ dysfunction in a subject. The method involves administering an effective amount of a composition selected from the group consisting of Lipocalin 10 (SEQ ID NO: 1), a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2, LcnlO-expressing vectors for full length/truncated LcnlO, or combinations thereof to the subject. In one embodiment, the subject is administered the composition at a dosage of 50-200 ng/g body weight. In another embodiment, the subject is administered with the composition via vein injection.

[0056] SEQ ID NO : 1

10 20 30 40 50

MRQGLLVLAL VLVLVLVLAA GSQVQEWYPR ESHALNWNKF SGFWYILATA

60 70 80 90 100

TDAQGFLPAR DKRKLGASVV KVNKVGQLRV LLAFRRGQGC GRAQPRHPGT 110 120 130 140 150

SGHLWASLSV KGVKAFHVLS TDYSYGLVYL RLGRATQNYK NLLLFHRQNV 160 170 180

SSFQSLKEFM DACDILGLSK AAVILPKDAS RTHTILP

[0057] SEQ ID NO:2 (Truncated human lipocalin 10 protein (aa 20-140)

20 30 40 50

A GSQVQEWYPR ESHALNWNKF SGFWYILATA

60 70 80 90 100

TDAQGFLPAR DKRKLGAS VV KVNKVGQLRV LLAFRRGQGC GRAQPRHPGT 110 120 130 140

SGHLWASLSV KGVKAFHVLS TDYSYGLVYL RLGRATQNYK

[0058] In another embodiment of the present invention, a method of reducing the risk of a heart attack-induced cardiac dysfunction, atherosclerosis, inflammatory bowel disease or diabetes- induced cardiomyopathy in a subject is provided. The method involves administering an effective amount of a composition selected from the group consisting of Lipocalin 10 (SEQ ID NO: 1), a truncated Lipocalin 10 (LcnlO) protein having the amino acid sequence SEQ ID NO: 2, LcnlO- expressing vectors, modified mRNA of full length/truncated LcnlO, or combinations thereof to the subject. In one embodiment, the subject is administered the composition at a dosage of 50-200 ng/g body weight. In another embodiment, the subject is administered with the composition via vein injection.

[0059] In another embodiment of the present invention, a pharmaceutical composition comprising a truncated Lipocalin 10 protein having the amino acid sequence SEQ ID NO: 2 is provided. In one embodiment, the composition is for use in a method of reducing the risk of a sepsis-induced vascular leak, tissue edema or organ dysfunction in a subject. In another embodiment, the composition is for use in a method of reducing the risk of a heart attack-induced cardiac dysfunction, atherosclerosis, inflammatory bowel disease or diabetes-induced cardiomyopathy in a subject.

LcnlO as novel regulator for correcting the imbalanced macrophage polarization in diabetic hearts [0060] Diabetic cardiomyopathy (DCM) is characterized by ventricular dysfunction that may be ascribed to abnormal macrophage function in the heart. How to modulate macrophage activity in diabetic hearts remains elusive. Recent meta-analysis data reveal that lipocalin 10 (LcnlO) is significantly downregulated in cardiac tissue of patients with heart failure. However, the functional roles of LcnlO in macrophages under diabetic condition has never been explored.

[0061] A study was conducted to examine the role of LcnlO in DCM (see Example 26). The data shows that LcnlO plays an essential role in modulating macrophage phenotypic change under stress conditions. Loss of LcnlO aggravates pro-inflammatory phenotypes in macrophages through damping of Nr4al pathway, leading to impaired cardiac function in diabetic mouse model.

LcnlO as a novel protector against sepsis-induced cardiovascular leakage

[0062] Despite recent advances in antibiotic therapy and supportive critical care, sepsis remains a leading cause of death in intensive care units. The increased vascular leakage seen in sepsis patients is well-recognized to be responsible for sepsis-triggered organ failure and patient mortality. Recently, we and others have shown a positive correlation between cardiovascular permeability, cardiac depression and mortality during sepsis. However, the mechanisms underlying sepsis- induced cardiovascular leakage remain obscure. Prior work suggests that lipocalin 10 (LcnlO), a member of the lipocalin superfamily, is significantly downregulated in the blood of non-survival septic patients when comparing to the survival group. Nonetheless, whether circulating LcnlO affects endothelial barrier integrity in sepsis remains unknown.

[0063] A study was conducted to examine the role of LcnlO in sepsis-induced cardiovascular leakage (Example 27). The results show that endothelial LcnlO is critical for protecting against sepsis-induced cardiovascular leakage via the activation of the Sshl-Cofilin pathway. The study results indicate that LcnlO could be a novel regulator of vascular barrier integrity and as a new protector against sepsis-induced organ failure.

LcnlO as a novel regulator of macrophage efferocytosis in ischemic/reperfused hearts

[0064] Efficient clearance of dead cells by macrophages (termed as efferocytosis) is critical for timely repairing the injured heart after ischemia/reperfusion (I/R), as defective removal of dying cells could cause secondary necrosis and infarct expansion. Recent studies have shown that lipocalin 10 (LcnlO) is significantly downregulated in cardiac tissue from patients with heart failure. However, the role of LcnlO is virtually unknown during cardiac I/R, and its function in macrophage efferocytosis has never been explored. Our initial data showed that LcnlO expression was upregulated in bone marrow-derived macrophages (BMDMs) during efferocytosis. Thus, we hypothesized that knockout (KO) of LcnlO would diminish macrophage efferocytosis, leading to exacerbated cardiac I/R injury.

[0065] A study was conducted to examine the role of LcnlO in macrophage efferocytosis (Example 28). Our data indicate that LcnlO is pivotal for macrophage efferocytosis to remove cardiac dead cells during I/R. Thus, LcnlO could be used as a new mediator for the treatment of cardiac I/R injury.

EXAMPLES

Genetic mouse models

[0066] A global LcnlO-knockout (KO) mouse model was purchased from UC-Davis KOMP Repository (LcnlOtml. l) (FIG. 2). Two novel transgenic mouse lines (EC-LcnlO-Tg, #A and #B) were also generated in which LcnlO is overexpressed specifically in endothelial cells (ECs), using an EC-specific Tie2 promoter (FIG. 3 A and 3B). Sshl-KO mouse model was acquired and was bred with EC-LcnlO-Tg mice to generate a new mouse model (EC-LcnlOTg/Sshl-/-) in which Sshl is ablated and LcnlO is overexpressed in ECs. The three genetic mouse models (LcnlO-KO, EC-LcnlO-Tg, and EC-LcnlOTg/Sshl-/-) are on the C57BL/6 background. All animal procedures have been approved by our Institutional Animal Care and Use Committee.

Example 1 - Expression levels of LcnlO are altered upon LPS treatment and CLP surgery

[0067] LcnlO was initially identified as an epididymis-specific gene. Later RNA-seq. analysis showed that LcnlO is ubiquitously expressed and enriched in the blood, spleen and heart. While prior studies reported that the LcnlO expression in blood cells was significantly lower in nonsurvival sepsis patients than survivals, it remains unclear whether cardiac LcnlO expression is altered under sepsis conditions. To test this, we first measured expression levels of cardiac LcnlO in mice (2-month old) after LPS injection (lOpg/g). RT-qPCR results showed that LcnlO expression was greatly increased in mouse hearts at 3 and 6 h after LPS treatment, but dramatically reduced at 24 h post-LPS injection (FIG. 4A). Next, endothelial cells (ECs), fibroblasts, and myocytes were isolated from these LPS-treated hearts to further assess LcnlO-mRNA levels. Interestingly, we observed that LPS-caused alterations of cardiac LcnlO occurred only in ECs but not in fibroblasts or myocytes (FIG. 4B). Similarly, cardiac LcnlO expression was remarkably upregulated in mice (2-month old) at 12 h post-CLP surgery, whereas it was significantly downregulated at 48 h post-CLP (FIG. 4C). Consistently, such CLP -induced up-/down-regulations of cardiac LcnlO expression occurred only in ECs but not in either fibroblasts or cardiomyocytes (FIG. 4D). Taken together, these data indicate that LcnlO is involved in the regulation of vascular permeability during sepsis. Its reduction at the late phase of sepsis may directly contribute to sepsis-induced cardiovascular leak, whereas its elevation at the early phase of sepsis could act as a compensatory mechanism.

Example 2 - Knockdown of LcnlO in cardiac ECs increases LPS-triggered permeability

[0068] To test whether reduced LcnlO expression in ECs contributes to sepsis-induced vascular leak, we next transfected mouse cardiac ECs (MCECs, purchased from CELLutions Biosystems Inc) with LcnlO-siRNA (siLcnlO) or control siRNA (SiCon) for 48 h. Then, these cells were harvested and seeded onto the upper chamber of a transwell system until a monolayer was formed, followed by addition of LPS (Ipg/ml) to induce EC leak. Using three different methods [trans- endothelial electrical resistance (TEER), FITC-labeled dextran and Evans blue dye (EB)-albumin flux assays], we observed that addition of LPS to siRNA-control cells caused remarkable drops in TEER values, which were further exaggerated in siLcnlO-transfected cells. Consistently, LPS- induced flux of FITC-dextran and EB-bound albumin was significantly increased in siLcnlO-cells, compared to siRNA-control cells. These results indicate that down-regulation of LcnlO can promote endothelial permeability during sepsis.

Example 3 - LcnlO-null mice exhibit the increased cardiovascular leak upon LPS exposure [0069] To evaluate whether LcnlO-deficiency affects vascular permeability in vivo during sepsis, an endotoxemia model was utilized by intraperitoneal (i.p.) injection of LPS (lOpg/g body weight) for 24 h, followed by the tail vein injection of 1% Evans blue dye (200 pl). The LcnlO-KO mice used for the study were healthy and did not show any obvious pathological abnormalities. Upon LPS challenge, however, these LcnlO-KO mice showed severe vascular leak, as evidenced by: 1) increased extravasation of EB dye in the aorta and the heart, edema was further confirmed by the ratio of wet/dry weight), and 2) increased release of EB dye to the extravascular compartment of frozen aorta sections and heart sections (note: EB is red under a fluorescence microscope, compared to LPS-treated WT controls. Therefore, these results show that knockout of LcnlO promotes sepsis-induced cardiovascular leak and tissue edema.

Example 4 - LcnlO-KO exaggerates cardiac depression and mortality in LPS-treated mice [0070] A test was conducted to determine whether increased vascular permeability in LPS-treated LcnlO mice could affect cardiac function and animal survival. Echocardiography analysis (FIGs 5 A, 5B and 5C) showed that absence of LcnlO did not affect cardiac function under basal PBS conditions, but it did exaggerate LPS-induced myocardial depression, as evidenced by significant decreases in the left ventricular ej ection fraction (EF%) and fractional shortening (FS%), compared to LPS-treated WTs (p < 0.05). In addition, loss of LcnlO had major impact on mortality during endotoxemia. Nine out of 10 KO mice (90%) died at 4 days post-LPS injection, whereas 40% of WT mice (n=10) remained alive (FIG. 5D, p < 0.05). Collectively, these data indicate that LcnlO- deficiency contributes significantly to LPS-triggered myocardial dysfunction and lethality.

Example 5 - Overexpression of LcnlO in cardiac ECs decreases permeability upon LPS treatment [0071] The data above strongly indicate that reduction of LcnlO aggravates LPS-induced vascular leakage, cardiac depression and mortality. However, it remains unclear whether forced overexpression of LcnlO in ECs attenuates vascular leakage during sepsis. To this end, we performed studies using cultured mouse cardiac ECs (MCECs), which were infected with recombinant adenovirus vector Ad.LcnlO expressing both LcnlO and GFP for 48 h. MCECs infected with Ad. GFP encoding GFP alone were used as controls. Overexpression of LcnlO in Ad.LcnlO-ECs was validated by RT-qPCR. In parallel, these Ad-infected cells were harvested and seeded onto the upper chamber of a transwell system until an EC monolayer was formed. Then, LPS (Ipg/ml) was added, followed by measurement of EC leakage as described above. We observed that LPS-caused a drop of TEER in Ad.GFP-cells, which was greatly suppressed in Ad.LcnlO-cells. Similarly, the flux analysis of FITC-dextran and EB-albumin both showed that Ad.LcnlO-cells had a lower permeability than Ad.GFP-cells upon LPS challenge. Collectively, these findings indicate that the elevation of LcnlO in ECs could decrease endotoxin-triggered vascular leakage.

Example 6 - RNA-seq analysis

[0072] Our RNA-seq analysis revealed that a total of 147 genes were differentially expressed including 102 up-regulated and 45 down-regulated in Ad.LcnlO-ECs, compared to Ad.GFP-cells (FIG. 6). Furthermore, gene ontology enrichment analysis results showed that elevation of LcnlO in ECs greatly activated two major signaling pathways involved in the regulation of actin cytoskeleton and cell junction, which were confirmed with RT-qPCR (data not shown). Of interest, Sshl is the most significantly upregulated gene in LcnlO-cells (FIG. 6). Prior work has indicated that Sshl is a phosphatase which activates Cofilin, a key actin-binding protein that plays an essential role in modulating actin filament dynamics. Indeed, our latest data (below) revealed that overexpression of LcnlO in ECs yielded less stress fibers and more cortical actin filaments than control cells upon LPS insult. Accordingly, knockdown of Sshl in LcnlO-ECs by siRNAs was found to greatly suppress LcnlO-elicted effects on actin filament turnover, leading to increased permeability, compared to control cells following LPS insult. Hence, these data indicate that LcnlO-elicited inhibition of vascular leakage during sepsis acts through Sshl-mediated actin filament re-organization. Example 7 - LcnlO regulates Sshl expression in ECs via LRP2

[0073] To identify possible mediators for the LcnlO-induced upregulation of Sshl in ECs, we first analyzed the expression data for membrane receptors since LcnlO is a secreted protein. Available data shows that lipocalin family members interact with LDL receptor-related proteins (LRPs). As implicated here, our RNA-seq data revealed that the expression of LRP2 was significantly elevated in LcnlO-cells compared to control GFP-cells (FIG. 6). This indicates a positive feedback loop involving LcnlO. Consistently, addition of recombinant LcnlO protein (rLcnlO, 200ng/ml) to cultured ECs promoted expression of both LRP2 and Sshl, compared to BSA-controls (FIG. 7A). Furthermore, when LRP2 was knocked down by siRNA (si-LRP2), the Sshl expression was not up-regulated in either Ad. LcnlO- infected (FIG. 7B) or rLcnlO-treated ECs (FIG. 7C). These data indicate that LcnlO upregulates Sshl expression in ECs through its interaction with LRP2.

Example 8 - LPS injection and CLP surgery both result in downregulation of Sshl in cardiac ECs [0074] Next, to determine whether sepsis conditions affect Sshl expression in ECs, we performed RT-qPCR, and observed that expression levels of Sshl were greatly reduced in cardiac ECs of mice at 24h post-LPS injection, compared to PBS-controls. Likewise, expression levels of LRP2, as an upstream regulator of Sshl, were remarkably lower in LPS-treated cardiac ECs than in control cells. Consequently, VE-cadherin levels on the surface of LPS-treated cardiac ECs were significantly decreased, compared to PBS-controls, as measured by flow cytometry analysis. Similar findings were observed in cardiac ECs isolated from mice at 48h post-CLP. These data correlate well with the reduced expression of LcnlO in cardiac ECs of mice upon LPS-injection and CLP surgery (FIG. 4), indicating that sepsis-induced vascular leakage is associated with a reduction in LcnlO-LRP2-Sshl signaling.

Example 9 - Overexpression of LcnlO in ECs

[0075] Available data has shown that Sshl activates Cofilin by dephosphorylation at Ser-3. Accordingly, we measured Cofilin and its Ser-3 -phos-phorylated levels in Ad.LcnlO-infected ECs and control cells by Western-blotting. We observed that Ad.LcnlO-ECs displayed higher levels of Sshl than Ad. GFP-cells, consistent with our RNA-seq data above. The phosphorylated levels of Cofilin were significantly lower in Ad. LcnlO-cells than in Ad. GFP-cells, suggesting its dephosphorylation and activation by Sshl. Given that Cofilin is well known to control actin filament reorganization by stimulating the severance and depolymerization of actin filaments, we next examined cytoskeletal structures (i.e., stress fibers and cortical actin networks), using Alexa- Fluor 594 phalloidin (F -actin probe) staining. Under basal conditions, GFP-ECs showed a cortical organization of cytoskeletal F-actin along the cell periphery. After LPS stimulation, peripherally located F-actin is substituted by centralized and irregular stress fibers running across the cytoplasm. However, overexpression of LcnlO strongly reinforced peripheral actin bands, both in unstimulated and LPS-treated cells. Importantly, LPS-triggered formation of stress fibers was less pronounced in LcnlO-cells than in control GFP-cells. Given that reorganization of the actin cytoskeleton affects the stability of EC junctions and paracellular gap formation, we lastly evaluated the integrity of cell peripheral membrane by staining these ECs with antibody to ZO-1, a tight junction protein. The linear shape of cell-cell junctions was displayed in both GFP- and LcnlO-cells under basal conditions. However, LPS treatment greatly impaired the integrity of cell membranes, as evidenced by jagged and disconnected ZO-1 staining in GFP-cells, but not in LcnlO-cells. Together, these data indicate that forced elevation ofLcnlO in ECs activates the Sshl- Cofilin pathway and reinforces the cortical actin network upon LPS insult, leading to reduced stress fiber formation and opening of cell junctions.

Example 10 - Knockdown of Sshl in ECs

[0076] To test whether LcnlO-elicited action is dependent on Sshl signaling, we first utilized sequence-specific siRNAs to knockdown Sshl expression in Ad.LcnlO-infected ECs, followed by LPS challenge for EC permeability analysis. Western-blotting data revealed that elevation of Sshl in Ad.LcnlO-ECs was greatly reduced by siRNA-Sshl to a similar level as in GFP-cells. Consequently, LcnlO- induced decrease in the levels of phosphorylated Cofilin was greatly blocked by siRNA-Sshl transfection, as revealed by similar degree of Cofilin phosphorylation between siRNA-Sshl/Ad.LcnlO-ECs and control siRNA-Sshl/Ad. GFP-cells. EC permeability analysis further indicated that FITC-dextran flux was greatly reduced in LcnlO-cells, compared to GFP-cells following LPS challenge (FIG. 8A). However, such LPS-caused leakage was significantly increased in LcnlO-cells when Sshl was knocked down by siRNAs (FIG.8A). Similar findings were observed using the EB-albumin flux assay (FIG. 8B). Taken together, these initial mechanistic data indicate that LcnlO-mediated effects in LPS-triggered EC leakage may be largely dependent on the Sshl signaling. Therefore, these data show that elevation of LcnlO in ECs increases Sshl expression through autocrine mechanisms via the LRP2 receptor, leading to dephosphorylation of Cofilin (activation, FIG. 9). Consequently, the formation of cortical actin filaments is enhanced, and stress fiber formation is suppressed upon LPS insult (FIG. 9). Importantly, knockdown of Sshl in ECs by siRNAs significantly limits the LcnlO-elicited reduction of monolayer leakage. Without being bound by theory, we hypothesize that the mechanism underlying LcnlO-induced protection against cardiovascular leak during sepsis is dependent on Sshl -mediated actin filament re-arrangement.

Example 11 - rLcnlO inhibits stress fiber formation and decreases leakage in LPS-treated ECs [0077] To address whether exogenous LcnlO protein has therapeutic effects against sepsis- triggered vascular leak, we conducted studies using cardiac ECs, and treated these cells with rLcnlO protein (purchased from MyBioSource Inc.) or control BSA for 12h, followed by the addition of LPS (Ipg/ml) for barrier integrity and permeability assays. (Note: endotoxin levels in rLcnlO and BSA proteins are < O.OlEU/pg, low enough to exclude its effects). We observed that treatment with rLcnlOa significantly increased trans-endothelial electrical resistance (TEER) in a time-dependent manner, compared to control BSA-treated cells after LPS insult (FIG. 10A). Accordingly, the leakage of FITC-dextran (FIG. 10B) and EB-albumin (FIG.10C) was greatly inhibited in rLcnlO-ECs, compared to control cells following LPS challenge. In follow-up studies, we selected the time point of 2 h post-LPS treatment for actin filament analysis and stained these ECs with Alexa Fluor 488 phalloidin. In control BSA-cells after LPS stimulation, peripherally located F-actin filament was re-organized into stress fiber bundles, spreading across the cytoplasm. However, treatment with rLcnlO strongly reinforced peripheral actin (cortical actin) bands and inhibited stress-fiber formation upon LPS insult. Together, these data indicate that exogenous addition of rLcnlO promotes the assembly of cortical actin filaments and counters stress fiber formation, thereby inhibiting LPS-triggered vascular leak.

Example 12 - rLcnlO-mediated protective effects against LPS-caused EC leakage

[0078] Given that rLcnlO upregulates endothelial Sshl expression, we next tested whether rLcnlO-elicited decrease of EC monolayer leakage is dependent on Sshl. ECs were transfected with siRNA-Sshl (Si-Sshl) or siRNA-control (Si-Con) for 48 h, followed by addition of rLcnlO (200ng/ml) or BSA control. Then, EC leakage was measured at 2 h post-LPS challenge. We observed that rLcnlO-induced reduction of EC permeability was significantly blocked by siRNA- Sshl transfection, as evidenced by similar degrees of TEER and leakage of FITC-dextran and EB dye between BSA- and rLcnlO-treated cells (FIGs 11 A, 11B and 11C). These data indicate that Sshl plays an essential role in LcnlO-induced benefits in reducing vascular leakage during sepsis.

Example 13 - rLcnlO decreases LPS-induced cardiovascular leak in vivo

[0079] To test whether rLcnlO has therapeutic effect in reducing vascular leakage during sepsis, the following studies were conducted: 8-week old mice were received rLcnlO (200ng/g) or BSA control via the tail veil injection at Ih post-LPS treatment. 24h later, EB dye was injected intravenously for the analysis of cardiovascular permeability, as described previously. We observed that treatment of LPS-mice with rLcnlO reduced EB extravasation to a greater degree in the heart and aorta, compared to BSA-treated controls. These data indicate that rLcnlO can decrease cardiovascular leak during sepsis.

Example 14 - The expression levels of LcnlO in tissues of septic mice

[0080] mRNA levels of LcnlO in different tissues of C57/6J mice were analyzed by RT-qPCR (FIG. 12A). After intraperitoneal injection of LPS (10 mg/kg), mice were sacrificed at 0, 3, 6 and 24 h, and the mRNA levels of LcnlO in the blood (FIG. 12B) and the spleen (FIG. 12C) were assessed. At the time points of 0, 6 and 24 h after CLP-operation in mice, mRNA levels of LcnlO in the blood (FIG 12D) and the spleen (FIG 12E) were determined by RT-qPCR. n=4-6 for each group at the indicated time. #, P<0.05.

Example 15 - LcnlO deficiency aggravates CLP-induced multiple organ injury

[0081] Western-blot was used to detect LcnlO expression in the spleens of WT and LcnlO KO mice. GAPDH was used as a loading control. Kaplan-Meier survival curves and the log-rank (Mantel-Cox) test were used to detect survival rate. LcnlO-KO mice showed a lower survival rate than WT mice within 96 hours after CLP operation. At 24 h post-CLP surgery, WT and LcnlO- KO mice were sacrificed for collecting the livers and kidneys, and then stained with hematoxylin and eosin. Representative images of the liver and kidney were shown. Scale bars, 20 pm. At 16 h after CLP operation, levels of ALT and Cr in the serum in WT and LcnlO-KO mice were analyzed by ELISA. #, P<0.05; n=4-6 mice per group. Example 16 - LcnlO deficiency increases inflammatory response

[0082] Serum and peritoneal lavage fluid were harvested at 16 h after CLP-operation (FIG. 13). The pro-inflammatory cytokines TNF-a (FIG. 13A, FIG. 13E), IL-6 (FIG. 13B, FIG. 13F) and MCP-1 (FIG. 13C, FIG. 13G) and anti-inflammatory cytokine IL- 10 (FIG. 13D, FIG. 13H) were measured using ELISA kits. #, P<0.05; n=6-8 mice per group. The data shows that LcnlO deficiency increases inflammatory response locally and systemically in sepsis.

Example 17 - LcnlO deficiency attenuates bacterial clearance and suppressed phagocytic capability of peritoneal macrophages (PMs) from septic mice

[0083] The bacterial burden in both blood (FIG. 14 A) and peritoneal lavage fluid (PLF, FIG. 14B) in LcnlO-KO mice were compared with WT mice after CLP operation. (FIG. 14 C) The PMs obtained from WT and LcnlO-KO CLP mice were incubated with the fluorescent labeled E. coli particles for 1.5 h and then examined under confocal microscopy for phagocytosis analysis by measuring the mean fluorescence intensity (FIG. 14C). In addition, the entry of living E. coli by PMs was determined by bacterial CFU counts after incubating with living E. coli for 1.5 h (FIG. 14D). The ratio of CFU at 4 h to CFU at 1.5 h after incubating with living E. coli was assessed, and the percentage of bacterial killing was calculated (FIG. 14F). #, P<0.05, n=6-8 per group.

Example 18 - Dynamic expression of LcnlO in septic models

[0084] At Oh, 3h, 6h and 24 h after intraperitoneal injection of LPS (10 mg/kg BW), mouse hearts were harvested for mRNA measurement by RT-qPCR (n=6-8). Hearts were harvested at Oh, 12h, and 48h after CLP and LcnlO mRNA levels were determined by RT-qPCR (n=5-9). (FIG. 15C, FIG. 15D) Endothelial cells, cardiomyocytes, and fibroblasts were respectively isolated from hearts harvested at appropriate time points both in LPS (Oh, 6h and 24h) (FIG. 15 A) and CLP (Oh, 12h and 48h) (FIG. 15B) models, then total RNAs were collected and assessed by RT-qPCR (n-5- 6). (FIG. 15E, FIG. 15F) mRNA expression levels of LCN10 in MCECs (mouse cardiac endothelial cells) at Ih, 3h, 6h and 24h after LPS (lug/ml) (FIG. 15E) and TNF- a (lOng/ml) (FIG. 15F) stimulation, 18s gene expression was used as the internal control (n-6-7). All results are presented as mean ± SEM and analyzed by student’s t test. (*, p < 0.05). Example 19 - Knockdown of LcnlO augments the permeability in mouse cardiac endothelial cells (MCECs)

[0085] MCECs were transfected with siRNA-LcnlO (siLcnlO) and negative control siRNA (siCon) was used as control. 60h later, cultured cells were treated with PBS, LPS (1 pg/ml) or TNF- a (lOng/ml) for 3h. Then the resulting MCECs were harvested to isolate total RNA. (FIG. 16A, FIG. 16B) mRNA levels of LcnlO were measured by RT-qPCR (n=5). Results are presented as mean ± SEM and analyzed by student’s t test. (*, p < 0.05). (FIG. 16C- FIG. 16E) Compared to negative control group, siLcnlO-transfected MCECs displayed significantly increased leakage after LPS treatment, as assessed by three measures: trans-endothelial electrical resistance (TEER) (FIG. 16C) (n=3), FITC-dextran (FIG. 16D) (n=6), and EB-BSA flux (FIG. 16E) (n=6). (FIG. 16F, FIG. 16H) Similar results were observed after treatment with TNF- a including decreased TEER (FIG. 16F) (n=3), increased FITC-dextran (FIG. 16G) (n=6) and EB-BSA flux (H) (n=6). Similar results were obtained in other two independent experiments. Results are presented as mean ± SD and analyzed by two-way ANOVA (*, p < 0.05).

Example 20 - LcnlO deficiency aggravates vascular permeability, organ injury and animal mortality in septic mice

[0086] Genotyping of Lcnl0+/+ (+/+), Lcnl0+/- (+/—), and LcnlO-/- (-/-) mice. LcnlO-KO mice were confirmed by Western blot using heart tissue. LcnlO-KO and WT mice were received LPS injection (lOmg/kg BW, i.p.). Twenty hours later, Evans blue dye (4mg/kg BW) was intravenously injected into these mice. KO mice displayed a worse peripheral circulation and increased vascular leakage in the heart and aorta. Vascular leakage within the heart and aorta was further quantitative analyzed by incubating these organs with formamide at 55 °C for 48h, then, the elution was measured the optical density at 620nm on a spectrophotometer (n=4). EB dye in the extravascular compartment of frozen sections of heart and aorta was examined under a confocal LSM 710 microscope. The intensity of red fluorescence within the heart and aorta was quantified with Image J software (n=5). 20h after LPS or PBS injection, the cardiac function of LCN10-KO and WT mice was determined by echocardiography. Representative M-mode echocardiography recordings for cardiac function measurement in WT and KO mice after LPS treatment. Left ventricular ejection fraction (EF %) (P) and fractional shortening (FS %) were calculated (n = 5- 7). (*, p < 0.05). Kaplan-Meier survival curves were generated to compare mortality between 2 groups, significance was determined by log-rank (Mantel-Cox) test. (*, p < 0.05; n = 20 per group).

Example 21 - Overexpression of full-length LcnlO reduces LPS-caused endothelial cell leakage [0087] Mouse cardiac ECs (MCECs) were infected with recombinant adenovirus vector Ad.LcnlO expressing both full-length LcnlO and GFP for 48 h. MCECs infected with Ad.GFP encoding GFP alone were used as controls. (FIG. 17A) Overexpression of full-length LcnlO in Ad.LcnlO-ECs was validated by RT-qPCR. (*, p < 0.05, n=6). (FIG. 17B-FIG. 17D) At 48h post-transfection, LPS (1 pg/ml) was added and the permeability of MCECs was measured by three methods: TEER (FIG. 17B), FITC-dextran leakage (FIG. 17C) and EB-BSA flux (FIG. 17D). LPS-caused a drop of TEER in Ad.GFP-cells, which was greatly suppressed in Ad.LcnlO-cells (FIG. 17B). Similarly, the flux analysis of FITC-dextran (FIG. 17C) and EB-albumin (FIG. 17D) both showed that Ad.LcnlO-cells had a lower permeability than Ad.GFP-cells upon LPS challenge. Likewise, higher TEER (FIG. 17E), lower FITC-dextran (FIG. 17F) and EB-BSA flux (FIG. 17G) were observed in Ad. LCNIO-transfected MCECs upon TNF-a challenge, compared to controls (*, p < 0.05). Collectively, these findings indicate that elevation of LcnlO in ECs could decrease endotoxin-triggered vascular leakage.

Example 22

[0088] Cofilin and its Ser-3 -phos-phorylated levels in Ad.LcnlO-infected ECs and control cells were measured by Western-blotting. We observed that Ad.LcnlO-ECs displayed higher levels of Sshl than Ad.GFP-cells. The phosphorylated levels of Cofilin were significantly lower in Ad.LcnlO-cells than in Ad.GFP-cells, suggesting its dephosphorylation and activation by Sshl. Given that Cofilin is well known to control actin filament reorganization by stimulating the severance and depolymerization of actin filaments, we next examined cytoskeletal structures (i.e., stress fibers and cortical actin networks), using Alexa-Fluor 594 phalloidin (F-actin probe) staining. Under basal conditions, GFP-ECs showed a cortical organization of cytoskeletal F-actin along the cell periphery. After LPS stimulation, peripherally located F-actin is substituted by centralized and irregular stress fibers running across the cytoplasm. However, overexpression of LcnlO strongly reinforced peripheral actin bands, both in unstimulated and LPS-treated cells. Importantly, LPS-triggered formation of stress fibers was less pronounced in LcnlO-cells than in control GFP-cells. Given that reorganization of the actin cytoskeleton affects the stability of EC junctions and paracellular gap formation, we lastly evaluated the integrity of cell peripheral membrane by staining these ECs with antibody to ZO-1, a tight junction protein. The linear shape of cell-cell junctions was displayed in both GFP- and LcnlO-cells under basal conditions. However, LPS treatment greatly impaired the integrity of cell membranes, as evidenced by jagged and disconnected ZO-1 staining in GFP-cells, but not in LcnlO-cells. Together, these data indicate that forced elevation of LcnlO in ECs activates the Sshl -Cofilin pathway and reinforces the cortical actin network upon LPS insult, leading to reduced stress fiber formation and opening of cell junctions.

Example 23

[0089] To test whether LcnlO-eli cited action is dependent on Sshl signaling, sequence-specific siRNAs were first utilized to knockdown Sshl expression in Ad.LcnlO-infected ECs, followed by LPS challenge for EC permeability analysis. Western-blotting data revealed that elevation of Sshl in Ad.LcnlO-ECs was greatly reduced by siRNA-Sshl to a similar level as in GFP-cells. Consequently, LcnlO-induced decrease in the levels of phosphorylated Cofilin was greatly blocked by siRNA-Sshl transfection, as revealed by a similar degree of Cofilin phosphorylation between siRNA-Sshl/Ad.LcnlO-ECs and control siRNA-Sshl/Ad. GFP-cells.

Example 24

[0090] Given that rLcnlO upregulates endothelial Sshl expression, we next tested whether rLcnlO-elicited decrease of EC monolayer leakage is dependent on Sshl. ECs were transfected with siRNA-Sshl (Si-Sshl) or siRNA-control (Si-Con) for 48 h, followed by addition of rLcnlO (200ng/ml) or BSA control. Then, EC leakage was measured at 2 h post-LPS challenge. We observed that rLcnlO-induced reduction of EC permeability was significantly blocked by siRNA- Sshl transfection, as evidenced by similar degrees of TEER and FITCZEB dye flux between BSA- and rLcnlO-treated groups. Consistently, rLcnlO-induced inhibition of stress fiber formation was greatly suppressed by Sshl knockdown. These data indicate that Sshl plays an essential role in LcnlO-induced benefits in reducing vascular leakage during sepsis. Example 25

[0091] To test if exogenous rLcnlO protein can rescue such a leakage in LcnlO-KO cells, we added rLcnlO (200ng/ml) or control BSA to cultured cardiac ECs that were isolated from WT and LcnlO- KO mice, followed by permeability analysis at 2 h post-LPS insult. Our results showed that the leakage of FITC-dextran and EB-dye in KO-ECs was greatly inhibited by addition of rLcnlO, compared to BSA-cells. These data indicate that rLcnlO can be used as an effective agent to block vascular leak during sepsis. In conclusion, loss of LcnlO exacerbated sepsis-induced systemic and local inflammation, aggravated multiple organ injury and reduced the survival rate of polymicrobial septic mice. One crucial factor seemed to be the impaired phagocytic function of LcnlO KO macrophages. The present invention presents the first evidence on the correlation between LcnlO and phagocytosis/autophagy pathway in macrophages.

Example 26

[0092] Type 2 diabetes (T2D) was induced in wild-type (WT) and LcnlO knockout (KO, C57BL6 background) mice by combination of high-fat diet (HFD, 60%) feeding and streptozotocin (STZ, lOOmg/kg body weight) injection. Flow cytometry was performed to characterize cardiac immune cell composition and phenotype. The expression levels of various macrophage marker genes were measured by qPCR. Bone marrow-derived macrophages (BMDMs) were isolated from WT and LcnlO-KO mice to test macrophage function under stress conditions.

[0093] When BMDMs were treated with palmitate, it was observed that LcnlO gene expression was significantly downregulated. Similar result was noted in BMDMs treated with high glucose (25mM), compared to low glucose (5mM) group. Consistently, LcnlO gene expression was 40% lower in cardiac macrophages from diabetic mice, comparing to those from mice fed with chow diet (CD). These results indicate that LcnlO may play a role in regulating macrophage function. To test this hypothesis, we treated WT and LcnlO-KO BMDMs with palmitate and assessed phenotypic changes using qPCR. The results showed that LcnlO-KO BMDMs had significantly higher expression of pro-inflammatory marker genes (i.e. iNOS, IL-6 and CCL2) in response to palmitate treatment, compared to WT group. Importantly, the macrophages in the hearts of LcnlO- KO diabetic (KO-T2D) mice exhibited stronger pro-inflammatory phenotype, as evidenced by higher ratio of Ly6C+ population to CD206+ population, compared to WT-T2D mice. Consequently, this led to reduced cardiac contractile function (10% decrease in ejection fraction, n=6-8, p<0.05). Mechanistically, RNA sequencing analysis using WT and LcnlO-KO BMDMs suggested that loss of LcnlO disrupted Nr4al signaling pathway, resulting in downregulation of Nr4al -targeted genes (i.e. CX3CR1, GDF3, MIDI). Accordingly, LcnlO-KO BMDMs failed to respond to Nr4al agonist, which showed strong anti-inflammatory effects in WT BMDMs.

Example 27

[0094] Sepsis models were induced in mice by both intraperitoneal (IP) injection of endotoxin LPS (lOmg/kg body weight) and cecal ligation and puncture (CLP). Using RT-qPCR analysis, we observed that LcnlO was significantly down-regulated in the hearts of both LPS- and CLP-treated mice, compared to their controls. Interestingly, further analysis of LcnlO expression in different cell types isolated from LPS- and CLP-treated hearts showed that reduction of LcnlO occurred only in cardiac endothelial cells (ECs) but not in cardiomyocytes or fibroblasts. These data suggest that LcnlO may be involved in sepsis-induced cardiovascular leakage. Using a global LcnlO- knockout (KO) mouse model, we found that loss of LcnlO greatly increased vascular permeability, which correlated with more severe cardiac depression and higher mortality following LPS challenge or CLP surgery, compared to LPS- or CLP-treated wild-type (WT) mice. By contrast, in vitro overexpression of LcnlO in ECs provided greater resistance to LPS-caused monolayer leak, compared to control cells. A mechanistic analysis by RNA-sequencing and RT-qPCR revealed that both endogenous and exogenous elevation of LcnlO in ECs could significantly upregulate slingshot homolog 1 (Sshl) expression. Sshl is a phosphatase known to activate Cofilin, a key actin-binding protein that plays an essential role in controlling actin filament dynamics. Accordingly, phosphorylated Cofilin levels were significantly reduced and thereby, reorganized F-actin to cortical actin for stabilizing tight junction molecules in LcnlO-treated ECs, compared to control cells. Finally, knockdown of Sshl in ECs by siRNA greatly offsets LcnlO-induced reduction of monolayer leakage upon LPS insult.

Example 28

[0095] Since LcnlO is a secreted protein, a global LcnlO-KO mouse model was generated. After co-culturing BMDMs with GFP-labeled dead myocytes for 2h, a 25% reduction on efferocytosis by KO-BMDMs, compared to wild-type (WT) controls was observed. Using LcnlO-KOmCherry mouse model (cardiac-specific overexpression of mCherry in LcnlO-KO mice), it was detected 1 that KO cardiac macrophages ingested fewer dead myocytes at 3 days post-I/R, compared to WT- mCherry controls. Accordingly, cardiac contractile function, measured by echocardiography at the same time, was significantly declined in KO-mice, together with increased accumulation of apoptotic cells and higher serum levels of troponin I, a marker of cardiac damage, compared with WTs (n=7-10, p<0.05). In addition, pre-treatment of KO-BMDMs with recombinant Lcn 10 protein could rescue the impaired efferocytosis. Mechanistically, RNA-seq analysis showed that many downregulated genes (i.e., ItgaV, Itga6, Cx3crl, and Msr) in KO-macrophages are associated with efferocytosis. Interestingly, it was identified that LcnlO contains a potential phosphatidylserine (PS)-binding motif (RxKRK), located at a helix-turn-helix structure, which is the primary “find- me” signal for efferocytosis.

[0096] All documents cited are incorporated herein by reference; the citation of any document is not to be construed as an admission that it is prior art with respect to the present invention.

[0097] It is to be further understood that where descriptions of various embodiments use the term “comprising,” and / or “including” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of’ or "consisting of.”

[0098] While particular embodiments of the present invention have been illustrated and described, it would be obvious to one skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.