IL-10 KO Mice Research Articles

MicroRNA-21 Knockout Improve the Survival Rate in DSS Induced Fatal Colitis through Protecting against Inflammation and Tissue Injury

BackgroundMicroRNA-21 (miR-21) is overexpressed in most inflammatory diseases, but its physiological role in gut inflammation and tissue injury is poorly understood. The goal of this work is to understand the role of miR-21 in colitis and damage progression of intestine in a genetically modified murine model.MethodsExperimental colitis was induced in miR-21 KO and wild-type (WT) mice by 3.5% dextran sulphate sodium (DSS) administration for 7 days. Disease activity index(DAI), blood parameters, intestinal permeability, histopathologic injury, cytokine and chemokine production, and epithelial cells apoptosis were examined in colons of miR-21 KO and WT mice.ResultsmiR-21 was overexpressed in intestine of inflammatory bowel diseases (IBD) and acute intestinal obstruction (AIO) patients when compared with normal intestinal tissues. Likewise, miR-21 was up-regulated in colon of IL-10 KO mice when compared with control mice. WT mice rapidly lost weight and were moribund 5 days after treatment with 3.5% DSS, while miR-21 KO mice survived for at least 6 days. Elevated leukocytes and more severe histopathology were observed in WT mice when compared with miR-21 KO mice. Elevated levels of TNF-α and macrophage inflammatory protein-2(MIP-2) in colon culture supernatants from WT mice exhibited significant higher than miR-21 KO mice. Furthermore, CD3 and CD68 positive cells, intestinal permeability and apoptosis of epithelial cells were significantly increased in WT mice when compared with miR-21 KO mice. Finally, we found that miR-21 regulated the intestinal barrier function through modulating the expression of RhoB and CDC42.ConclusionOur results suggest that miR-21 is overexpressed in intestinal inflammation and tissue injury, while knockout of miR-21 in mice improve the survival rate in DSS-induced fatal colitis through protecting against inflammation and tissue injury. Therefore, attenuated expression of miR-21 in gut may prevent the onset or progression of inflammatory bowel disease in patients.

Ferritin L is the Sole Serum Ferritin Constituent and a Positive Hepatic Acute-Phase Protein

Ferritin L (FTL) and ferritin H (FTH) subunits are responsible for intracellular iron storage. Serum ferritin levels are not only dependant on body iron stores. Aims of the present study are to demonstrate nature, source, and major regulatory mediators of serum ferritin in an animal model of acute-phase (AP) response. Animals (rats, wild-type [WT] mice, and interleukin [IL]-6ko mice) were injected with turpentine oil (TO) intra-muscularity to induce a sterile abscess and sacrificed at different time points afterward. Rat hepatocytes were isolated for cell culture and, after reaching confluence, stimulated with major AP cytokines to induce AP conditions. We found a significantly increased expression of both ferritin subunits in liver at mRNA and protein levels during AP response. In the serum of both control and TO-injected rats, only FTL was detectable by Western blotting, whereas no increase in serum FTL was measured by Western blot or enzyme-linked immunosorbent assay. An increase in protein expression of FTL and FTH was observed in lysates of rat hepatocytes after treatment with IL-6, IL-1β, and tumor necrosis factor-α; however, only FTL was increasingly released into supernatant. In both TO-injected rats and WT mice, a dramatic increase in serum IL-6 levels was observed, along with an increased amount of hepatic ferritin subunits. However, an increase of hepatic FTL but not of FTH protein expression was observed in IL-6ko mice after TO injection. Our data demonstrate that FTL is the only rat serum ferritin whose release into circulation from the hepatocytes is increased by the effect of AP cytokines (e.g., IL-6). In contrast, FTH expression is intracellular in both under physiological and AP conditions.

Increased apoptosis of intrahepatic central memory CD8 T cells in the absence of IL-15 is attributed to the loss of long-lasting protection against malaria infection (P3334)

Abstract Ag-specific memory T cell responses elicited by infections or vaccinations are inextricably linked to long-lasting protective immunity. Studies of protective immunity amongst residents of malaria endemic areas indicate that memory responses to Plasmodia antigens are not adequately developed or maintained. In contrast, multiple exposures to radiation-attenuated Plasmodia sporozoites (γ-spz) induce long-lasting protective immunity to experimental sporozoite challenge. In the P. berghei (Pb) γ-spz mouse model, lasting protection is associated with the presence of intrahepatic CD8 T cells that comprise two phenotypically and functionally discrete sets: effector/effector memory (TE/EM) cells that produce IFN-γ, and central memory (TCM) cells which express elevated levels of IL-15Rβ, suggesting that CD8 TCM cells depend upon IL-15 for maintenance and/or function. We asked whether immunization with Pb γ-spz could protect IL-15 KO mice against sporozoite challenge. The results demonstrate that although protective immunity is inducible in IL-15 KO mice, protection is short-lived, owing to reduced expression of Bcl-2 and increased apoptosis of proliferating intrahepatic CD8 TCM in the absence of IL-15. Therefore, we hypothesize that the maintenance of long-lasting protection induced by Pb γ-spz depends on a process whereby intrahepatic CD8 TCM cells, maintained by IL-15-mediated survival and basal proliferation, are conscripted into CD8 TE/EM cell pool during subsequent infections.

IL-9 strategy to perturb Treg function and enhance anti-tumor immunity (P4273)

Abstract IL-9 is a cytokine that is primarily associated with mast cells and airway inflammation, however it also plays a role in T regulatory cell (Treg) survival and recruitment to tumors. We previously reported that 4-1BB treatment of Tregs inhibits both Treg function and IL-9 secretion. Neutralization of IL-9 with an anti-IL-9 antibody inhibits the suppressive function of Tregs without affecting the function of CD4+ and CD8+ T effector cells. Furthermore, the combination of intra-tumoral CpG and anti-IL-9 induced tumor rejection in BALB-neuT and MUC1 tolerant transgenic mice. Here we show that the lack of IL-9 inhibits Treg suppression of T cell proliferation, using IL-9 knockout (IL-9ko) mice. Phenotypic analysis of Treg markers showed that tumor draining lymph nodes of 4T1 bearing mice contain fewer CTLA-4+ and CD103+ Tregs, and that these also have reduced of CTLA-4 and CD103 expression. We also observed that IL-9ko mice spontaneously reject TUBO and 4T1 mammary carcinoma cells and that rejection is abrogated by CD8+ T cell depletion. Finally, preliminary studies of human CD4+ T cell proliferation revealed that IL-9 neutralization led to greater proliferation than isotype control treated cells. Taken together our results suggest that IL-9 can have an immunosuppressive function that comes into play in the tumor microenvironment, and that the blockade of IL-9 could serve as a novel strategy to perturb the function of Tregs to enhance the antitumor effect of tumor vaccines.

Shp1 regulates T cell homeostasis by antagonizing IL-4 signalling (P1312)

Abstract The tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulator. Null mutations in Ptpn6, which encodes Shp1, result in the motheaten phenotype, characterized by widespread inflammation that causes death by 3-4 weeks of age. Previous studies of motheaten mice identified Shp1 as a negative regulator of TCR signaling, but the severe inflammation in these mice could have confounded our understanding of Shp1 function in T cell biology. To define the T cell intrinsic role of Shp1, we have characterized T cell-specific conditional knockout mice. Surprisingly, we detect no changes in TCR responsiveness in Shp1-deficient thymocytes or peripheral T cells. Instead, T cell Shp1 KO mice are enriched for memory-phenotype T-cells that express elevated levels of CD44 in the periphery. In addition, activated Shp1-deficient CD4+ T cells skew to Th2 and produce elevated levels of IL-4. Following IL-4 stimulation, Stat 6 activation is sustained in Shp1 deficient T-cells, leading to enhanced IL-4 production. Elimination of IL-4, through in vivo administration of a neutralizing antibody or by crossing to IL-4 KO mice, results in marked reduction of the excessive memory-like population in T cell specific Shp1 KO mice. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in and is critical for the maintainance of T cell homeostasis.

CCN1 expression in interleukin-6 deficient mouse kidney in experimental model of heart failure

Chronic heart failure often leads to worsening of the renal function. Mediators of this process include inflammatory and neuroendocrine factors. CCN1 (Cyr 61), a member of growth factor-inducible immediate early genes, which modulates inflammation and fibrogenesis, is excreted with urine in the early phase of acute renal injury and may be involved in the pathogenesis of the cardiorenal syndrome. The aim of the study was to evaluate CCN1 protein abundance and localization in the kidney of IL-6-deficient C57BL/6J (IL-6 KO) mice and respective wild-type (WT) animals in basal conditions and in animals with chronic heart failure twelve weeks after myocardial infarction. Age- and sex-matched mice from both strains subjected to sham operation served as controls. One group of WT animals subjected to myocardial infarction was treated with antagonist of AT1 receptor telmisartan over 12 weeks. Abundance and localization of CCN1 protein in kidney were assessed with Western blotting and immunohistochemistry, respectively. In all groups the strongest immunohistochemical reaction for CCN1 was observed in distal convoluted tubules and in smaller arteries, however, the total expression of CCN1 protein was lower in IL-6 KO mice in comparison to WT animals. The main difference in CCN1 distribution between the examined genotypes was lack of reaction in internal renal medulla and very weak reaction in proximal convoluted tubules in IL-6 KO mice. Experimental heart failure only slightly attenuated the expression of CCN1 protein in the kidney of WT mice and had no effect in IL-6 KO mice. Although, blockade of AT1 receptor did not alter CCN1 protein expression in kidneys of WT mice after myocardial infarction, it significantly changed its CCN1 distribution in the renal tubular system.

737 A Crucial Role of PU.1 Expressing Th9 Cells in Chronic Intestinal Inflammation

Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cytokine mainly produced by T cells, beside B cells and mast cells. Mounting evidence suggests that there may be a specialized subset of T cells dedicated to produce IL-9 which are so called Th9 cells. In these cells the IL-9 gene is regulated by transcription factors like PU.1 and IRF4. Also many cytokines have been investigated so far in experimental models of chronic intestinal inflammation, the role of IL-9 is largely unidentified. But high concentrations of IL-9 in the colon tissue during colitis reveal the important role of this cytokine. Methods: For the analysis of IL-9 in the development of chronic intestinal inflammation IL-9 deficient mice were used in oxazolone-colitis model. Miniendoscopic analysis has been done to monitor the manifestation of the colitis. The inflamed colon was isolated and histological sections were taken for immunohistofluorescent staining and real-time PCR analysis. For therapeutically treatment wildtype mice were given 40μg of specific anti IL-9 antibody to prevent the emergence of colitis. Results: In the experimental oxazolone-colitis model the IL-9KO mice were protected. This became evident in the miniendoscopic analysis and in the HE staining. Immunofluorescence staining shows a decrease of the IL-9 regulating transcription factor PU.1 in the IL-9 deficient mice, indicating the involvement of PU.1 in the IL-9 production. This is consistent with the fact that PU.1 is higher expressed in human biopsies of colitis patients, which illustrates the pro-inflammatory role of IL-9 in patients suffering from inflammatory bowel disease. Double staining of CD4+ and PU.1 and EPCAM and PU.1 respectively suggests that there are specialized T cells expressing the transcription factor PU.1 implying a role of Th9 cells in colitis. Further analysis of the pro-inflammatory effect of IL-9 showed that the blockage of high IL-9 concentrations with a specific anti IL-9 antibody in wildtype mice lead to a protection in oxazolone-colitis model. Conclusion: Here, we have identified a central pathogenic role for Th9 cells in chronic intestinal inflammation. This is based on the fact that IL-9 is increased in inflamed colon tissue and IL-9KO mice are protected in the experimental oxazolone-colitis model. Furthermore, administration of a blocking anti IL-9 antibody before the manifestation of colitis has a protective effect. Thus IL-9 emerges as a potentially new therapeutic target for inflammatory bowel diseases.

740 The Role of SMAD Signaling in the Pathogenesis of Pain in Chronic Pancreatitis

Introduction: Interleukin 9 is a pleiotropic pro-inflammatory cytokine mainly produced by T cells, beside B cells and mast cells. Mounting evidence suggests that there may be a specialized subset of T cells dedicated to produce IL-9 which are so called Th9 cells. In these cells the IL-9 gene is regulated by transcription factors like PU.1 and IRF4. Also many cytokines have been investigated so far in experimental models of chronic intestinal inflammation, the role of IL-9 is largely unidentified. But high concentrations of IL-9 in the colon tissue during colitis reveal the important role of this cytokine. Methods: For the analysis of IL-9 in the development of chronic intestinal inflammation IL-9 deficient mice were used in oxazolone-colitis model. Miniendoscopic analysis has been done to monitor the manifestation of the colitis. The inflamed colon was isolated and histological sections were taken for immunohistofluorescent staining and real-time PCR analysis. For therapeutically treatment wildtype mice were given 40μg of specific anti IL-9 antibody to prevent the emergence of colitis. Results: In the experimental oxazolone-colitis model the IL-9KO mice were protected. This became evident in the miniendoscopic analysis and in the HE staining. Immunofluorescence staining shows a decrease of the IL-9 regulating transcription factor PU.1 in the IL-9 deficient mice, indicating the involvement of PU.1 in the IL-9 production. This is consistent with the fact that PU.1 is higher expressed in human biopsies of colitis patients, which illustrates the pro-inflammatory role of IL-9 in patients suffering from inflammatory bowel disease. Double staining of CD4+ and PU.1 and EPCAM and PU.1 respectively suggests that there are specialized T cells expressing the transcription factor PU.1 implying a role of Th9 cells in colitis. Further analysis of the pro-inflammatory effect of IL-9 showed that the blockage of high IL-9 concentrations with a specific anti IL-9 antibody in wildtype mice lead to a protection in oxazolone-colitis model. Conclusion: Here, we have identified a central pathogenic role for Th9 cells in chronic intestinal inflammation. This is based on the fact that IL-9 is increased in inflamed colon tissue and IL-9KO mice are protected in the experimental oxazolone-colitis model. Furthermore, administration of a blocking anti IL-9 antibody before the manifestation of colitis has a protective effect. Thus IL-9 emerges as a potentially new therapeutic target for inflammatory bowel diseases.

P023 Therapy with FREMS (Frequency Rhythmic Electrical Modulation System)

Background: Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation with unknown etiology. Interleukin-10 Knockout (IL-10 KO) Mice are known to develop intestinal inflammation that resemble CD. We investigated if a human adipose-derived stem cells (h-ASCs) are capable to attenuated colon inflammation in IL-10 KO mice. Methods: h-ASCs were isolated from adipose tissues obtained from human donors. Colitis was induced in the mice by administration of piroxicam orally. Mice treated with intraperitoneal h-ASCs injection (5×105 cells) or normal saline as control after induction of colitis. All mice were euthanized 14 days after colitis. Histopathologic score and level of inflammation related cytokines were evaluated. Comparisons were made between h-ASCs treated or control non-treated IL-10 KO mice and wild type littermate. Results: The flow cytometry showed higher expression of CD105, CD73, CD90, CD44, however, hematopoietic stem cell markers such as CD45, CD34 and CD31 were not expressed in ADSC. Clinical score was lower in h-ASCs treated IL-10 KO mice than in control. Histopathologic assesment of large intestine showed lower inflammatory cells infiltration and less mucosal damage in h-ASCs treated mice than in control. Tissue score was lower in h-ASCs treated IL-10 KO mice than in control. The level of Monocyte chemoattractant protein-1 and Toll-like receptor 4 expression in the large intestine was lower and anti human IL-10 and anti mouse IL-4 in the serum was higher in h-ASCs treated mice. Conclusions: These findings suggested that administration of h-ASCs show beneficial effect on T-cell induced spontaneous colitis model and emerge a therapeutic option in inflammatory bowel disease.

P022 Therapeutic effect of adipose tissue-derived stem cell in Interleukin-10 knockout mice

Background: Inflammatory Bowel Disease (IBD) is characterized by chronic intestinal inflammation with unknown etiology. Interleukin-10 Knockout (IL-10 KO) Mice are known to develop intestinal inflammation that resemble CD. We investigated if a human adipose-derived stem cells (h-ASCs) are capable to attenuated colon inflammation in IL-10 KO mice. Methods: h-ASCs were isolated from adipose tissues obtained from human donors. Colitis was induced in the mice by administration of piroxicam orally. Mice treated with intraperitoneal h-ASCs injection (5×105 cells) or normal saline as control after induction of colitis. All mice were euthanized 14 days after colitis. Histopathologic score and level of inflammation related cytokines were evaluated. Comparisons were made between h-ASCs treated or control non-treated IL-10 KO mice and wild type littermate. Results: The flow cytometry showed higher expression of CD105, CD73, CD90, CD44, however, hematopoietic stem cell markers such as CD45, CD34 and CD31 were not expressed in ADSC. Clinical score was lower in h-ASCs treated IL-10 KO mice than in control. Histopathologic assesment of large intestine showed lower inflammatory cells infiltration and less mucosal damage in h-ASCs treated mice than in control. Tissue score was lower in h-ASCs treated IL-10 KO mice than in control. The level of Monocyte chemoattractant protein-1 and Toll-like receptor 4 expression in the large intestine was lower and anti human IL-10 and anti mouse IL-4 in the serum was higher in h-ASCs treated mice. Conclusions: These findings suggested that administration of h-ASCs show beneficial effect on T-cell induced spontaneous colitis model and emerge a therapeutic option in inflammatory bowel disease.

Induction of atypical EAE mediated by transgenic production of IL‐6 in astrocytes in the absence of systemic IL‐6

Interleukin (IL)-6 is crucial for the induction of many murine models of autoimmunity including experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. While IL-6-deficient mice (IL-6 KO) are resistant to EAE, we showed previously that in transgenic mice with astrocyte-targeted production of IL-6-restricted to the cerebellum (GFAP-IL6), EAE induced with MOG(35-55) was redirected away from the spinal cord to the cerebellum. To further establish the importance of IL-6 produced in the central nervous system, we have generated mice producing IL-6 essentially only in the brain by crossing the GFAP-IL6 mice with IL-6 KO mice. Interestingly, GFAP-IL6-IL-6 KO mice showed a milder but almost identical phenotype as the GFAP-IL6 mice, which correlated with a lower load of inflammatory cells and decreased microglial reactivity. These results indicate that not only is cerebellar IL-6 production and eventual leakage into the peripheral compartment the dominating factor controlling this type of EAE but that it can also facilitate induction of autoimmunity in the absence of normal systemic IL-6 production.

Distinct IFN-Gamma Dependent Effect of the TLR5 Agonist, Flagellin On NK and CD4+ T Cell Immunity Against CMV

AbstractAbstract 255 Background:Opportunistic CMV infection remains a major clinical complication in allogeneic BMT (allo-BMT) recipients. We previously published that peri-transplant treatment of highly purified flagellin, a TLR5 agonist extracted from S. typhimurium reduced GvHD in lethally irradiated allo-BMT recipients by reducing early activation and proliferation of donor T cells. Flagellin-treated recipients had enhanced lymphoid immune reconstitution and were protected from lethal MCMV infection. CMV initiates innate immunity through the activation of Toll-like receptors (TLR) 9, 3 and 2 but direct enhancement of anti-CMV immunity through TLR5 has not been studied. Methods:To further explore the effects of a TLR5 agonist on anti-CMV immunity, we infected flagellin-treated C57BL/6 (B6), TLR5KO and Myd88KO mice (B6 background) with sub lethal dose (1×105pfu/mouse i.p) of MCMV. Anti-CMV immunity of NK and T-cells were determined by measuring surface activation markers on the NK and T-cells. Flagellin-treated and MCMV infected IL-12KO, IFN-γRKO and IDO KO mice were used to study the role of IL-12, IFN-γ and IDO on anti-CMV immunity. The relationship between the MCMV lethality and flagellin signaling in hematopoietic or epithelial tissues expressing TLR5 was further investigated by generating radiation chimeras, using WT mice engrafted with TLR5 KO bone marrow (BM) and TLR5 KO mice engrafted with WT B6 BM. Chimeric recipients with WT or TLR5KO hematopoietic cells (or the reverse) were then treated with flagellin or PBS and infected with a sub lethal dose of MCMV infection. Results:Without MCMV infection, flagellin treatment significantly increased numbers of NK cells (p= 0.001) and KLRG1+ NK cells (p=0.009) in WT B6 mice compared with the PBS-treated control mice. Following MCMV infection (day 3), there were significantly increased numbers of splenic NK cells (p= 0.01) and KLRG1+ NK cells (p=0.0008) in flagellin-treated mice compared with the control mice. Flagellin-treatment also enhanced viral clearance, tending to decrease liver viral loads in WT B6 mice. In contrast, MCMV-infected and flagellin-treated TLR5 KO or Myd88KO mice had significantly increased liver viral loads (p=00001) compared with the flagellin- and PBS-treated B6 mice on day 3 post MCMV infection. While flagellin-treatment increased the numbers and activation status of NK cells, it decreased the numbers of activated (CD69+, ICOS+ and KLRG1+) CD4+ T cells on day 3 post MCMV infection. On day 10 post MCMV infection, enhanced numbers of splenic CD8+ T cells and CMV-specific tetramer+ CD8+ T cells (p<0.05) were detected in flagellin-treated mice compared with controls. The numbers of splenic CD4+ T cells increased >2 fold (p= 0.008) associated with significantly increased numbers of splenic CD4+CD25+ Foxp3+ regulatory T cells (p<0.05) on day 10 post MCMV infection in flagellin-treated mice compared with the control mice. Flagellin-induced anti-CMV immunity required IFN-γ signaling as flagellin-treated IFN-γRKO mice had markedly increased susceptibility to MCMV infection whereas flagellin-treated IL-12 KO and IDO KO mice had significantly reduced liver viral loads compared to the PBS-treated IL-12 KO and IDO KO mice on day 10 post MCMV infection. To explore interaction of flagellin with TLR5+ host hematopoietic cells and TLR5+host gut epithelium on CMV immunity, we next infected radiation chimeras expressing either TLR5 on hematopoietic cells or on gut epithelium with sub lethal i.p dose of MCMV. Flagellin-treated TLR5 KO BM → B6 radiation chimeras (TLR5+ host epithelium) had increased mortality compared with other groups following MCMV infection indicating that TLR5+ hematopoietic cells are required for protective immunity against CMV infection.In summary, flagellin caused selective early increased activation of NK cells and reduced activation of CD4+ T cells in presence of MCMV infection. The protective effect of flagellin on anti-CMV immune responses are IFN-γ dependent but independent of IL-12 and IDO. The TLR5+ hematopoietic cells are required for the protective immunity against CMV infection. These data suggest the clinical importance of flagellin in modulating both innate and adaptive immunity against CMV infection and also as an alternative GvHD prophylaxis in allo-BMT recipients. Disclosures:No relevant conflicts of interest to declare.

Pressure overload-induced cardiac remodeling and dysfunction in the absence of interleukin 6 in mice

Congestive heart failure is associated with increased expression of pro-inflammatory cytokines, and the levels of these cytokines correlate with heart failure severity and prognosis. Chronic interleukin 6 (IL-6) stimulation leads to left ventricular (LV) hypertrophy and dysfunction, and deletion of IL-6 reduces LV hypertrophy after angiotensin II infusion. In this study, we tested the hypothesis that IL-6 deletion has favorable effects on pressure-overloaded hearts. We performed transverse aortic constriction on IL-6-deleted (IL6KO) mice and C57BL/6J mice (CON) to induce pressure overload. Pressure overload was associated with similar LV hypertrophy, dilation, and dysfunction in CON and IL6KO mice. Re-activation of the fetal gene program was also similar in pressure-overloaded CON and IL6KO mice. There were no differences between CON and IL6KO mice in LV fibrosis or expression of extracellular matrix proteins after pressure overload. In addition, no group differences in apoptosis or autophagy were seen. These data indicate that IL-6 deletion does not block LV remodeling and dysfunction induced by pressure overload. Attenuated content of IL-11 appears to be a compensatory mechanism for IL-6 deletion in pressure-overloaded hearts. We infer from these data that limiting availability of IL-6 alone is not sufficient to attenuate LV remodeling and dysfunction in failing hearts.

Obesity and IL-6 interact in modulating the response to endotoxemia in mice

Obesity is associated with elevated levels of IL-6. High IL-6 is prognostic of mortality in sepsis, while controversial data link obesity to sepsis outcome. We used Lean and diet-induced obese (DIO) WT and IL-6 KO mice to investigate the interaction between obesity and IL-6 in endotoxemia. Circulating levels of IL-6 were significantly higher in WT DIO versus WT Lean mice receiving LPS (2.5μg/mouse, ip). Obesity lead to greater weight loss in response to LPS, with IL-6 deficiency being partially protective. Plasma TNFα, IFNγ, Galectin-3 and leptin were significantly elevated in response to LPS and were each differentially affected by obesity and/or IL-6 deficiency. Plasma Galectin-1 and adiponectin were significantly suppressed by LPS, with obesity and IL-6 deficiency modulating the response. However, LPS comparably increased IL-10 levels in each group. Leukopenia with relative neutrophilia and thrombocytopenia developed in each group after injection of LPS, with obesity and genotype affecting the kinetics, but not the magnitude, of the response. Hepatic induction of the acute-phase protein SAA by LPS was not affected by obesity or IL-6 deficiency, although baseline levels were highest in WT DIO mice. Injection of LPS significantly increased hepatic mRNA expression of PAI-1 in Lean WT and Lean KO mice, while it suppressed the high baseline levels observed in the liver of DIO WT and DIO KO mice. Thus, both IL-6 and obesity modulate the response to endotoxemia, suggesting a complex interaction that needs to be considered when evaluating the effect of obesity on the outcome of septic patients.

IL-6 deficiency exacerbates skin inflammation in a murine model of irritant dermatitis

Contact dermatitis is the second most reported occupational injury associated with workers compensation. Inflammatory cytokines are closely involved with the development of dermatitis, and their modulation could exacerbate skin damage, thus contributing to increased irritancy. IL-6 is a pro-inflammatory cytokine paradoxically associated with both skin healing and inflammation. To determine what role this pleiotropic cytokine plays in chemically-induced irritant dermatitis, IL-6 deficient (KO), IL-6 over-expressing transgenic (TgIL6), and corresponding wild-type (WT) mice were exposed to acetone or the irritants JP-8 jet fuel or benzalkonium chloride (BKC) daily for 7 days. Histological analysis of exposed skin was performed, as was tissue mRNA and protein expression patterns of inflammatory cytokines via QPCR and multiplex ELISA. The results indicated that, following JP-8 exposure, IL-6KO mice had greatly increased skin IL-1β, TNFα, CCL2, CCL3, and CXCL1 mRNA and corresponding product protein expression when compared to that of samples from WT counterparts and acetone-exposed control mice. BKC treatment induced the expression of all cytokines examined as compared to acetone, with CCL2 significantly higher in skin from IL-6KO mice. Histological analysis showed that IL-6KO mice displayed significantly more inflammatory cell infiltration as compared to WT and TgIL6 mice in response to jet fuel. Analysis of mRNA for the M2 macrophage marker CD206 indicated a 4-fold decrease in skin of IL-6KO mice treated with either irritant as compared to WT. Taken together, these observations suggest that IL-6 acts in an anti-inflammatory manner during irritant dermatitis, and these effects are dependent on the chemical nature of the irritant.

O035 IL-33 and ST2 KO mice display different phenotypes in K/BxN serum transfer-induced arthritis

Introduction IL-33 is the most recently described member of the IL-1 family of cytokines. Expression studies and data obtained with ST2 KO mice, ST2 blockade and recombinant IL-33 have suggested implication of the IL-33/ST2 axis in the pathogenesis of human and mouse arthritis. In the present study we investigated the role of endogenous IL-33 in K/BxN serum transfer-induced arthritis by using IL-33 KO mice and compared the results to those obtained using ST2 KO mice. Methods Arthritis was induced in IL-33 KO and ST2 KO mice, backcrossed to the C57BL/6 background, or in WT C57BL/6 control mice, by injection of arthritogenic K/BxN serum or purified total IgG. The development of arthritis was followed by clinical scoring of the paws and confirmed by histological evaluation. Cytokine mRNA expression was assessed by RT-qPCR. Cytokine protein levels were monitored by ELISA or Milliplex. Results IL-33 mRNA and protein expression in synovial tissues, as well as circulating IL-33 levels, were similar in arthritic WT and ST2 KO mice, and absent in IL-33 KO mice. The incidence and severity of K/BxN serum transfer-induced arthritis was identical in IL-33 KO and WT mice, while disease development was significantly reduced in ST2 KO mice. Similar data were obtained when injecting purified arthritogenic total IgG instead of complete K/BxN serum, excluding a potential contribution of IL-33 contained in the serum of donor mice to explain this difference. Conclusion IL-33 is expressed locally in the inflamed synovium and is detected in the circulation during serum transfer-induced arthritis. However IL-33 KO mice displayed identical arthritis development and severity as compared to WT controls. In contrast, disease severity was reduced in ST2 KO mice. The reasons for the difference between IL-33 KO and ST2 KO mice, which might suggest IL-33 independent effects of ST2, or reveal the existence of confounding variables in these KO strains, are currently under investigation.

Abstract 15: Remote Ischemic Preconditioning Confers Late Protection Against Myocardial Ischemia-Reperfusion Injury in Mice by Upregulating Interleukin-10

Remote ischemic preconditioning (RIPC) induces a prolonged late phase of multi-organ protection against ischemia-reperfusion (IR) injury. In the present study, we tested the hypothesis that RIPC confers late protection against myocardial IR injury by upregulating expression of IL-10. Mice were exposed to lower limb RIPC or sham leg ischemia. After 24 h, mice with RIPC demonstrated decreased myocardial infarct size and improved cardiac contractility following 30-min ischemia and 120-min reperfusion (I-30/R-120), accompanied by increased phosphorylation of Akt and eNOS in the heart. These effects of RIPC were completely blocked by anti-IL-10 receptor antibodies. Moreover, in IL-10 KO mice, RIPC was no longer able to provide cardioprotection against IR injury. IL-10 receptors were expressed in cardiomyocytes under basal conditions. Stimulation with exogenous IL-10 increased Akt phosphorylation and decreased myocardial infarct size after I-30/R-120 in isolated perfused hearts. Wildtype mice with RIPC demonstrated increased plasma IL-10 levels, while in the preconditioned gastrocnemius muscle, the phosphatase PTEN was inactivated and expression of IL-10 was increased through Stat3. Myocyte-specific PTEN inactivation led to increased Stat3 phosphorylation and IL-10 protein expression in the gastrocnemius muscle. Taken together, these results suggest that RIPC induces late protection against myocardial IR injury by increasing expression of IL-10 in the remote muscle, followed by release of IL-10 into the circulation, and activation of protective signaling pathways in the heart. This study provides a scientific basis for the use of RIPC to confer systemic protection against IR injury.

Early IL-10 production is essential for syngeneic graft acceptance

We performed a comparative study and evaluated cellular infiltrates and anti-inflammatory cytokine production at different time-points after syngeneic or allogeneic skin transplantation. We observed an early IL-10 production in syngeneic grafts compared with allografts. This observation prompted us to investigate the role of IL-10 in isograft acceptance. For this, we used IL-10 KO and WT mice to perform syngeneic transplantation, where IL-10 was absent in the graft or in the recipient. The majority of syngeneic grafts derived from IL-10 KO donors did not engraft or was only partially accepted, whereas IL-10 KO mice transplanted with skin from WT donors accepted the graft. We evaluated IL-10 producers in the transplanted skin and observed that epithelial cells were the major source. Taken together, our data show that production of IL-10 by donor cells, but not by the recipient, is determinant for graft acceptance and strongly suggest that production of this cytokine by keratinocytes immediately upon transplantation is necessary for isograft survival.

Role of IL-6 in the resolution of pancreatitis in obese mice

Obesity increases severity of acute pancreatitis and risk of pancreatic cancer. Pancreatitis and obesity are associated with elevated IL-6, a cytokine involved in inflammation and tumorigenesis. We studied the role of IL-6 in the response of lean and obese mice to pancreatitis induced by IL-12 + IL-18. Lean and diet-induced obese (DIO) WT and IL-6 KO mice and ob/ob mice pretreated with anti-IL-6 antibodies were evaluated at Days 1, 7, and 15 after induction of pancreatitis. Prolonged elevation of IL-6 in serum and visceral adipose tissue was observed in DIO versus lean WT mice, whereas circulating sIL-6R declined in DIO but not lean mice with pancreatitis. The severe inflammation and lethality of DIO mice were also observed in IL-6 KO mice. However, the delayed resolution of neutrophil infiltration; sustained production of CXCL1, CXCL2, and CCL2; prolonged activation of STAT-3; and induction of MMP-7 in the pancreas, as well as heightened induction of serum amylase A of DIO mice, were blunted significantly in DIO IL-6 KO mice. In DIO mice, production of OPN and TIMP-1 was increased for a prolonged period, and this was mediated by IL-6 in the liver but not the pancreas. Results obtained in IL-6 KO mice were confirmed in ob/ob mice pretreated with anti-IL-6 antibodies. In conclusion, IL-6 does not contribute to the increased severity of pancreatitis of obese mice but participates in delayed recovery from acute inflammation and may favor development of a protumorigenic environment through prolonged activation of STAT-3, induction of MMP-7, and sustained production of chemokines.

A Single Bout of Exercise Increases the Expression of Glucose but not Fatty Acid Transporters in Skeletal Muscle of IL‐6 KO Mice

IL-6 is a biologically active cytokine released during exercise by contracting skeletal muscles. It appears to be highly involved in the regulation of muscles energy substrate utilization. Whether an ablation of IL-6 (IL-6 KO) in mice subjected to a single bout of exercise affects lipid and/or glucose metabolism is currently unknown. In the present study we examined fatty acid (FAT/CD36, FABPpm, FATP-1, FATP-4) as well as glucose (GLUT-1, GLUT-4) transporters expression in IL-6 KO mice. In addition, intramuscular glycogen and lipid content was also evaluated. The expression of all fatty acid transporters (FAT/CD36: +25 %; FATP-1: +31 %; FABPpm: +12.7 %; FATP-4: +7.2 %) was increased in muscles from IL-6 KO mice compared to wild type (WT) mice. Accordingly intramuscular lipid content was also increased in these muscles (FFA: +38 %; DAG: +36 % and TAG: +160 %). Interestingly, IL-6 deficiency had only minor effect on glucose transporters expression (GLUT-1: −4 %, and GLUT-4: −5.1 %), with no apparent difference in muscular glycogen content. A single bout of exercise increased the glucose transporters (GLUT-1: +8 %; GLUT-4: +15 %) as well as FA transporters (FAT/CD36: +13 %; FABPpm: +4.5 %; FATP: +2.5 %, FATP-4: +10 %) expression but only in WT skeletal muscles. In muscles from IL-6 KO mice exercise induced changes only in glucose (GLUT-1: +20 %; GLUT-4: +35 %) but not in the content of FA transporters. Concomitantly, IL-6 KO mice displayed shorter time toward exhaustion with more pronounced reductions in intramuscular lipid and glycogen content. We may speculate, that IL-6 deficiency provokes more pronounced glucose utilization over lipid oxidation during a single bout of exhausting exercise.