IL-10 Knockout Mice Research Articles

Abstract 522: IL-10 Knockout Bone Marrow Fibroblast Progenitor Cells-derived Exosomes Activate Cardiac Fibroblast and Exaggerate Pressure Overload-induced Fibrosis in Mice Heart

Background: Inflammatory mediators play important role in cardiac remodeling. In heart, fibrosis is mainly mediated by activated fibroblasts (myofibroblasts), however, origin of myofibroblasts in diseased heart remains unclear. Recently, we have shown that bone marrow fibroblast progenitor cells (FPCs) significantly contribute in cardiac fibrosis in IL10 KO (KO) mice, but its molecular mechanism is not known. Here, we hypothesize that BM-FPCs activate resident cardiac fibroblast via their paracrine mediators (packaged in exosomes) to exaggerate the cardiac fibrosis in KO mice post- transverse aortic constriction (TAC). Method and Results: Cardiac hypertrophy and fibrosis was induced in Wild-type (WT) and IL10-knockout (IL10KO) mice by TAC surgery. TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Recombinant IL10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO enhanced FPC mobilization and homing in KO mice compared to WT mice. Furthermore, to identify the paracrine signaling, exosomes were isolated from WT and KO-FPCs culture media and characterized for fibrotic miRNA and proteins. Our data suggests that KO-FPC-exosomes are enriched with profibrotic miRs (miR21 and miR27) and proteins (RhoA). To explore whether KO-FPC-exosomes modulates resident fibroblasts biology and activation, adult cardiac fibroblasts (WT) were isolated and treated with WT and IL10KO-BMFPC-exosomes for 48 hrs. myoFB-associated gene (Collagen1α, Collagen3α and α- SMA) expression was notably increased in KO-FPC-exosome treated cells. Finally, immunostaining data further corroborated with our gene expression data and showed significantly enhanced α-SMA expression after KO-FPCs exosome treatments in cFBs. Conclusion: In conclusion, our data suggest that fibrotic remodeling factors (miRs and/or proteins) packaged in KO-FPC exosomes are sufficient to enhance the resident cardiac fibroblast activation and mediate cardiac fibrotic remodeling. Acknowledgement: NIH (RO1-135060) and AHA (14SDG20480104) SKV

Consequences of the Lack of IL-10 in Different Endotoxin Effects and its Relationship With Glucocorticoids.

Sepsis constitutes one of the major causes of death in ICUs. In sepsis induced by gram-negative, although lipopolysaccharide (LPS) initially induces an exacerbated secretion of proinflammatory cytokines leading to endotoxic shock and death resembling a septic shock, it is also capable of inducing refractoriness to subsequent challenge with LPS, a state known as endotoxin tolerance, which is considered the initial step of the immunosuppression found in septic patients. As we previously demonstrated the importance of glucocorticoids in endotoxin tolerance, the aim of this study was to evaluate the contribution of Interleukin-10 (IL-10) both in the endotoxic shock and in the development of the tolerance and its relationship with glucocorticoids. Our results show that, upon LPS challenge, IL-10 knockout mice (KO) mice had an enhanced LPS sensitivity, along with elevated levels of proinflammatory cytokines as tumor necrosis factor-α, IL-12 and interferon-γ, and enhanced tissue damage, despite the high levels of glucocorticoids. This effect may be because, in part, of the higher expression of tumor necrosis factor receptors in IL-10 KO mice. Further, the injection of dexamethasone did not protect IL-10 KO mice from a LPS lethal challenge. Although tolerance was achieved in the absence of IL-10, it was weaker and the elevated levels of glucocorticoids were not able to reverse the high sensitivity of IL-10 KO mice to LPS. Nevertheless, glucocorticoids would play a pivotal role in the establishment and maintenance of this partial tolerance in IL-10 KO mice. Finally, our results show that IL-10 and glucocorticoids could act in a bidirectional way influencing the inflammatory and anti-inflammatory periods.

IL-6/p-BTK/p-ERK signaling mediates calcium phosphate-induced pruritus.

Uremic pruritus with elevated levels of calcium phosphate (CaP) in skin is a common symptom in patients with chronic kidney disease (CKD). In this study, we demonstrate that intradermal injection of CaP into mice triggered scratching by up-regulating the IL-6 in skin and phosphorylation of ERKs in dorsal root ganglion (DRG) in a dose-dependent manner. IL-6 is essential because the CaP-induced up-regulation of phosphorylated (p)-ERK in DRG was considerably reduced in the IL-6 knockout mice. Microarray analysis in conjunction with real-time PCR revealed a higher mRNA expression of Bruton's tyrosine kinase (BTK) gene in DRG after CaP injection. The inhibition of BTK by ibrutinib noticeably diminish the CaP-induced up-regulation of IL-6 and p-ERK in mice. A high amount of IL-6 was detected in itchy skin and blood of patients with CKD. The expressions of p-BTK and p-ERK in DRG primary cells reached maximum levels at 1 and 10 min, respectively, after treatment of recombinant IL-6 and were significantly reduced by treatment of IL-6 along with ibrutinib. The mechanism by which the CaP-induced pruritus mediated by the IL-6/p-BTK/p-ERK signaling was revealed.-Keshari, S., Sipayung, A. D., Hsieh, C.-C., Su, L.-J., Chiang, Y.-R., Chang, H.-C., Yang, W.-C., Chuang, T.-H., Chen, C.-L., Huang, C.-M. IL-6/p-BTK/p-ERK signaling mediates calcium phosphate-induced pruritus.

Anti-CD45RB Antibody Therapy Attenuates Renal Ischemia-Reperfusion Injury by Inducing Regulatory B Cells.

Regulatory B cells are a newly discovered B cell subset that suppresses immune responses. Recent studies found that both anti-CD45RB and anti-Tim-1 treatments regulate immune responses by inducing regulatory B cells; however, the role of these cells in renal ischemia-reperfusion injury (IRI) is unknown. Using mouse models, including T cell-deficient (RAG1 knockout and TCRα knockout) mice and B cell-deficient (μMT) mice, we investigated the effects of regulatory B cells and anti-CD45RB on IRI and the mechanisms underlying these effects. Adoptive transfer of regulatory B cells before or after IRI attenuated renal IRI. Anti-CD45RB treatment with or without anti-Tim-1 before IRI increased renal infiltration of CD19+Tim-1+ regulatory B and regulatory T cells. Anti-CD45RB decreased serum creatinine levels, pathologic injury score, tubular apoptosis, and proinflammatory cytokines levels, whereas IL-10 levels increased. Following IRI, anti-CD45RB with or without anti-Tim-1 also induced regulatory B cells, improving renal function and tubular regeneration. In RAG1 knockout mice with B cell transfer, TCRα knockout mice, and wild-type mice with T cell depletion, anti-CD45RB increased regulatory B cells and attenuated IRI. However, anti-CD45RB did not attenuate IRI in RAG1 knockout mice with T cell transfer or μMT mice and induced only mild improvement in wild-type mice with B cell depletion. Furthermore, B cell-deficient mice receiving B cells from IL-10 knockout mice (but not from wild-type mice) did not show renal protection against IRI when treated with anti-CD45RB. Anti-CD45RB treatment attenuated acute renal injury and facilitated renal recovery after IRI through induction of IL-10+ regulatory B cells, pointing to anti-CD45RB as a potential therapeutic strategy in renal IRI.

Roles of T Follicular Helper Cells and T Follicular Regulatory Cells in Autoantibody Production in IL-2–Deficient Mice

Autoantibodies can result from excessive T follicular helper (Tfh) cell activity, whereas T follicular regulatory (Tfr) cells negatively regulate autoantibody production. IL-2 knockout (KO) mice on the BALB/c background have elevated Tfh responses, produce autoantibodies, and develop lethal autoimmunity. We analyzed Tfh and Tfr cells in IL-2 KO mice on the C57BL/6 (B6) genetic background. In B6 IL-2 KO mice, the spontaneous formation of Tfh cells and germinal center B cells was greatly enhanced, along with production of anti-DNA autoantibodies. IL-2 has been reported to repress Tfr cell differentiation; however, Tfr cells were not increased over wild-type levels in the B6 IL-2 KO mice. To assess Tfh and Tfr cell regulation of autoantibody production in IL-2 KO mice, we generated IL-2 KO mice with a T cell-specific deletion of the master Tfh cell transcription factor Bcl6. In IL-2 KO Bcl6 conditional KO (2KO-Bcl6TC) mice, Tfh cells, Tfr cells, and germinal center B cells were ablated. In contrast to expectations, autoantibody IgG titers in 2KO-Bcl6TC mice were significantly elevated over autoantibody IgG titers in IL-2 KO mice. Specific deletion of Tfr cells with Foxp3-cre Bcl6-flox alleles in IL-2 KO mice led to early lethality, before high levels of autoantibodies could develop. We found IL-2+/+ Tfr cell-deficient mice produce significant levels of autoantibodies. Our overall findings provide evidence that Tfh cells are dispensable for high-level production of autoantibodies and also reveal a complex interplay between Tfh and Tfr cells in autoantibody production and autoimmune disease.

IL-10/STAT3/SOCS3 Axis Is Involved in the Anti-inflammatory Effect of Benznidazole.

Anti-parasitic treatment for Chagas disease mainly relies on benznidazole, which is virtually the only drug available in the market. Besides its anti-parasitic effects, benznidazole has anti-inflammatory properties. In this work we studied the mechanisms involved in the latter, demonstrating the participation of the IL-10/STAT3/SOCS3 pathway. To achieve this goal, the anti-inflammatory properties of benznidazole were studied using an in vitro model of cardiomyocyte primary culture stimulated with LPS. LPS increased both SOCS3 expression and STAT3 phosphorylation. The addition of benznidazole increased their expression even further. Specific inhibition of STAT3 precluded this effect, suggesting a role for STAT3 in the increase of SOCS3 expression induced by benznidazole. To assess the participation of SOCS3 in the anti-inflammatory effect of benznidazole, we accomplished specific knockdown of SOCS3 with siRNA. Silencing of SOCS3 in cardiomyocytes precluded the inhibitory effects of benznidazole on TNF-α, IL-6, iNOS expression and NO release. Moreover, in the absence of SOCS3, benznidazole could neither prevent IKK phosphorylation nor IκBα degradation, supporting the notion that SOCS3 is required for the benznidazole-mediated inhibition of the NF-κB pathway. Previously, we demonstrated that IL-10 increases the expression of SOCS3 in cultured cardiomyocytes. Here, we found that benznidazole shows a trend to increased IL-10 expression. To evaluate whether benznidazole increased SOCS3 in an IL-10-dependent manner, cardiomyocytes from IL-10 knockout mice were pre-treated with benznidazole and stimulated with LPS. Benznidazole neither inhibited NO release nor avoid IKK phosphorylation or IκBα degradation, showing that IL-10 is required for benznidazole-mediated inhibition of NF-κB. Moreover, exogenous addition of IL-10 to IL-10 knockout cardiomyocytes restored the inhibitory effect of benznidazole on NO release. The results reported herein show, for the first time, that the IL-10/STAT3/SOCS3 axis is involved in the anti-inflammatory effects of benznidazole. These findings may add up to new therapeutic strategies for chronic Chagas disease given its inflammatory nature.

Regulation of breathing pattern by IL-10.

Proinflammatory cytokines like interleukin-1β (IL-1β) affect the control of breathing. Our aim is to determine the effect of the anti-inflammatory cytokine IL-10 οn the control of breathing. IL-10 knockout mice (IL-10-/-, n = 10) and wild-type mice (IL-10+/+, n = 10) were exposed to the following test gases: hyperoxic hypercapnia 7% CO2-93% O2, normoxic hypercapnia 7% CO2-21% O2, hypoxic hypercapnia 7% CO2-10% O2, and hypoxic normocapnia 3% CO2-10% O2. The ventilatory function was assessed using whole body plethysmography. Recombinant mouse IL-10 (rIL-10; 10 μg/kg) was administered intraperitoneally to wild-type mice (n = 10) 30 min before the onset of gas challenge. IL-10 was administered in neonatal medullary slices (10-30 ng/ml, n = 8). We found that IL-10-/- mice exhibited consistently increased frequency and reduced tidal volume compared with IL-10+/+ mice during room air breathing and in all test gases (by 23.62 to 33.2%, P < 0.05 and -36.23 to -41.69%, P < 0.05, respectively). In all inspired gases, the minute ventilation of IL-10-/- mice was lower than IL-10+/+ (by -15.67 to -22.74%, P < 0.05). The rapid shallow breathing index was higher in IL-10-/- mice compared with IL-10+/+ mice in all inspired gases (by 50.25 to 57.5%, P < 0.05). The intraperitoneal injection of rIL-10 caused reduction of the respiratory rate and augmentation of the tidal volume in room air and also in all inspired gases (by -12.22 to -29.53 and 32.18 to 45.11%, P < 0.05, respectively). IL-10 administration in neonatal rat (n = 8) in vitro rhythmically active medullary slice preparations did not affect either rhythmicity or peak amplitude of hypoglossal nerve discharge. In conclusion, IL-10 may induce a slower and deeper pattern of breathing.

IL-33 promotes antiviral immunity and protects against congenital Zika disease in mice

Abstract Zika virus (ZIKV) infection during pregnancy can cause congenital Zika syndrome and other birth defects. The alarmin IL-33 can evoke strong antiviral immune responses during viral infection. However, whether IL-33 is involved in immune responses during ZIKV infection is not clear. In this study, we infected three-week-old wildtype (WT) and IL-33 knockout (KO) mice with ZIKV and analyzed the ZIKV-specific cytotoxic T lymphocyte (CTL) responses at 6 days post infection. We found that IL-33 deficiency resulted in decreased ZIKV-specific CTL responses as evidenced by lower numbers of IFN-g+TNF-a+ CD8+ T effectors. To determine if IL-33 contributes to anti-ZIKV immunity during pregnancy, we infected WT pregnant mice with ZIKV and found increased IL-33 in the placenta at a mid-gestational period (embryo E13.5) compared with that of uninfected ones. Likewise, elevated IL-33 expression was also observed in human trophoblast cell lines JEG-3 and HTR-8 upon ZIKV infection. To determine if IL-33 plays a protective role in congenital Zika disease, we infected pregnant WT and IL-33 KO mice by i.p. injection of ZIKV at E6.5 and found higher percentages of fetal demise and absorption in IL-33 KO mice compared with that in WT mice (16.7 % vs. 3.3 %) from necropsy on E13.5. In addition, the fetuses from IL-33 KO mice displayed smaller sizes than those of WT fetuses (55 mm2 vs. 48 mm2). Moreover, IL-33 deficient mice had lower numbers of virus-specific CD8+ T cells in the placenta, uterine and decidua. Importantly, IL-33 deficiency resulted in higher viral loads in both the placenta and fetus. In summary, our study highlights IL-33 as a key immune regulator in inducing anti-ZIKV CTL responses and protecting against congenital Zika disease.

Blocking the Pro-Inflammatory Response Associated with Inflammatory Bowel Disease with 7-Ketocholesterol

Abstract Inflammatory bowel disease (IBD) affects more than one million people in the United States alone, and currently has no cure. IBD is a chronic inflammatory condition, perpetrated by dysregulation of the adaptive immune system’s response to nonpathogenic flora in the gastrointestinal tract. CD4+ T cells, critical components of the adaptive immune response, misperceive commensal microbiota in the gut as threats and persistent stimulators of CD4+ T cells, resulting in uncontrolled and episodic responses. Regulatory T cells, through secretion of interleukin 10 (IL-10) cytokine, suppress the pro-inflammatory environment in the gut. In the absence of this suppression a chronic inflamed state can be reached. Previous studies have shown that 7-ketocholesterol (7-KC), a natural metabolite of cholesterol, can successfully disrupt lipid raft-based membrane order in vitro, promoting a disordered state. This forces the question of whether pro-inflammatory responses can be disrupted through this method. We investigated the potential of 7-KC to suppress gut inflammation using the IL-10 KO animal model. Mice deficient in IL-10 have inflamed guts and symptoms of IBD as observed in humans. IL-10 knockout mice were orally gavaged twice daily, 3 days a week for 3 weeks to deliver 7-KC. Treated and control mice were observed for 4 weeks and necropsies were performed on day 27 to dissect their colons for immunohistochemistry. We observed a change in tissue constitution, stool quality, and fatty pockets in the treatment group in comparison with the controls receiving saline. These preliminary findings with IL-10 knockout mouse model reflect the potential of 7-KC as immunomodulatory agent for suppressing the inflammatory environment in the gut.

IL17 depletion enhances the DNA vaccine efficacy in pancreatic ductal adenocarcinoma treatment

Abstract Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive cancer with a 5-year survival rate of ~8%. PDA is considered an “immunologically-cold” tumor because of the abundant desmoplastic immune-suppressive microenvironment and low presence of neo-antigens. Moreover, it is characterised by a complex inflammatory response in which the role of the IL17 and T helper-17 remain controversial. We have demonstrated that a DNA-based vaccination strategy targeting a PDA-associated antigen, namely alpha-enolase (ENO1), significantly prolongs survival in genetically engineered mice (GEM). We have also observed that the absence of IL17 strongly modified PDA microenvironment, improving CD8 T cell recruitment. Therefore, the aim of this study is to assess the role of IL17-targeting in combination with the DNA-vaccination. GEM crossed with IL17 knockout mice (GEM/IL17−/−) were vaccinated and monitored weekly to obtain a Kaplan-Meier survival curve. GEM vaccinated in absence of IL17 showed higher level of anti-ENO1 antibodies and IFNγ-secreting ENO1-specific T cells. The analysis of the tumor infiltrating cells revealed an increase of antigen-presenting cells and effector/memory T cells. To translate the experimental model into a clinical setting, mice orthotopically injected with PDA cells, were treated with anti-IL17 mAb in combination or not with ENO1-DNA vaccine. Only the combination elicited a significantly reduction in tumor growth that paralleled the induction of cytotoxic T cells not observed after ENO1-vaccine or anti-IL17 Ab alone. Overall, the depletion of IL17 opens new possibilities for combination to design more effective immunotherapeutic strategies to be associated with conventional one.

Protective effect and mechanism of IL-10 on renal ischemia–reperfusion injury

Interleukin (IL)-10, a cytokine with anti-inflammatory effects, is produced by blood cells and cells of various organs. Ischemia–reperfusion injury (IRI) is a systemic inflammatory disease caused by a systemic circulation of pro-inflammatory cytokines and chemokines produced from blood cells or organs damaged by ischemia. Apoptosis, a key event after IRI, is correlated with the degree of injury. Here we investigated the effects and mechanism of IL-10 in renal IRI. Compared to wild-type (WT) mice with a renal IRI, IL-10 knockout (IL-10 KO) mice with IRI demonstrated decreased renal function as represented by blood urea nitrogen and serum creatinine, upregulated early acute kidney injury (AKI) biomarkers such as kidney injury molecule-1 (Kim-1), increased mRNA expression of the pro-inflammatory cytokines IL-1β, IL-6, and IL-18 and a chemokine (regulated on activation, normal T cell expressed and secreted; RANTES), and increased expression of the pro-apoptosis factors Bax and cleaved caspase-3. When tubular epithelial cells (TECs) from IL-10 KO mice were put in a hypoxic state and added with recombinant IL-10, their expression of Bax decreased. Our findings demonstrated that IL-10 suppressed the production of pro-inflammatory cytokines, renal dysfunction, and the expression of pro-apoptosis factors after IRI.

Enhanced responsiveness of platelets to vicagrel in IL‐10‐deficient mice through STAT3‐dependent up‐regulation of the hydrolase arylacetamide deacetylase in the intestine

Vicagrel is a novel promising antiplatelet drug designed for overcoming clopidogrel resistance. There is limited evidence indicating that exogenous IL-10 suppresses CYP3A4 activity in healthy subjects and that IL-10 knockout (KO) mice exhibit increased clopidogrel bioactivation compared with wild-type (WT) mice. In this study, we sought to determine whether IL-10 could play an important role in the metabolism of and platelet response to vicagrel in mice. IL-10 KO and WT mice were administered vicagrel, then their plasma H4 (active metabolite of vicagrel) concentrations were determined by LC-MS/MS, and inhibition of ADP-induced whole-blood platelet aggregation by vicagrel was assessed with an aggregometer. The mRNA and protein levels of several relevant genes between IL-10 KO and WT mice were measured by qRT-PCR and Western blots, respectively. Intestinal Aadac protein levels were measured in IL-10 WT mice injected i.p. with vehicle control, Stattic, or BAY 11-7082. Compared with WT mice, IL-10 KO mice exhibited significantly increased plasma levels of H4 and enhanced platelet responses to vicagrel, as well as significantly higher mRNA and protein levels of arylacetamide deacetylase (Aadac) in the intestine. In WT mice, STAT3, not NF-κB, mediated Aadac expression in the intestine. IL-10 suppresses metabolic activation of vicagrel through down-regulation of Aadac in mouse intestine in a STAT3-dependent manner and, consequently, attenuates platelet responses to vicagrel, suggesting that the antiplatelet effect of vicagrel may be modulated by changes in plasma IL-10 levels in relevant clinical settings.

Interleukin-6 contributes to initiation of neuronal regeneration program in the remote dorsal root ganglia neurons after sciatic nerve injury.

To assess the potential role of IL-6 in sciatic nerve injury-induced activation of a pro-regenerative state in remote dorsal root ganglia (DRG) neurons, we compared protein levels of SCG-10 and activated STAT3, as well as axon regeneration in IL-6 knockout (IL-6ko) mice and their wild-type (WT) counterparts. Unilateral sciatic nerve compression and transection upregulated SCG-10 protein levels and activated STAT3 in DRG neurons not only in lumbar but also in cervical segments of WT mice. A pro-regenerative state induced by prior sciatic nerve lesion in cervical DRG neurons of WT mice was also shown by testing for axon regeneration in crushed ulnar nerve. DRG neurons from IL-6ko mice also displayed bilaterally increased levels of SCG-10 and STAT3 in both lumbar and cervical segments after sciatic nerve lesions. However, levels of SCG-10 protein in lumbar and cervical DRG of IL-6ko mice were significantly lower than those of their WT counterparts. Sciatic nerve injury induced a lower level of SCG-10 in cervical DRG of IL-6ko than WT mice, and this correlates with significantly shorter regeneration of axons distal to the crushed ulnar nerve. These results suggest that IL-6 contributes, at the very least, to initiation of the neuronal regeneration program in remote DRG neurons after unilateral sciatic nerve injury.

Interleukin 17A promotes diabetic kidney injury

The role of the pro-inflammatory cytokine IL-17 in the pathogenesis of numerous inflammatory disorders is well-documented, but conflicting results are reported for its role in diabetic nephropathy. Here we examined the role of IL-17 signalling in a model of streptozotocin-induced diabetic nephropathy through IL-17 knockout mice, administration of neutralising monoclonal anti-IL-17 antibody and in vitro examination of gene expression of renal tubular cells and podocytes under high glucose conditions with or without recombinant IL-17. IL-17 deficient mice were protected against progression of diabetic nephropathy, exhibiting reduced albuminuria, glomerular damage, macrophage accumulation and renal fibrosis at 12 weeks and 24 weeks. Administration of anti-IL-17 monoclonal antibody to diabetic wild-type mice was similarly protective. IL-17 deficiency also attenuated up-regulation of pro-inflammatory and pro-fibrotic genes including IL-6, TNF-α, CCL2, CXCL10 and TGF-β in diabetic kidneys. In vitro co-stimulation with recombinant IL-17 and high glucose were synergistic in increasing the expression of pro-inflammatory genes in both cultured renal tubular cells and podocytes. We conclude that absence of IL-17 signalling is protective against streptozotocin-induced diabetic nephropathy, thus implying a pro-inflammatory role of IL-17 in its pathogenesis. Targeting the IL-17 axis may represent a novel therapeutic approach in the treatment of this disorder.

Immune Cell Infiltration into the Eye Is Controlled by IL-10 in Recoverin-Induced Autoimmune Retinopathy.

Autoimmune retinopathy (AIR) is a treatable condition that manifests in acute and progressive vision loss in patients. It has recently been determined that AIR is associated with an imbalance of TH1 versus regulatory T cell immunity toward the retinal protein, recoverin. This study describes a new murine model to understand the immunopathology of AIR and its association with T cell responses toward recoverin. Immunization of C57BL/6 mice with recoverin resulted in ocular inflammation including infiltration of CD4+ and CD8+ T lymphocytes, B cells, and CD11b+Ly6C+ inflammatory monocytes in the eyes. Production of IFN-γ and IL-17 from T cells was exacerbated in IL-10 knockout (KO) mice and kinetics of disease development was accelerated. Infiltration of T cells and inflammatory monocytes into the eyes dramatically increased in recoverin-immunized IL-10 KO mice. An immunodominant peptide of recoverin, AG-16, was capable of inducing disease in IL-10 KO mice and resulted in expansion of AG-16 tetramer-specific CD4+ T cells in lymphoid organs and eyes. Adoptive transfer of recoverin-stimulated cells into naive mice was sufficient to induce AIR, and immunization of B cell-deficient mice led to a milder form of the disease. This model supports the hypothesis that recoverin-specific T cell responses are major drivers of AIR pathogenesis and that IL-10 is an important factor in protection.

Interleukin-18 exacerbates skin inflammation and affects microabscesses and scale formation in a mouse model of imiquimod-induced psoriasis.

Background:As a potent pro-inflammatory cytokine of the interleukin (IL)-1 family, IL-18 was elevated in early active and progressive plaque-type psoriatic lesions and that serum or plasma levels of IL-18 correlated with the Psoriasis Area and Severity Index (PASI). Although results from previous studies have established that IL-18 may aggravate psoriatic inflammation, the mechanisms of this process remain unknown. In this study, IL-18 knock out (KO) mice and wild-type (WT) mice were used to investigate the effects of IL-18 within a mouse model of psoriasis.Methods:WT and IL-18 KO mice were divided into four groups, including imiquimod (IMQ)-treated IL-18 KO group (n = 11) and WT group (n = 13) as well as their respectively gene-matched control mice (receiving vaseline; n = 12). PASI scores were used to evaluate psoriatic lesions in IMQ-treated mice. Pathological features and dermal cellular infiltration were investigated by hematoxylin and eosin staining. The levels of psoriasis-related cytokines including IL-23, IL-17, IL-12, IL-1β, IFNγ, IL-15, IL-27, and IL-4 were tested by real-time polymerase chain reaction (PCR). The protein level of IL-1β, IL-27, CXCL1, and Ly6 g were investigated by immunohistochemistry (IHC).Results:Acanthosis (98.46 ± 14.12 vs. 222.68 ± 71.10 μm, P < 0.01) and dermal cell infiltration (572.25 ± 47.45 vs. 762.47 ± 59.59 cells/field, P < 0.01) were significantly milder in IMQ-induced IL-18 KO mice compared with that in WT mice. IMQ-induced IL-18 KO mice manifested larger areas of Munro microabscesses (11,467.83 ± 5112.09 vs. 4093.19 ± 2591.88 μm2, P < 0.01) and scales (100,935.24 ± 41,167.77 vs. 41,604.41 ± 14,184.10 μm2, P < 0.01) as compared with WT mice. In skin lesions of IL-18 KO mice, the expressions of IL-1β, IL-4, and IL-27 were all significantly upregulated but IL-17 was decreased. Histologically, strong positive signals of Ly6g were observed within the epidermis of IL-18 KO mice but expressions of CXCL1 were decreased.Conclusions:IL-18 may exacerbate prominent inflammation and influence pathological features in IMQ-induced mouse model of psoriasis. IL-18 may upregulate pro-inflammatory cytokines and reduce protective cytokines, thus aggravating psoriatic inflammation. In addition, IL-18 may be involved in the formation of Munro microabscesses and scales.

IL-6/Smad2 signaling mediates acute kidney injury and regeneration in a murine model of neonatal hyperoxia.

Prematurity is linked to incomplete nephrogenesis and risk of chronic kidney diseases (CKDs). Oxygen is life-saving in that context but induces injury in numerous organs. Here, we studied the structural and functional impact of hyperoxia on renal injury and its IL-6 dependency. Newborn wild-type (WT) and IL-6 knockout (IL-6-/-) mice were exposed to 85% O2 for 28 d, followed by room air until postnatal d (P) 70. Controls were in room air throughout life. At P28, hyperoxia reduced estimated kidney cortex area (KCA) in WT; at P70, KCA was greater, number of glomeruli was fewer, fractional potassium excretion was higher, and glomerular filtration rate was slightly lower than in controls. IL-6-/- mice were protected from these changes after hyperoxia. Mechanistically, the acute renal injury phase (P28) showed in WT but not in IL-6-/- mice an activation of IL-6 (signal transducer and activator of transcription 3) and TGF-β [mothers against decapentaplegic homolog (Smad)2] signaling, increased inflammatory markers, disrupted mitochondrial biogenesis, and reduced tubular proliferation. Regenerative phase at P70 was characterized by tubular proliferation in WT but not in IL-6-/- mice. These data demonstrate that hyperoxia increases the risk of CKD through a novel IL-6-Smad2 axis. The amenability of these pathways to pharmacological approaches may offer new avenues to protect premature infants from CKD.-Mohr, J., Voggel, J., Vohlen, C., Dinger, K., Dafinger, C., Fink, G., Göbel, H., Liebau, M. C., Dötsch, J., Alejandre Alcazar, M. A. IL-6/Smad2 signaling mediates acute kidney injury and regeneration in a murine model of neonatal hyperoxia.

Abstract 55: Interleukin 4 Improves White Matter Repair After Stroke By Promoting Oligodendrocyte Differentiation

Introduction: White matter (WM) injury is an important cause of long-term deficits after stroke. This study explores a novel action of IL-4 on promoting WM integrity after stroke and elucidates the underlying mechanisms. Hypothesis: IL-4 promotes WM repair and enhances long-term neurological recovery after ischemic stroke by targeting both OPC differentiation and microglial polarization. Methods: Cerebral ischemia was induced by transient (60 min) middle cerebral artery occlusion (tMCAO) in wild type, IL-4 knockout and oligodendrocyte precursor cell (OPC)-specific PPARγ KO mice. The animals were then randomly assigned to vehicle or IL-4 treatment. IL-4 was delivered intranasally 6h after stroke and repeated every day at 1-7d, 14d, 21d, and 28d after tMCAO. Sensorimotor deficits were determined by adhesive removal, rotarod test and corner test. Cognitive deficits were determined by water maze test. A combined approach including cell-specific depletion, immunohistology staining, electron microscopy (EM), diffusion tensor imaging (DTI) and electrophysiology, was applied to evaluate WM integrity. Results: IL-4 knockout (KO) mice exhibited worse sensorimotor deficits and deteriorated WM injury over 14 days after tMCAO. In contrast, intraventricular infusion of IL-4 beginning 6h after MCAO attenuated long-term sensorimotor and cognitive deficits and improved WM integrity, as revealed by immunohistology staining, EM, DTI and electrophysiology. In addition to promoting microglia/macrophage M2 polarization, IL-4 treatment enhanced OPC differentiation in the ischemic brain and in organotypic cultures. Interestingly, IL-4-afforded oligodendrogenesis was still potent in microglia depleted (with PLX5662) mice. In vitro study confirmed that IL-4 directly induced the differentiation of primary OPCs into mature oligodendrocytes. This effect was mediated through the activation of PPARγ. The therapeutic efficacy of IL-4 on OPC differentiation and white matter protection was significantly reduced in OPC-specific PPARγ KO mice. Conclusions: IL-4 promotes oligodendrogenesis and white matter repair in an PPARγ-dependent manner. IL-4 may represent a novel recovery-enhancing strategy to promote functional recovery after ischemic stroke.

Autophagy Induction by Rapamycin Ameliorates Experimental Colitis and Improves Intestinal Epithelial Barrier Function in IL-10 Knockout Mice

Autophagy Induction by Rapamycin Ameliorates Experimental Colitis and Improves Intestinal Epithelial Barrier Function in IL-10 Knockout Mice

Caffeine-stimulated muscle IL-6 mediates alleviation of non-alcoholic fatty liver disease

Caffeine intake is associated with a reduced risk developing non-alcoholic fatty liver disease (NAFLD), but the underlying molecular mechanisms remain to be fully elucidated. We report here that caffeine markedly improved high fat diet-induced NAFLD in mice resulting in a 10-fold increase in circulating IL-6 levels, leading to STAT3 activation in the liver. Interestingly, the expression of IL-6 mRNA was not increased in the liver, but increased substantially in the muscles of caffeine-treated mice. Caffeine was found to stimulate IL-6 production in cultured myotubes but not in hepatocytes, adipocytes, or macrophages. The inhibition of p38/MAPK abrogated caffeine-induced IL-6 production in muscle cells. Caffeine failed to improve NAFLD in IL-6 and hepatocyte-specific STAT3 knockout mice, indicating that the IL-6/STAT3 pathway is vital for the hepatoprotective effects of caffeine in NAFLD. The possibility that IL-6/STAT3-mediated hepatic autophagosome induction and hepatocytic oxygen consumption are involved in the anti-NAFLD effects of caffeine cannot be excluded, based on the findings presented here. Our results reveal that caffeine ameliorates NAFLD via crosstalk between muscle IL-6 production and liver STAT3 activation.