IL-10 Knockout Mice Research Articles

Ovariectomy-induced bone loss in TNFα and IL6 gene knockout mice is regulated by different mechanisms.

We examined the effects of tumor necrosis factor-α (TNFα) and interleukin-6 (IL6) gene knockout in preserving the bone loss induced by ovariectomy (OVX) and the mechanisms involved in bone metabolism. Twenty female wild-type (WT), TNFα-knockout (TNFα-/-) or IL6-knockout (IL6-/-) mice aged 12 weeks were sham-operated (SHAM) or subjected to OVX and killed after 4 weeks. Bone mass and skeletal microarchitecture were determined using micro-CT. Bone marrow stromal cells (BMSCs) from all three groups (WT, TNFα-/- and IL6-/-) were induced to differentiate into osteoblasts or osteoclasts and treated with 17-β-estradiol. Bone metabolism was assessed by histological analysis, serum analyses and qRT-PCR. OVX successfully induced a high turnover in all mice, but a repair effect was observed in TNFα-/- and IL6-/- mice. The ratio of femoral trabecular bone volume to tissue volume, trabecular number and trabecular thickness were significantly decreased in WT mice subjected to OVX, but increased in TNFα-/- mice (1.62, 1.34, 0.27-fold respectively; P < 0.01) and IL6-/- mice (1.34, 0.80, 0.22-fold respectively; P < 0.01). Furthermore, we observed a 29.6% increase in the trabecular number in TNFα-/- mice when compared to the IL6-/- mice. Both, TNFα-/- and IL6-/- BMSCs exhibited decreased numbers of TRAP-positive cells and an increase in ALP-positive cells, with or without E2 treatment (P < 0.05). While the knockout of TNFα or IL6 significantly upregulated mRNA expressions of osteoblast-related genes (Runx2 and Col1a1) and downregulated osteoclast-related mRNA for TRAP, MMP9 and CTSK in vivo and in vitro, TNFα knockout appeared to have roles beyond IL6 knockout in upregulating Col1a1 mRNA expression and downregulating mRNA expressions of WNT-related genes (DKK1 and Sost) and TNF-related activation-induced genes (TRAF6). TNFα seemed to be more potentially invasive in inhibiting bone formation and enhancing TRAF6-mediated osteoclastogenesis than IL6, implying that the regulatory mechanisms of TNFα and IL6 in bone metabolism may be different.

Exercise Protects Against Olanzapine-Induced Hyperglycemia in Male C57BL/6J Mice

Olanzapine is a widely prescribed antipsychotic drug. While effective in reducing psychoses, treatment with olanzapine causes rapid increases in blood glucose. We wanted to determine if a single bout of exercise, immediately prior to treatment, would attenuate the olanzapine-induced rise in blood glucose and if this occurred in an IL-6 dependent manner. We found that exhaustive, but not moderate exercise, immediately prior to treatment, prevented olanzapine-induced hyperglycemia and this occurred in parallel with increases in serum IL-6. To determine if IL-6 was involved in the mechanisms through which exhaustive exercise protected against olanzapine-induced hyperglycemia several additional experiments were completed. Treatment with IL-6 (3 ng/g bw, IP) alone did not protect against olanzapine-induced increases in blood glucose. The protective effects of exhaustive exercise against olanzapine-induced increases in blood glucose were intact in whole body IL-6 knockout mice. Similarly, treating mice with an IL-6 neutralizing antibody prior to exhaustive exercise did not negate the protective effect of exercise against olanzapine-induced hyperglycemia. Our findings provide evidence that a single bout of exhaustive exercise protects against acute olanzapine-induced hyperglycemia and that IL-6 is neither sufficient, nor required for exercise to protect against increases in blood glucose with olanzapine treatment.

PM2.5-induced oxidative stress increases intercellular adhesion molecule-1 expression in lung epithelial cells through the IL-6/AKT/STAT3/NF-\u03baB-dependent pathway

BackgroundEpidemiological studies have shown that ambient air pollution is closely associated with increased respiratory inflammation and decreased lung function. Particulate matters (PMs) are major components of air pollution that damages lung cells. However, the mechanisms remain to be elucidated. This study examines the effects of PMs on intercellular adhesion molecule-1 (ICAM-1) expression and the related mechanisms in vitro and in vivo.ResultThe cytotoxicity, reactive oxygen species (ROS) generation, and monocyte adherence to A549 cells were more severely affected by treatment with O-PMs (organic solvent-extractable fraction of SRM1649b) than with W-PMs (water-soluble fraction of SRM1649b). We observed a significant increase in ICAM-1 expression by O-PMs, but not W-PMs. O-PMs also induced the phosphorylation of AKT, p65, and STAT3. Pretreating A549 cells with N-acetyl cysteine (NAC), an antioxidant, attenuated O-PMs-induced ROS generation, the phosphorylation of the mentioned kinases, and the expression of ICAM-1. Furthermore, an AKT inhibitor (LY294002), NF-κB inhibitor (BAY11–7082), and STAT3 inhibitor (Stattic) significantly down-regulated O-PMs-induced ICAM-1 expression as well as the adhesion of U937 cells to epithelial cells. Interleukin-6 (IL-6) was the most significantly changed cytokine in O-PMs-treated A549 cells according to the analysis of the cytokine antibody array. The IL-6 receptor inhibitor tocilizumab (TCZ) and small interfering RNA for IL-6 significantly reduced ICAM-1 secretion and expression as well as the reduction of the AKT, p65, and STAT3 phosphorylation in O-PMs-treated A549 cells. In addition, the intratracheal instillation of PMs significantly increased the levels of the ICAM-1 and IL-6 in lung tissues and plasma in WT mice, but not in IL-6 knockout mice. Pre-administration of NAC attenuated those PMs-induced adverse effects in WT mice. Furthermore, patients with chronic obstructive pulmonary disease (COPD) had higher plasma levels of ICAM-1 and IL-6 compared to healthy subjects.ConclusionThese results suggest that PMs increase ICAM-1 expression in pulmonary epithelial cells in vitro and in vivo through the IL-6/AKT/STAT3/NF-κB signaling pathway.

Deficiency of IL-18 Aggravates Esophageal Carcinoma Through Inhibiting IFN-γ Production by CD8+T Cells and NK Cells.

To investigate the potential role of interleukin-18 (IL-18) in immunomodulation during tumorigenesis of esophageal carcinoma and elucidate the underlying molecular mechanism, we employed IL-18 knockout mice for this purpose. Carcinogen 4-nitroquinoline 1-oxide (4NQO) was administrated in drinking water to induce occurrence of esophageal squamous cell carcinoma (ESCC). T cell activation as indicated by the surface CD molecules was analyzed with flow cytometry. The serous content of interferon-γ (IFN-γ) along with other cytokines was determined by inflammatory human cytokine cytometric bead array. The cytotoxicity assay was performed by co-culture of tumor cells with immune cells and relative cell viability was determined by lactate dehydrogenase (LDH) assay. Apoptotic cells were stained with Annexin-V/propidium iodide (PI) and analyzed by flow cytometry. Cell proliferation was measured with Cell Counting Kit-8 (CCK-8) assay. Our data demonstrated that deficiency of IL-18 promoted the progression and development of 4NQO-induced ESCC. Loss of IL-18 suppressed the activation of T cells in the esophagus. Deficiency of IL-18 inhibited the IFN-γ production by CD8+ T cells and natural killer (NK) cells. Absence of IL-18 inhibited the cytotoxicity of CD8+ T cells and NK cell in vitro. Moreover, deficiency of IL-18 promoted the apoptosis of CD8+ T cells and inhibited the proliferation of CD8+ T cells in vitro. Our data elucidated the immunomodulatory role of IL-18 during tumorigenesis of ESCC, whose deficiency compromised antitumor immunity and contributed to immune escape of esophageal carcinoma. Our results also indicated the therapeutic potential of exogenous IL-18 against ESCC, which warrants further investigations.

Skeletal muscle IL-6 regulates muscle substrate utilization and adipose tissue metabolism during recovery from an acute bout of exercise.

An acute bout of exercise imposes a major challenge on whole-body metabolism and metabolic adjustments are needed in multiple tissues during recovery to reestablish metabolic homeostasis. It is currently unresolved how this regulation is orchestrated between tissues. This study was undertaken to clarify the role of skeletal muscle derived interleukin 6 (IL-6) in the coordination of the metabolic responses during recovery from acute exercise. Skeletal muscle specific IL-6 knockout (IL-6 MKO) and littermate Control mice were rested or ran on a treadmill for 2h. Plasma, skeletal muscle, liver and adipose tissue were obtained after 6 and 10h of recovery. Non-exercised IL-6 MKO mice had higher plasma lactate and lower plasma non-esterified fatty acids than Controls. The activity of pyruvate dehydrogenase in the active form was, in skeletal muscle, higher in IL-6 MKO mice than Controls in non-exercised mice and 6h after exercise. IL-6 MKO mice had lower glucose transporter 4 protein content in inguinal adipose tissue (WAT) than Control in non-exercised mice and 10h after treadmill running. Epididymal WAT hormone sensitive lipase phosphorylation and inguinal WAT mitogen activated kinase P38 phosphorylation were higher in IL-6 MKO than Control mice 6h after exercise. These findings indicate that skeletal muscle IL-6 may play an important role in the regulation of substrate utilization in skeletal muscle, basal and exercise-induced adaptations in adipose tissue glucose uptake and lipolysis during recovery from exercise. Together this indicates that skeletal muscle IL-6 contributes to reestablishing metabolic homeostasis during recovery from exercise by regulating WAT and skeletal muscle metabolism.

Interleukin-6 Affects Aging-Related Changes of the PPARα-PGC-1α Axis in the Myocardium.

Aging is related to gradual increase of interleukin 6 (IL-6) plasma level that affects peroxisome proliferator-activated receptor (PPAR) expression. We evaluated age-related changes in cardiac expression of PPARα, its coactivator PGC-1α, and selected downstream proteins in mice with systemic IL-6 knockout (IL6KO). Male C57BL6/J wild-type (WT) and IL6KO mice were used at the age of 16-20 weeks (young) and 24-30 months (senescent). Echocardiography and electron microscopy were applied to assess the function and ultrastructure of the heart. Western blotting and quantitative real-time PCR were used to estimate protein and mRNA levels of selected genes. PPARα expression in the myocardium of young IL6KO animals is lower and remains unchanged with aging, whereas in WT mice it declines with age and in both senescent groups it is equal. We observed aging-related upregulation of PGC-1α and less pronounced decline of Sirt3 in IL6KO animals; the level of cytochrome C was significantly decreased in IL6KO group only, suggesting disturbed mitochondrial function, which was not sufficient to evoke obvious changes in cardiac performance and function assessed by echocardiography. IL-6 and aging are involved in regulation of PPARα and PGC-1α expression and may influence the mitochondrial function.

The axis IL-10/claudin-10 is implicated in the modulation of aggressiveness of melanoma cells by B-1 lymphocytes.

B-1 lymphocytes are known to increase the metastatic potential of B16F10 melanoma cells via the extracellular signal-regulated kinase (ERK) pathway. Since IL-10 is associated with B-1 cells performance, we hypothesized that IL-10 could be implicated in the progression of melanoma. In the present work, we found that the C57BL/6 mice, inoculated with B16F10 cells that were co-cultivated with B-1 lymphocytes from IL-10 knockout mice, developed fewer metastatic nodules than the ones which were injected with the melanoma cells that were cultivated in the presence of wild-type B-1 cells. The impairment of metastatic potential of the B16F10 cells was correlated with low activation of the ERK signaling pathway, supporting the idea that the production of IL-10 by B-1 cells influences the behavior of the tumor. A microarray analysis of the B-1 lymphocytes revealed that IL-10 deficiency is associated with down-regulation of the genes that code for claudin-10, a protein that is involved in cell-to-cell contact and that has been linked to lung adenocarcinoma. In order to determine the impact of claudin-10 in the cross-talk between B-1 lymphocytes and the B16F10 tumor cells, we took advantage of small interfering RNA. The silencing of claudin-10 gene in B-1 lymphocytes inhibited activation of the ERK pathway and abrogated the B-1-induced aggressive behavior of the B16F10 cells. Thus, our findings suggest that the axis IL-10/claudin-10 is a promising target for the development of therapeutic agents against aggressive melanoma.

Interleukin-4 Supports the Suppressive Immune Responses Elicited by Regulatory T Cells.

Interleukin-4 (IL-4) has been considered as one of the tolerogenic cytokines in many autoimmune animal models and clinical settings. Despite its role in antagonizing pathogenic Th1 responses, little is known about whether IL-4 possesses functions that affect regulatory T cells (Tregs). Tregs are specialized cells responsible for the maintenance of peripheral tolerance through their immune modulatory capabilities. Interestingly, it has been suggested that IL-4 supplement at a high concentration protects responder T cells (Tresps) from Treg-mediated immune suppression. In addition, such supplement also impedes TGF-β-induced Treg differentiation in vitro. However, these phenomena may contradict the tolerogenic role of IL-4, and the effects of IL-4 on Tregs are therefore needed to be further elucidated. In this study, we utilized IL-4 knockout (KO) mice to validate the role of IL-4 on Treg-mediated immune suppression. Although IL-4 KO and control animals harbor similar frequencies of Tregs, Tregs from IL-4 KO mice weakly suppressed autologous Tresp activation. In addition, IL-4 deprivation impaired the ability of Tregs to modulate immune response, whereas IL-4 supplementation reinforced IL-4 KO Tregs in their function in suppressing Tresps. Finally, the presence of IL-4 was associated with increased cell survival and granzyme expression of Tregs. These results suggest the essential role of IL-4 in supporting Treg-mediated immune suppression, which may benefit the development of therapeutic strategies for autoimmune diseases.

IL-10 targets myofibroblasts and dampens cardiac fibrosis

In a recent issue of Circulation, Verma et al. (1) uncover a new interleukin-10 (IL-10)-dependent mechanism inhibiting fibrosis in pressure overloaded mouse myocardium. In this study, pressure overload was achieved in wild-type (WT) and IL-10 knockout (IL-10KO) mice by transverse aortic constriction (TAC), a widely used experimental mouse model that creates pressure overload-induced left ventricular hypertrophy, leading to heart failure. IL-10KO mice displayed more bone marrow (BM)-derived fibroblast progenitor cell mobilization and homing to the heart. Chimeric IL-10KO mice with WT BM were protected against TAC-induced cardiac fibrosis and presented improved heart function. Verma et al. (1) found out that IL-10 inhibits homing and trans-differentiation of BM-derived fibroblast progenitor cells (FPC) in myofibroblasts, major effector cells of cardiac fibrosis. In vitro experiments performed on FPC isolated from mouse BM (prominin 1+ CD45+ cells) indicated that IL-10 could suppress the expression of the fibrosis-associated miR-21 in response to transforming growth factor-β (TGF-)β.

Impairment of Hepcidin Upregulation by Lipopolysaccharide in the Interleukin-6 Knockout Mouse Brain.

To find out whether the Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is involved in the expression of hepcidin in the mouse brain in vivo, we investigated the phosphorylation of STAT3, as well as the expression of hepcidin mRNA, ferroportin 1 (Fpn1) and ferritin light chain (Ft-L) proteins in the cortex and hippocampus of LPS-treated wild type (IL-6+/+) and IL-6 knockout (IL-6-/-) mice. We demonstrated that IL-6 knockout could significantly reduce the response of hepcidin mRNA, phospho-STAT3, Fpn1 and Ft-L protein expression to LPS treatment, in both the cortex and hippocampus of mice. Also, Stattic, an inhibitor of STAT3, significantly reduced the expression of phospho-STAT3 and hepcidin mRNA in the cortex and hippocampus of the LPS-treated wild type mice. These findings provide in vivo evidence for the involvement of the IL-6/STAT3 signaling pathway in the expression of hepcidin.

Commensal-infected macrophages induce dedifferentiation and reprogramming of epithelial cells during colorectal carcinogenesis.

The colonic microbiome contributes to the initiation of colorectal cancer through poorly characterized mechanisms. We have shown that commensal-polarized macrophages induce gene mutation, chromosomal instability, and endogenous transformation through microbiome-induced bystander effects (MIBE). In this study we show that MIBE activates Wnt/β-catenin signaling and pluripotent transcription factors associated with dedifferentiation, reprogramming, and the development of colorectal cancer stem cells (CSCs). Exposure of murine primary colon epithelial cells (YAMC) to Enterococcus faecalis-infected macrophages increased Wnt3α expression while suppressing Wnt inhibitor factor 1 (Wif1). Wnt/β-catenin activation was confirmed by increased active β-catenin and Tcf4. in vivo, active β-catenin was evident in colon biopsies from E. faecalis-colonized Il10 knockout mice compared to sham-colonized mice. This effect was mediated, in part, by 4-hydroxy-2-nonenal and tumor necrosis factor α. MIBE also activated pluripotent transcription factors c-Myc, Klf4, Oct4, and Sox2 in YAMC cells and colons from E. faecalis-colonized Il10 knockout mice. These transcription factors are associated with cellular reprogramming, dedifferentiation, and induction of colorectal CSC progenitors. In support of this was an increase in the expression of Dclk1 and CD44, two colorectal CSC markers, in YAMC cells that were exposed to MIBE. Finally, compared to normal colon biopsies and hyperplastic polyps, DCLK1 expression increased in human tubular adenomas and invasive colorectal cancers. Blocking β-catenin/TCF4 signaling using FH535 and CTNNB1-specific small interfering RNA decreased DCLK1 expression in HCT116 human colon cancer cells. These findings provide mechanism for microbiome-induced colorectal cancer and identify new potential targets for colorectal cancer prevention.

Interleukin-6 deficiency attenuates angiotensin II-induced cardiac pathogenesis with increased myocyte hypertrophy

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 μg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation.

Characterization of Growth Hormone Resistance in Experimental and Ulcerative Colitis.

Growth hormone (GH) resistance may develop as a consequence of inflammation during conditions such as inflammatory bowel disease, encompassing ulcerative colitis (UC). However, the specific role of the GH–insulin growth factor (IGF)-1-axis and/or the functional consequences of GH resistance in this condition are unclear. In situ hybridization targeting the GH receptor (GHR) and relevant transcriptional analyses were performed in patients with UC and in IL-10 knock-out mice with piroxicam accelerated colitis (PAC). Using cultured primary epithelial cells, the effects of inflammation on the molecular mechanisms governing GH resistance was verified. Also, the therapeutic potential of GH on mucosal healing was tested in the PAC model. Inflammation induced intestinal GH resistance in UC and experimental colitis by down-regulating GHR expression and up-regulating suppressor of cytokine signalling (SOCS) proteins. These effects are driven by pro-inflammatory mediators (tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6) as confirmed using primary epithelial cells. Treatment of experimental colitis with GH increased IGF-1 and body weight of the mice, but had no effects on colonic inflammation or mucosal healing. The high transcriptional similarity between UC and experimental colitis accentuates the formation of intestinal GH resistance during inflammation. Inflammation-induced GH resistance not only impairs general growth but induces a state of local resistance, which potentially impairs the actions of GH on mucosal healing during colitis when using long-acting GH therapy.

Ginsenoside Re Protects Trimethyltin-Induced Neurotoxicity via Activation of IL-6-Mediated Phosphoinositol 3-Kinase/Akt Signaling in Mice.

Ginseng (Panax ginseng), an herbal medicine, has been used to prevent neurodegenerative disorders. Ginsenosides (e.g., Re, Rb1, or Rg1) were obtained from Korean mountain cultivated ginseng. The anticonvulsant activity of ginsenoside Re (20mg/kg/day × 3) against trimethyltin (TMT) insult was the most pronounced out of ginsenosides (e.g., Re, Rb1, and Rg1). Re itself did not significantly alter tumor necrosis factor-α (TNF-α), interferon-ϒ (IFN-ϒ), and interleukin-1β (IL-1β) expression, however, it significantly increases the interleukin-6 (IL-6) expression. In addition, Re attenuated the TMT-induced decreases in IL-6 protein level. Therefore, IL-6 knockout (-/-) mice were employed to investigate whether Re requires IL-6-dependent neuroprotective activity against TMT toxicity. Re significantly attenuated TMT-induced lipid peroxidation, protein peroxidation, and reactive oxygen species in the hippocampus. Re-mediated antioxidant effects were more pronounced in IL-6 (-/-) mice than in WT mice. Consistently, TMT-induced increase in c-Fos-immunoreactivity (c-Fos-IR), TUNEL-positive cells, and nuclear chromatin clumping in the dentate gyrus of the hippocampus were significantly attenuated by Re. Furthermore, Re attenuated TMT-induced proapoptotic changes. Protective potentials by Re were comparable to those by recombinant IL-6 protein (rIL-6) against TMT-insult in IL-6 (-/-) mice. Moreover, treatment with a phosphoinositol 3-kinase (PI3K) inhibitor, LY294002 (1.6µg, i.c.v) counteracted the protective potential mediated by Re or rIL-6 against TMT insult. The results suggest that ginsenoside Re requires IL-6-dependent PI3K/Akt signaling for its protective potential against TMT-induced neurotoxicity.

Interleukin-6 deficiency facilitates myocardial dysfunction during high fat diet-induced obesity by promoting lipotoxicity and inflammation

ObjectiveObesity is associated with metabolic disorder and chronic inflammation that plays a crucial role in cardiovascular diseases. IL-6 is involved in regulating obesity-related lipid metabolism and inflammation. In this study, we sought to determine the role of IL-6 in high-fat diet (HFD)-induced cardiomyopathy and explore the signaling pathway. MethodsFemale, 5-week-old IL-6 knockout (KO) and littermate mice were fed a normal diet (ND, 10% fat) or HFD (45% fat) for 14 weeks. At the end of treatment, cardiac function was assessed by echocardiography. Adipose tissues and plasma were collected for further measurement. Immunohistology of CD68 was performed to detect inflammation in the heart. Masson's trichrome staining and Oil Red O staining was applied to evaluated cardiac fibrosis and lipid accumulation. Real-time PCR and Western immunoblotting analyses on heart tissue were used to explore the underlying mechanism. ResultsIL-6 KO mice displayed increased insulin resistance compared to WT mice at baseline. When fed HFD, IL-6 KO mice showed decreased gains in body weight and fat mass, increased insulin resistance relative to IL-6 KO mice feed ND. Furthermore, IL-6 KO mice developed cardiac dysfunction during HFD-induced obesity. Histological analysis suggested increased lipid accumulation, fibrosis and inflammation without affecting cardiac morphology during HFD treatment in the heart of IL-6 KO mice. Finally, IL-6 deficiency increased the phosphorylation of AMPK and ACC in the heart during HFD-induced obesity. ConclusionOur results suggest that IL-6 contributes to limit lipid metabolic disorder, cardiac hypertrophy, fibrosis, inflammation and myocardium lipotoxicity during HFD-induced obesity.

Ascaris suum infection modulates inflammation: Implication of CD4+ CD25high Foxp3+ T cells and IL-10.

Helminth infections have the ability to modulate host's immune response through mechanisms that allow the chronic persistence of the worms in the host. Here, we investigated the mechanisms involved on the suppressive effect of Ascaris suum infection using a murine experimental model of LPS-induced inflammation. We found that infection with A.suum markedly inhibited leucocyte influx induced by LPS into air pouches, suppressed secretion of pro-inflammatory cytokines (IL-1β, TNF-α and IL-6) and induced high levels of IL-10 and TGF-β. Augmented frequency of CD4+ CD25high Foxp3+ T cells was observed in the mesenteric lymph nodes of infected mice. Adoptive transfer of purified CD4+ CD25+ T cells to recipient uninfected mice demonstrated that these cells were able to induce a suppressive effect in the LPS-induced inflammation in air pouch model. In addition, adoptive transfer of CD4+ CD25+ T cells derived from IL-10 knockout mice suggests that this suppressive effect of A.suum infection involves IL-10 cytokine. In conclusion, our results demonstrated that A.suum experimental infection was capable of suppressing LPS-induced inflammation by mechanisms, which seem to be dependent on responses of CD4+ CD25+ T cells and secretion of IL-10 cytokine.

An ALPHA7 Nicotinic Acetylcholine Receptor Agonist (GTS-21) Promotes C2C12 Myonuclear Accretion in Association with Release of Interleukin-6 (IL-6) and Improves Survival in Burned Mice.

The role of interleukin-6 (IL-6) in physiological processes and disease is poorly understood. The hypothesis tested in this study was that selective alpha7 acetylcholine receptor (α7AChR) agonist, GTS-21, releases IL-6 in association with myonuclear accretion and enhances insulin signaling in muscle cells, and improves survival of burn injured (BI) mice. The in vitro effects of GTS-21 were determined in C2C12 myoblasts and 7-day differentiated myotubes (myotubes). The in vivo effects of GTS-21 were tested in BI wild-type (WT) and IL-6 knockout (IL6KO) mice. GTS-21 dose-dependently (0 μM, 100 μM, and 200 μM) significantly increased IL-6 levels in myoblasts and myotubes at 6 and 9 h. GTS-21-induced IL-6 release in myotubes was attenuated by methyllycaconitine (α7AChR antagonist), and by Stat-3 or Stat-5 inhibitors. GTS-21 increased MyoD and Pax7 protein expression, myonuclear accretion, and insulin-induced phosphorylation of Akt, GSK-3β, and Glut4 in myotubes. The glucose levels of burned IL6KO mice receiving GTS-21 decreased significantly compared with sham-burn IL6KO mice. Superimposition of BI on IL6KO mice caused 100% mortality; GTS-21 reduced mortality to 75% in the IL6KO mice. The 75% mortality in burned WT mice was reduced to 0% with GTS-21. The in vitro findings suggest that GTS-21-induced IL-6 release from muscle is mediated via α7AChRs upstream of Stat-3 and -5 pathways and is associated with myonuclear accretion, possibly via MyoD and Pax7 expression. GTS-21 in vivo improves survival in burned WT mice and IL6KO mice, suggesting a potential therapeutic application of α7AChR agonists in the treatment of BI.

Interleukin-6 Plays a Detrimental Role in LV Remodeling after an Acute Myocardial Infarction

Introduction: Interleukin-6 (IL-6), a proinflammatory cytokine, has been implicated in various cardiovascular diseases, including myocardial ischemic injury. Although its expression has been shown to rise in the aftermath of an acute myocardial infarction (MI), its exact role in left ventricular (LV) remodeling and pathogenesis of ischemic cardiomyopathy remains unclear. Hypothesis: We hypothesized that the absence of IL-6 will have a favorable impact on LV remodeling after a reperfused MI. Methods: To simulate a human myocardial infarction, we used a model of reperfused MI because most MIs in humans are followed by spontaneous or interventional reperfusion. Age-matched male C57BL/6 control mice and IL-6 knockout (KO) mice were subjected to a 30-min occlusion of the LAD coronary artery followed by reperfusion. Cardiac structure and function were serially assessed by echocardiography at baseline (4 d before MI), at 48 h and 35 d after MI. Mice were sacrificed after 35 days for histological and biochemical assessments. Results: At 48 h post-MI, ejection fraction (EF) was similarly and significantly reduced in both groups compared with baseline, indicating that the ischemic injury was similar in both groups. However, 35 d after MI, IL-6 KO mice exhibited significantly greater EF compared with control mice (49.5 ± 2.2% in IL-6 KO mice vs 42.6 ± 1.3% in control mice). IL-6 KO mice had significantly smaller LV mass and cardiomyocyte size. Moreover, LV end-diastolic volume was significantly smaller in IL-6 KO mice, indicating superior remodeling. Conclusions: Genetic deletion of IL-6 ameliorates LV remodeling and systolic dysfunction after a reperfused myocardial infarction, indicating a deleterious role of IL-6 in LV remodeling after acute myocardial ischemia/reperfusion injury. Modulation of IL-6 signaling may therefore have therapeutic potential for patients after MI at risk for adverse remodeling and development of heart failure.

The role of peripheral interleukin-6 in the development of acute seizures following virus encephalitis.

Seizure disorders are often associated with infectious etiologies. Infection, via the intracerebral (i.c.) route, of C57BL/6J mice with the Daniels (DA) strain of Theiler's murine encephalomyelitis virus (TMEV) results in approximately 50% of the mice developing acute behavioral seizures. TMEV-DA is the wild-type strain of the virus that replicates within the parenchyma of the brain. A variant of TMEV-DA, TMEV-H101, does not replicate within the parenchyma of the brain. However, infection with TMEV-H101 via the i.c. route still results in approximately 40% of the mice developing acute behavioral seizures. Infiltrating macrophages producing interleukin-6 (IL-6) have been implicated in the induction of acute seizures following TMEV-DA infection. We examined macrophage infiltration and microglial activation within the brain and cytokine levels in the periphery in mice infected with TMEV-DA or TMEV-H101 and assessed the effects of the addition of recombinant IL-6 to the periphery in wild-type and IL-6 knockout mice infected with TMEV-DA. We found that pathologic levels of IL-6 in the periphery may play a role in the development of seizures when viral replication within the brain is limited. Examination of the role played by the peripheral immune system in the development of seizures/epilepsy in the TMEV-induced seizure model, the first viral infection driven model for epilepsy, could lead to the elucidation of novel therapeutics.

Abstract 353: Bone Marrow Fibroblast Progenitor Cell-derived Exosomes Activate Resident Fibroblast and Augment Pressure Overload Induced Cardiac Fibrosis in IL10KO Mice

Background: Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Previous studies suggest the contribution of bone marrow fibroblasts progenitor cells (FPC) in pressure overload (PO)-induced cardiac fibrosis and inflammation acts as catalyst in this process. Recently others and we have shown that paracrine mediators packaged in exosomes play important role in cardiac pathophysiology. Thus, we hypothesized that exosome-derived from IL10KO-FPC augments PO-induced resident cardiac fibroblast activation and therefore, aggravate cardiac fibrosis. Methods and Results: Cardiac fibrosis was induced in Wild-type (WT) and IL10-knockout (IL10KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in IL10KO mice. PO-enhanced FPC (Prominin1 + cells) mobilization and homing in IL10KO mice compared to WT mice. To establish the IL10KO-FPC paracrine signaling, exosomes were isolated from WT and IL10KO BM-FPC culture media and characterized for proteins/miRNA. IL10 KO FPC-exosomes showed altered packaging of signature fibrotic miR and proteins. To explore whether FPC-exosomes modulate resident fibroblast activation, adult cardiac fibroblasts were treated with WT and IL10KO FPC-derived exosomes. IL10KO-FPC-derived exosomes exaggerate TGFβ 2 -induced activation of adult fibroblasts. These data suggest that fibrotic remodeling factors (miRs and/or proteins) packaged in IL10KO-FPC exosomes are sufficient to enhance the resident cardiac fibroblast activation and mediate cardiac fibrotic remodeling IL10 treatment significantly inhibits TGFβ 2 -induced FPC to myoFBs transition. Conclusion: Taken together, our findings suggest that paracrine factors secreted by BM-FPC augment resident cardiac fibroblast activation and fibrosis in pressure overloaded myocardium and IL10 negatively regulates this process. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.