IL-10 Knockout Mice Research Articles

Interleukin-6 mediated exercise-induced alleviation of adiposity and hepatic steatosis in mice

IntroductionExercise training has been shown to be the most effective strategy to combat obesity and non-alcoholic fatty liver disease. However, exercise promotes loss of adipose tissue mass and improves obesity-related...

Synergistic effect of the TLR5 agonist CBLB502 and its downstream effector IL-22 against liver injury

The toll-like receptor 5 (TLR5) agonist, CBLB502/Entolimod, is a peptide derived from bacterial flagellin and has been shown to protect against radiation-induced tissue damage in animal models. Here we investigated the protective mechanism of CBLB502 in the liver using models of ischemia-reperfusion injury and concanavalin A (ConA) induced immuno-hepatitis. We report that pretreatment of mice with CBLB502 provoked a concomitant activation of NF-κB and STAT3 signaling in the liver and reduced hepatic damage in both models. To understand the underlying mechanism, we screened for cytokines in the serum of CBLB502 treated animals and detected high levels of IL-22. There was no transcriptional upregulation of IL-22 in the liver, rather it was found in extrahepatic tissues, mainly the colon, mesenteric lymph nodes (MLN), and spleen. RNA-seq analysis on isolated hepatocytes demonstrated that the concomitant activation of NF-κB signaling by CBLB502 and STAT3 signaling by IL-22 produced a synergistic cytoprotective transcriptional signature. In IL-22 knockout mice, the loss of IL-22 resulted in a decrease of hepatic STAT3 activation, a reduction in the cytoprotective signature, and a loss of hepatoprotection following ischemia-reperfusion-induced liver injury. Taken together, these findings suggest that CBLB502 protects the liver by increasing hepatocyte resistance to acute liver injury through the cooperation of TLR5-NF-κB and IL-22-STAT3 signaling pathways.

Interleukin-6 ablation does not alter morphofunctional heart characteristics but modulates physiological and inflammatory markers after strenuous exercise

Interleukin-6 (IL-6) is associated with pathological cardiac hypertrophy and can be dramatically increased in serum after an acute strenuous exercise session. However, IL-6 is also associated with the increased production and release of anti-inflammatory cytokines and the inhibition of tumor necrosis factor-alpha (TNF-α) after chronic moderate exercise. To elucidate the relevance of IL-6 in inflammatory and hypertrophic signaling in the heart in response to an acute strenuous exercise session, we combined transcriptome analysis using the BXD mice database and exercised IL-6 knockout mice (IL-6KO). Bioinformatic analysis demonstrated that low or high-levels of Il6 mRNA in the heart did not change the inflammation- and hypertrophy-related genes in BXD mice strains. On the other hand, bioinformatic analysis revealed a strong positive correlation between Il6 gene expression in skeletal muscle with inflammation-related genes in cardiac tissue in several BXD mouse strains, suggesting that skeletal muscle-derived IL-6 could alter the heart's intracellular signals, particularly the inflammatory signaling. As expected, an acute strenuous exercise session increased IL-6 levels in wild-type, but not in IL-6KO mice. Despite not showing morphofunctional differences in the heart at rest, the IL-6KO group presented a reduction in physical performance and attenuated IL-6, TNF-α, and IL-1beta kinetics in serum, as well as lower p38MAPK phosphorylation, Ampkalpha expression, and higher Acta1 and Tnf gene expressions in the left ventricle in the basal condition. In response to strenuous exercise, IL-6 ablation was linked to a reduction in the pro-inflammatory response and higher activation of classical physiological cardiac hypertrophy proteins.

Interleukin-22 From Type 3 Innate Lymphoid Cells Aggravates Lupus Nephritis by Promoting Macrophage Infiltration in Lupus-Prone Mice.

Lupus nephritis (LN) is one of the most severe manifestations of systemic lupus erythematosus (SLE). Our previous studies demonstrated increased serum and renal Interleukin (IL)-22 in LN patients and MRL/lpr mice. This study investigated the role of IL-22 and its mechanism in LN. Here, we found that IL-22 was mainly produced by type 3 innate lymphoid cells (ILC3) in kidney of MRL/lpr mice. The systemic illness and local renal lesion were significantly alleviated in IL-22 or IL-22R gene knockout (KO) mice (IL-22 KO or IL-22R KO MRL/lpr mice) than control mice (MRL/lpr mice). IL-22 KO or IL-22R KO MRL/lpr mice had significantly slighter infiltration of macrophage in kidney than MRL/lpr mice. Consistently, by RNA-Seq, the expression of (CC motif) ligand 2 (CCL2) and (CXC motif) ligand 10 (CXCL10) was decreased in kidney of KO mice compared with control mice. By immunoblotting, significantly increased levels of STAT3 phosphorylation were found in the kidney of control mice compared to KO mice. In vitro, primary kidney epithelial cells from control mouse stimulated with recombinant IL-22 (rIL-22) expressed higher levels of CCL2, CXCL10, and phosphorylated STAT3. At the same time, when primary kidney epithelial cells were treated with rIL-22, transwell assay demonstrated their supernatant recruited more macrophages. In human kidney epithelial cell line (HK2) cells, when treated with rIL-22, we observed similar results with primary mouse kidney epithelial cells. Moreover, when cells were stimulated with rIL-22 following pre-treatment with STAT3 pathway inhibitor, the expression of CCL2 and CXCL10 were significantly reversed. Our findings demonstrate that IL-22 binding to IL-22R in kidney epithelial cells activated the STAT3 signaling pathway, enhanced the chemokine secretion and then promoted macrophage infiltration to the kidney of MRL/lpr mice, thus aggravated LN in lupus-prone mice. These findings indicate that IL-22 may play a pathogenic role in LN and may provide a promising novel therapeutic target for LN.

Interleukin-17 upregulation participates in the pathogenesis of heart failure in mice via NF-κB-dependent suppression of SERCA2a and Cav1.2 expression.

Interleukin-17 (IL-17), also called IL-17A, is an important regulator of cardiac diseases, but its role in calcium-related cardiac dysfunction remains to be explored. Thus, we investigated the influence of IL-17 on calcium handling process and its contribution to the development of heart failure. Mice were subjected to transaortic constriction (TAC) to induce heart failure. In these mice, the levels of IL-17 in the plasma and cardiac tissue were significantly increased compared with the sham group. In 77 heart failure patients, the plasma level of IL-17 was significantly higher than 49 non-failing subjects, and was negatively correlated with cardiac ejection fraction and fractional shortening. In IL-17 knockout mice, the shortening of isolated ventricular myocytes was increased compared with that in wild-type mice, which was accompanied by significantly increased amplitude of calcium transient and the upregulation of SERCA2a and Cav1.2. In cultured neonatal cardiac myocytes, treatment of with IL-17 (0.1, 1 ng/mL) concentration-dependently suppressed the amplitude of calcium transient and reduced the expression of SERCA2a and Cav1.2. Furthermore, IL-17 treatment increased the expression of the NF-κB subunits p50 and p65, whereas knockdown of p50 reversed the inhibitory effects of IL-17 on SERCA2a and Cav1.2 expression. In mice with TAC-induced mouse heart, IL-17 knockout restored the expression of SERCA2a and Cav1.2, increased the amplitude of calcium transient and cell shortening, and in turn improved cardiac function. In addition, IL-17 knockout attenuated cardiac hypertrophy with inhibition of calcium-related signaling pathway. In conclusion, upregulation of IL-17 impairs cardiac function through NF-κB-mediated disturbance of calcium handling and cardiac remodeling. Inhibition of IL-17 represents a potential therapeutic strategy for the treatment of heart failure.

IL-10 Deficiency Aggravates Renal Inflammation, Fibrosis and Functional Failure in High-Fat Dieted Obese Mice

BACKGROUND:High-fat diet-induced obesity is one of the major cause of chronic renal failure. This obesity-related renal failure is mainly caused by inflammatory processes. However, the role of the major anti-inflammatory cytokine interleukin (IL)-10 has not been researched intensively.METHODS:To evaluate the effect of IL-10 deficiency on obesity-related renal failure, the in vivo study was carried with four animal groups; (1) Low-fat dieted C57BL/6 mice, (2) Low-fat dieted IL-10 knockout (KO) mice, (3) High‐fat dieted C57BL/6 mice and (4) High‐fat dieted IL-10 KO mice group. The analysis was carried with blood/urine chemistry, H&E, Oil-Red-O, periodic acid-Schiff and Masson’s trichrome staining immunohistochemistry and real-time PCR methods.RESULTS:At week 12, high‐fat dieted IL-10 KO mice showed 1) severe lipid accumulation in kidneys, cholesterol elevation (in total, serum kidney) and low-density lipoprotein increasion through the SCAP-SREBP2-LDLr pathway; (2) serious histopathologic alterations showing glomerulosclerosis, tubulointerstitial fibrosis and immune cell infiltration; (3) increased pro‐inflammatory cytokines and chemokines expression; (4) enhanced renal fibrosis; and (5) serious functional failure with high serum creatinine and BUN and proteinuria excretion compared to other groups.CONCLUSION:IL-10 deficiency aggravates renal inflammation, fibrosis and functional failure in high-fat dieted obese mice, thus IL-10 therapy could be applied to obesity-related chronic renal failure.

STAT6 Pathway Is Critical for the Induction and Function of Regulatory T Cells Induced by Mucosal B Cells.

B cells could convert naïve T cells into regulatory T cells (so-called Treg-of-B cells) which have the ability to treat animal models of inflammatory diseases, including allergic asthma, collagen-induced arthritis and colitis; however, the mechanisms of Treg-of-B cell generation remain unclear. In this study, we investigated the role of STAT6 in the generation of Treg-of-B (P) cells, which Treg cells were generated by Peyer’s patch B cells (P stands for Peyer’s patch). CD4+CD25- T cells from wild type, STAT6 knockout and IL-4 knockout mice were cocultured with wild type Peyer’s patch B cells for Treg-of-B (P) cell generation. A murine asthmatic model was used to analyze the in vivo regulatory function of Treg-of-B (P) cells. The data demonstrated that STAT6 played a critical role in the generation of Treg-of-B (P) cells, which confirmed with STAT6-deficient T cells and the STAT6 inhibitor AS1517499. When STAT6 was lacking, Treg-of-B (P) cells exerted impaired suppressive ability with decreased LAG3 expression. Furthermore, Peyer’s patch B cells played an essential role in regulatory T cell generation. In the absence of Peyer’s patch B cells, T cells expressed decreased phosphorylated STAT6, which was followed by decreased LAG3 expression and impaired suppressive ability, suggesting that Peyer’s patch B cells provided the critical signal to activate STAT6 phosphorylation in T cells. Moreover, STAT6 deficient Treg-of-B (P) cells could not alleviate inflammation in an animal model of asthma in vivo. IL-4 was downstream of phosphorylated STAT6 and maintained Treg-of-B (P) cell survival with increased expression of Bcl-2 and BclXL. We reported a novel finding that the STAT6-LAG3 signaling axis is important for the induction and function of Treg-of-B (P) cells.

Mechanism of IL-6-related spontaneous atrial fibrillation after coronary artery grafting surgery: IL-6 knockout mouse study and human observation

We established a mouse model of cardiac surgery with nonpaced sPOAF. IL-6 knockout mice were protected from sPOAF compared with wild-type mice. At 48 hours after surgery, the heart was separated into 6 regions and cultured. IL-6 was expressed in all regions, with highest abundance in the left atrium (LA). In PHASE III, we demonstrated that IL-6 in the LA elicited early profibrotic properties in atria via the pSTAT3/STAT3 signaling pathway and contributed to sPOAF. In a translational prospective clinical study, we demonstrated that humans with POAF had a higher IL-6 concentration in pericardial drainage (PD). This study provides preliminary evidence of a causal relationship between IL-6 and POAF in a novel nonpaced sPOAF mouse model. IL-6 is a crucial prerequisite for eliciting profibrotic properties in cardiac myocytes via the pSTAT3 pathway during the early postoperative period, leading to an increased susceptibility to POAF. Measuring IL-6 in PD could be a new noninvasive biomarker for the clinical prediction of POAF.

Adenovirus Vector-Induced IL-6 Promotes Leaky Adenoviral Gene Expression, Leading to Acute Hepatotoxicity.

Adenovirus (Ad) vector-mediated transduction can cause hepatotoxicity during two phases, at ∼2 and 10 days after administration. Early hepatotoxicity is considered to involve inflammatory cytokines; however, the precise mechanism remains to be clarified. We examined the mechanism of early Ad vector-induced hepatotoxicity by using a conventional Ad vector, Ad-CAL2, and a modified Ad vector, Ad-E4-122aT-CAL2. Ad-E4-122aT-CAL2 harbors sequences complementary to the liver-specific miR-122a in the 3' untranslated region of E4, leading to significant suppression of leaky Ad gene expression in the liver via posttranscriptional gene silencing and a significant reduction in late-phase hepatotoxicity. We found that Ad-E4-122aT-CAL2 transduction significantly attenuated acute hepatotoxicity, although Ad-E4-122aT-CAL2 and Ad-CAL2 induced comparable cytokine expression levels in the liver and spleen. IL-6, a major inflammatory cytokine induced by Ad vectors, significantly enhanced leaky Ad gene expression and cytotoxicity in primary mouse hepatocytes following Ad-CAL2 but not Ad-E4-122aT-CAL2 transduction. Furthermore, leaky Ad gene expression and cytotoxicity in Ad-CAL2-treated hepatocytes in the presence of IL-6 were significantly suppressed upon inhibition of JAK and STAT3. Ad vector-mediated acute hepatotoxicities and leaky Ad expression were significantly reduced in IL-6 knockout mice compared with those in wild-type mice. Thus, Ad vector-induced IL-6 promotes leaky Ad gene expression, leading to acute hepatotoxicity.

Mouse Models of Intestinal Fibrosis.

Mouse models are essential for investigation of underlying disease mechanisms that drive intestinal fibrosis, as well as assessment of potential therapeutic approaches to either prevent or resolve fibrosis. Here we describe several common mouse models of intestinal inflammation and fibrosis, including chemically driven colitis models, a bacterially triggered colitis model, and spontaneous intestinal inflammation in genetically susceptible mouse strains. Detailed protocols are provided for dextran sodium sulfate (DSS) colitis, 2,4,6-trinitro-benzene sulfonic acid (TNBS) colitis, adherent-invasive Escherichia coli (AIEC)-triggered colitis, the interleukin-10 knockout (IL-10KO) mouse model of spontaneous colitis, and the SAMP/YitFc model of spontaneous ileocolitis.

Enterococcus faecalis Glucosamine Metabolism Exacerbates Experimental Colitis.

Background & AimsThe inflammatory bowel diseases (IBDs), Crohn’s disease and ulcerative colitis, are caused in part by aberrant immune responses to resident intestinal bacteria. Certain dietary components, including carbohydrates, are associated with IBDs and alter intestinal bacterial composition. However, the effects of luminal carbohydrates on the composition and colitogenic potential of intestinal bacteria are incompletely understood. We hypothesize that carbohydrate metabolism by resident proinflammatory intestinal bacteria enhances their growth and worsens intestinal inflammation.MethodsWe colonized germ-free, wild-type, and colitis-susceptible interleukin-10 knockout mice (Il10-/-) with a consortium of resident intestinal bacterial strains and quantified colon inflammation using blinded histologic scoring and spontaneous secretion of IL12/23p40 by colon explants. We measured luminal bacterial composition using real-time 16S polymerase chain reaction, bacterial gene expression using RNA sequencing and real-time polymerase chain reaction, and luminal glucosamine levels using gas chromatography–mass spectrometry.ResultsWe show that a consortium of 8 bacterial strains induces severe colitis in Il10-/- mice and up-regulates genes associated with carbohydrate metabolism during colitis. Specifically, Enterococcus faecalis strain OG1RF is proinflammatory and strongly up-regulates OG1RF_11616-11610, an operon that encodes genes of a previously undescribed phosphotransferase system that we show imports glucosamine. Experimental colitis is associated with increased levels of luminal glucosamine and OG1RF_11616 causes worse colitis, not by increasing E faecalis numbers, but rather by mechanisms that require the presence of complex microbiota.ConclusionsFurther studies of luminal carbohydrate levels and bacterial carbohydrate metabolism during intestinal inflammation will improve our understanding of the pathogenesis of IBDs and may lead to the development of novel therapies for these diseases.

Lack of Interleukin-6 Affects IFN-γ and TNF-α Production and Early In Vivo Control of Brucella abortus Infection.

Interleukin-6 (IL-6) is a pleiotropic cytokine promptly produced in response to infections, which contributes to host defense through the stimulation of acute phase immune responses. Brucella abortus is an intracellular bacterium that causes chronic disease in humans and domestic animals and triggers a robust immune response, characterized by the production of inflammatory cytokines. However, the mechanisms of IL-6-related immune responses in the context of Brucella infections are not completely understood. In this report, we describe an increased susceptibility of IL-6 knockout (KO) mice in the early phase of Brucella infection. Furthermore, we demonstrate that IL-6 is required for interferon (IFN)-γ and tumor necrosis factor (TNF)-α induction by infected splenocytes, indicating a protective role for IL-6 against B. abortus that parallels with Th1 type of immune response. Additionally, IL-6 KO mice exhibited reduced splenomegaly during the early phase of the infection. Corroborating this result, IL-6 KO mice displayed reduced numbers of macrophages, dendritic cells, and neutrophils in the spleen and reduced myeloperoxidase activity in the liver compared to wild-type infected mice. However, we demonstrate that IL-6 is not involved in B. abortus intracellular restriction in mouse macrophages. Taken together, our findings demonstrate that IL-6 contributes to host resistance during the early phase of B. abortus infection in vivo, and suggest that its protective role maybe partially mediated by proinflammatory immune responses and immune cell recruitment.

Bile is a promising gut nutrient that inhibits intestinal bacterial translocation and promotes gut motility via an interleukin-6-related pathway in an animal model of endotoxemia

ObjectivesPeople who are critically ill have high rates of endotoxemia that can significantly decrease bile flow and increase bile cytokines, the latter of which might worsen their condition. Bile acids are nutrient-signaling hormones that have a significant impact on gut barrier function and motility, and the gut is considered the origin of systemic inflammation. Therefore, healthy exogenous bile could be a promising gut nutrient for critical illness, so the biomedical role of bile in endotoxemia was investigated in this study. MethodsTwelve rats were injected with lipopolysaccharide (LPS) and randomized into a group with sham operation) and a group with bile external drainage (n = 6 for each group); six rats with sham operation served as the control group. In addition, interleukin-6 (IL-6) knockout mice and macrophages were treated with LPS. ResultsCompared to the control animals, the group with LPS injection and sham operation had significantly increased levels of gut permeability, gut bacterial translocation, gut mucosal tumor necrosis factor α, IL-6 transcripts, and serum tumor necrosis factor α and IL-6. Compared to group with sham operation and LPS injection, bile external drainage (in LPS-challenged rats) increased gut bacterial translocation by 10 times, and this detrimental effect was associated with prolonged intestinal transit time, increased serum IL-6 concentration, and up-regulated gut mucosal IL-6 transcripts. Moreover, bile selectively inhibited LPS-stimulated macrophages in IL-6 release, which can activate gastrointestinal submucosal neurons to promote motility. Knocking out IL-6 significantly reduced gut bacterial translocation in endotoxemic mice. ConclusionsBile is a promising gut nutrient that inhibits gut bacterial translocation and promotes gut motility via an IL-6-related pathway in experimental endotoxemia.

A Novel PAK1-Notch1 Axis Regulates Crypt Homeostasis in Intestinal Inflammation.

Background & Aimsp21-activated kinase-1 (PAK1) belongs to a family of serine-threonine kinases and contributes to cellular pathways such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), and Wingless-related integration site(Wnt)/β-catenin, all of which are involved in intestinal homeostasis. Overexpression of PAK1 is linked to inflammatory bowel disease as well as colitis-associated cancer (CAC), and similarly was observed in interleukin (IL)10 knockout (KO) mice, a model of colitis and CAC. Here, we tested the effects of PAK1 deletion on intestinal inflammation and carcinogenesis in IL10 KO mice.MethodsIL10/PAK1 double-knockout (DKO) mice were generated and development of colitis and CAC was analyzed. Large intestines were measured and prepared for histology or RNA isolation. Swiss rolls were stained with H&E and periodic acid-Schiff. Co-immunoprecipitation and immunofluorescence were performed using intestinal organoids, SW480, and normal human colon epithelial cells 1CT.ResultsWhen compared with IL10 KO mice, DKOs showed longer colons and prolonged crypts, despite having higher inflammation and numbers of dysplasia. Crypt hyperproliferation was associated with Notch1 activation and diminished crypt differentiation, indicated by a reduction of goblet cells. Gene expression analysis indicated up-regulation of the Notch1 target hairy and enhancer of split-1 and the stem cell receptor leucin-rich repeat-containing G-protein–coupled receptor 5 in DKO mice. Interestingly, the stem cell marker olfactomedin-4 was present in colonic tissue. Increased β-catenin messenger RNA and cytoplasmic accumulation indicated aberrant Wnt signaling. Co-localization and direct interaction of Notch1 and PAK1 was found in colon epithelial cells. Notch1 activation abrogated this effect whereas silencing of PAK1 led to Notch1 activation.ConclusionsPAK1 contributes to the regulation of crypt homeostasis under inflammatory conditions by controlling Notch1. This identifies a novel PAK1–Notch1 axis in intestinal pathophysiology of inflammatory bowel disease and CAC.

Interleukin-6 (IL-6) deficiency enhances intramembranous osteogenesis following stress fracture in mice.

Interleukin-6 (IL-6) is highly upregulated in response to skeletal injury, suggesting it plays a role in the inflammatory phase of fracture repair. However, the impact of IL-6 on successful repair remains incompletely defined. Therefore, we investigated the role of IL-6 in two models of fracture repair (full fracture and stress fracture) using 12-week old IL-6 global knockout mice (IL-6 KO) and wild type (WT) littermate controls. Callus morphology and mineral density 14days after full femur fracture did not differ between IL-6 knockout mice and controls. In contrast, IL-6 KO mice had an enhanced bone response 7days after ulnar stress fracture compared to WT, with increased total callus volume (p=0.020) and callus bone volume (p=0.045). IL-6 KO did not alter the recruitment of immune cells (Gr-1 or F4/80 positive) to the stress fracture callus. IL-6 KO also did not alter the number of osteoclasts in the stress fracture callus. Using RNA-seq, we identified differentially expressed genes in stress fracture vs. contralateral control ulnae, and observed that IL-6 KO resulted in only modest alterations to the gene expression response to stress fracture (SFx). Wnt1 was more highly upregulated in IL-6 KO SFx callus at both day 1 (fold change 12.5 in KO vs. 5.7 in WT) and day 3 (fold change 4.7 in KO vs. 1.9 in WT). Finally, using tibial compression to induce bone formation without bone injury, we found that IL-6 KO directly impacted osteoblast function, increasing the propensity for woven bone formation. In summary, we report that IL-6 knockout enhanced formation of callus and bone following stress fracture injury, likely through direct action on the osteoblast's ability to produce woven bone. This suggests a novel role of IL-6 as a suppressor of intramembranous bone formation.

Tissue-Derived IL-33 Is a Critical Local Signal That Targets Th1 Cells in the Small Intestine to Sustain Graft Versus Host Disease in the Absence of IL-12

Background: Graft vs. host disease (GVHD) remains a major complication of allogeneic stem cell transplantation (alloSCT). To create space for donor stem cells and prevent their rejection, alloSCT protocols rely on conditioning regimens involving chemotherapy, frequently combined with radiation. Interleukin (IL)-33 is a nuclear protein that is found in the secondary lymphoid organs and barrier tissues. IL-33 is augmented in recipient barrier tissues by conditioning. We have established that IL-33 is a clinically-relevant signal. While we have further elucidated that IL-33 is acting directly on donor T cells to augment GVHD, neither the critical place nor timing of IL-33 stimulation of donor T cells during the alloimmune response mediating GVHD have been defined. Methods: We used both IL-33 knockout mice as recipients and ST2 (IL33 receptor) knockout mice as T cell donors in MHC mismatch alloSCT models to define the spatial and temporal importance of IL-33 in GVHD tissue damage. More specifically, to establish the role of IL-33 in T cell effector function in the small intestine during GVHD, irradiated Il33-/- and Il33+/+ C57BL/6 (B6) recipients were given BALB/c bone marrow (BM) and T cells. Additionally, irradiated BALB/c mice were given St2+/+ B6 BM and Cd4 Cre x St2fl/fl or St2-/- and St2+/+ T cells. To determine the role of IL-33 on T cell persistence in GVHD, we used an MHCII-disparate model. Irradiated Il33-/- and Il33+/+ bm12 recipients were reconstituted with B6 BM, with or without CD90.1+ B6 T cells. Results: When donor T cell responses were characterized by flow cytometry at days 7, 14, and 28 post alloSCT, the bulk of these data suggest that IL-33 sustains CD4+ T helper Type 1 (Th1) cells in the barrier tissues at late points following GVHD initiation. Specifically, B6 Il33-/- recipients had fewer BALB/c CD4+Tbet+ cells in the small intestine lamina propria (SI LP; WT 15.9 ± 1.31, KO 9.53 ± 1.32; p=0.03; n=3/group), but not in the spleen (WT 54.8 ± 7.37, KO 57.68 ± 4.90; p=0.75; n=4/group) at day 7. Additionally, we found that when we used therapeutic doses of anti-IL-12p40 to neutralize IL-12 there was no impact on the CD4+Tbet+ cells in the SI of B6 il33-/- recipients (KO control IgG 7.29 ± 0.63, KO anti-IL-12p40 6.61 ± 0.75; p=0.52; n=4/group) suggesting that the decrease in the frequency of CD4+Tbet+ T cells in Il33-/- in the SI is independent of IL-12. In the CD4-dependent B6 to bm12 GVHD model, we find that IL-33-deficient recipients have reduced overall CD4+ donor T cells in the SI LP at both day 14 and day 28 (d14: WT 16.15 ± 2.55, KO 0.35 ± 0.07; p=0.003; n=3/group; d28: WT 33.07 ± 7.17, KO 6.27 ± 1.59; p=0.022; n=3/group). Consistent with our B6 recipients of BALB/c T cells above, this decrease was not observed in the spleen or lymph nodes. Reduced clusters of CD3+ cells were evident in immunohistochemistry of bm12 Il33-/- relative to bm12 Il33+/+ at day 28 post-alloSCT. In corroboration with our IL-33-deficient recipient results, B6 CD4+ T cells lacking the ST2 receptor when co-transferred with ST2 competent T cells were not sustained in the SI LP at day 7 (ST2 WT 18.55 ± 0.77, ST2 KO 6.35 ± 1.42; p=0.0003; n=4/group). ST2 deficient CD4+ donor cells were of equal frequency to the ST2 competent donor cells in the spleen of BALB/c recipients (ST2 WT 54.58 ± 1.914, ST2 KO 50 ± 2.53; p=0.19; n=4/group). Conclusions: Our data reveal that IL-33 is not required for donor Th1 responses in the spleen and LN, but IL-33 is critical for Th1 cells to persist in barrier tissues like the small intestine after alloSCT in the absence of IL-12 stimulation. These data suggest that targeting IL-33 signaling may be an effective therapy to diminish GVHD in target tissues. Disclosures Blazar: Fate Therapeutics Inc.: Research Funding; Childrens' Cancer Research Fund: Research Funding; BlueRock Therapeutics: Research Funding; BlueRock Therapeuetic: Consultancy; Magenta Therapeutics: Consultancy; KidsFirst Fund: Research Funding; Tmunity: Other: Co-founder. Shlomchik:Bluesphere Bio: Consultancy, Other: shareholder.

Lactococcus lactis subsp. Cremoris C60 restores T Cell Population in Small Intestinal Lamina Propria in Aged Interleukin-18 Deficient Mice.

Lactic acid bacteria (LAB), a major commensal bacterium in the small intestine, are well known beneficial bacteria which promote establishment of gut-centric immunity, such as anti-inflammation and anti-infection. In this report, we show that a LAB strain Lactococcus lactis subsp. Cremoris C60 possess an ability to activate antigen presenting cells, such as dendritic cells (DCs), and intestinal T cells which possibly support to maintain healthy intestinal immunological environment in aging process. We found that CD4+ T cells in the small intestine are dramatically decreased in aged Interleukin-18 knock out (IL-18KO) mice, associated with the impairment of IFN-γ production in the CD4+ T cells, especially in small intestinal lamina propria (LP). Surprisingly, heat killed-C60 (HK-C60) diet completely recovered the CD4+ T cells population and activity in SI-LP and over activated the population in Peyer’s patches (PPs) of IL-18KO mice. The HK-C60 diet was effective approach not only to restore the number of cells, but also to recover IFN-γ production in the CD4+ T cell population in the small intestine of IL-18-deficient mice. As a possible cause in the age-associated impairment of CD4+ T cells activity in IL-18KO mice, we found that the immunological activity was downregulated in the IL-18-deficient DCs. The cytokines production and cellular activation markers expression were downregulated in the IL-18-deficient bone marrow derived dendritic cells (BMDCs) at the basal level, however, both activities were highly upregulated in HK-C60 stimulation as compared to those of WT cells. Antigen uptake was also attenuated in the IL-18-deficient BMDCs, and it was significantly enhanced in the cells as compared to WT cells in HK-60 stimulation. An in vitro antigen presentation assay showed that IFN-γ production in the CD4+ T cells was significantly enhanced in the culture of IL-18-deficient BMDCs compared with WT cells in the presence of HK-C60. Thus, we conclude that HK-C60 diet possesses an ability to restore T cells impairment in the small intestine of IL-18-deficient environment. In addition, the positive effect is based on the immunological modification of DCs function which directory influences into the promotion of effector CD4+ T cells generation in the small intestine.

Inhibitory role of interleukin-10 in the cutaneous reverse Arthus reaction.

The formation and deposition of immune complexes (IC) containing immunoglobulin (Ig)G antibodies induces an acute inflammatory response with tissue injury. One of the experimental models of IC-related vasculitis is the cutaneous reverse passive Arthus reaction, in which IgG antibodies are injected i.d., followed immediately by the i.v. application of the corresponding antigen. This reaction is characterized by edema, hemorrhage and neutrophil infiltration. To assess the role of the anti-inflammatory cytokine interleukin (IL)-10 in IC-related vasculitis, we investigated the cutaneous Arthus reaction using IL-10 knockout (IL-10KO) mice. Edema, which was quantified macroscopically by measuring the vascular leakage of Evans blue dye at 4h after IC challenge, was significantly increased in IL-10KO mice compared with wild-type mice. In addition, hemorrhage, which was assessed by the average diameter of purpuric spots at 8h after IC challenge, was enhanced significantly in IL-10KO mice compared with wild-type mice. Histological examination showed that the number of extravascular neutrophils was significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. Analysis of pro-inflammatory cytokine mRNA expression showed that IL-6 mRNA levels were significantly increased in IL-10KO mice compared with wild-type mice at 4 and 8h after IC challenge. These results showed that IC-induced inflammation and vascular damage were significantly enhanced in the absence of IL-10. Thus, IL-10 may limit tissue disruption by suppressing the excessive infiltration of neutrophils and cytokine expression in a mouse model of type III vasculitis.

A new mouse model to study restoration of interleukin-6 (IL-6) expression in a Cre-dependent manner: microglial IL-6 regulation of experimental autoimmune encephalomyelitis

BackgroundInterleukin-6 (IL-6) is a pleiotropic cytokine that controls numerous physiological processes both in basal and neuroinflammatory conditions, including the inflammatory response to experimental autoimmune encephalomyelitis (EAE). IL-6 is produced by multiple peripheral and central cells, and until now, the putative roles of IL-6 from different cell types have been evaluated through conditional cell-specific IL-6 knockout mice. Nevertheless, these mice probably undergo compensatory responses of IL-6 from other cells, which makes it difficult to assess the role of each source of IL-6.MethodsTo give some insight into this problem, we have produced a novel mouse model: a conditional reversible IL-6 KO mouse (IL6-DIO-KO). By using double-inverted, open-reading-frame (DIO) technology, we created a mouse line with the loss of Il6 expression in all cells that can be restored by the action of Cre recombinase. Since microglia are one of the most important sources and targets of IL-6 into the central nervous system, we have recovered microglial Il6 expression in IL6-DIO-KO mice through breeding to Cx3cr1-CreER mice and subsequent injection of tamoxifen (TAM) when mice were 10–16 weeks old. Then, they were immunized with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) 7 weeks after TAM treatment to induce EAE. Clinical symptoms and demyelination, CD3 infiltration, and gliosis in the spinal cord were evaluated.ResultsIL6-DIO-KO mice were resistant to EAE, validating the new model. Restoration of microglial Il6 was sufficient to develop a mild version of EAE-related clinical symptoms and neuropathology.ConclusionsIL6-DIO-KO mouse is an excellent model to understand in detail the role of specific cellular sources of IL-6 within a recovery-of-function paradigm in EAE.

Chlorogenic Acid Improves Symptoms of Inflammatory Bowel Disease in Interleukin-10 Knockout Mice

Inflammatory bowel disease (IBD) is a chronic intestinal inflammation that is highly prevalent worldwide. Interleukin (IL)-10 can effectively inhibit negative cascades such as the production of inflammatory mediators (inducible nitric oxide synthase [iNOS], cyclooxygenase-2), accumulation of inflammatory infiltrates (macrophages, eosinophils, neutrophils), toxicity (lower T cell subsets), and secretion of pro-inflammatory cytokines (IL-1β, TNF-α) in tissues such as the spleen, mesenteric lymph nodes (MLN), Peyer's patch (PP), and colon. In this study, we investigated whether chlorogenic acid (CHA) can regulate inflammation in IL-10 knockout (KO) mice used as an IBD animal model. CHA significantly increased the ratio of CD4+/CD8+, T cell subsets in PP, and MLN of IL-10 KO mice. In addition, CHA also morphologically attenuated colon inflammation in IL-10 KO mice. We demonstrated that CHA significantly reduced the expression levels of iNOS, IL-1β, TNF-α, which were highly expressed in IL-10 KO mice. Therefore, CHA may provide beneficial effects for improving IBD by decreasing inflammations.