IL-10 Knockout Mice Research Articles

Piroxicam treatment augments bone abnormalities in interleukin-10 knockout mice.

Osteoporosis and fractures are common complications of inflammatory bowel disease. The pathogenesis is multifactorial and has been partly attributed to intestinal inflammation. The aim of this study was to evaluate bone status and assess the association between bone loss and gut inflammation in an experimental colitis model. Colitis was induced in interleukin-10 knockout mice (PAC IL-10 k.o.) by peroral administration of piroxicam for 12 days. The degree of colitis was assessed by clinical, macroscopic, and microscopic evaluation. Trabecular and cortical bone microarchitecture of tibia were determined using micro-computed tomography. Moreover, the serum levels of bone formation and bone resorption biomarkers were measured, and inflammatory protein profiling was performed on colons. PAC IL-10 k.o. mice developed severe colitis, characterized by hyperplasia and focal transmural inflammation, which was consistent with Crohn's disease-like pathology. The gut inflammation was accompanied by a 14% and 12% reduction in trabecular thickness relative to piroxicam-treated wild type and untreated wild type mice, respectively (P < 0.001). The trabecular bone structure was also changed in PAC IL-10 k.o. mice, whereas no differences in cortical bone geometry were observed. The trabecular thickness was inversely correlated with serum levels of CTX (r = -0.93, P = 0.006). Moreover, numerous inflammatory mediators, including RANKL and osteoprotegerin, were significantly increased in the colon of PAC IL-10 k.o. mice. PAC IL-10 k.o. mice develop bone loss and changed trabecular structure, as a result of increased bone resorption. Thus, the PAC IL-10 k.o. model could be a useful experimental model in preclinical research of inflammatory bowel disease-associated bone loss.

Endogenous interleukin 18 suppresses hyperglycemia and hyperinsulinemia during the acute phase of endotoxemia in mice.

Hyperglycemia associated with insulin resistance is common among critically ill patients. Interleukin (IL)-18 has been linked with hyperglycemia and insulin resistance in chronic disease, but the relation between IL-18 and insulin resistance during critical illness was unexplored. This study investigated whether IL-18 modulates hyperglycemia and insulin resistance during acute inflammation. We injected lipopolysaccharide (LPS) 40 mg/kg into wild-type (WT) and IL-18 knockout (KO) mice to induce endotoxemia and examined insulin resistance and insulin-dependent signaling pathways during the acute phase. During the first hour after LPS treatment, IL-18 KO mice showed higher blood glucose and insulin and less insulin receptor substrate-1 and less phosphorylated Akt in the liver compared with WT mice. Interleukin-18 KO mice exhibited better survival after LPS treatment. The findings suggest that endogenous IL-18 may attenuate hyperglycemia and modulate insulin signaling in liver. Accordingly, IL-18 may modulate glucose tolerance during acute inflammation.

Exercise increases pancreatic β-cell viability in a model of type 1 diabetes through IL-6 signaling.

Type 1 diabetes (T1D) is provoked by an autoimmune assault against pancreatic β cells. Exercise training enhances β-cell mass in T1D. Here, we investigated how exercise signals β cells in T1D condition. For this, we used several approaches. Wild-type and IL-6 knockout (KO) C57BL/6 mice were exercised. Afterward, islets from control and trained mice were exposed to inflammatory cytokines (IL-1β plus IFN-γ). Islets from control mice and β-cell lines (INS-1E and MIN6) were incubated with serum from control or trained mice or medium obtained from 5-aminoimidazole-4 carboxamide1-β-d-ribofuranoside (AICAR)-treated C2C12 skeletal muscle cells. Subsequently, islets and β cells were exposed to IL-1β plus IFN-γ. Proteins were assessed by immunoblotting, apoptosis was determined by DNA-binding dye propidium iodide fluorescence, and NO(•) was estimated by nitrite. Exercise reduced 25, 75, and 50% of the IL-1β plus IFN-γ-induced iNOS, nitrite, and cleaved caspase-3 content, respectively, in pancreatic islets. Serum from trained mice and medium from AICAR-treated C2C12 cells reduced β-cell death, induced by IL-1β plus IFN-γ treatment, in 15 and 38%, respectively. This effect was lost in samples treated with IL-6 inhibitor or with serum from exercised IL-6 KO mice. In conclusion, muscle contraction signals β-cell survival in T1D through IL-6.

Severe neutrophil-dominated inflammation and enhanced myelopoiesis in IL-33-overexpressing CMV/IL33 mice.

IL-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous studies emphasized a role for IL-33 in shaping innate and adaptive immune responses. IL-33 was also reported to modulate myelopoiesis and myeloid cell activity. In this article, we describe IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, which display an inflammatory phenotype associated with growth retardation and paw swelling. The phenotype of CMV/IL33 mice is dependent on activation of the ST2 receptor and is characterized by extensive neutrophil infiltration into different organs, including the paws. Local or systemic levels of proinflammatory mediators such as IL-1β, Cxcl-1, G-CSF, and IL-6 are increased. CMV/IL-33 mice also suffer from anemia, thrombocytosis, and a marked dysregulation of myelopoiesis, leading to an important increase in myeloid cell production or accumulation in bone marrow (BM), spleen, and peripheral blood. Consistently, recombinant IL-33 induced proliferation of myeloid lineage cells in BM-derived granulocyte cultures, whereas IL-33 knockout mice exhibited minor deficiencies in spleen and BM myeloid cell populations. Our observations reveal a neutrophil-dominated inflammatory phenotype in IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, and highlight important regulatory effects of IL-33 on myelopoiesis in vitro and in vivo, where excessive IL-33 signaling can translate into the occurrence of a myeloproliferative disorder.

Analysis of IL-22 contribution to hepcidin induction and hypoferremia during the response to LPS in vivo.

The anaemia of chronic disease (ACD) results from inflammation-mediated up-regulation of the iron regulatory hormone hepcidin, with the consequent sequestration of iron limiting its availability for erythropoiesis. The inflammatory cytokine IL-6, a regulator of hepcidin, has been implicated in this process. Recent in vivo and in vitro studies indicate that IL-22 is also able to stimulate hepcidin expression. We aimed to determine if IL-22 had a role in causing the hypoferremia associated with the inflammatory response. Wild-type and Il22-knockout mice were subjected to an acute inflammatory stimulus via administration of LPS and the response of hepcidin and iron homeostasis was analysed. In the absence of IL-22, there was a response of hepcidin, resulting in a reduction in serum iron levels. However, the hypoferremic response to LPS was slightly blunted in mice lacking IL-22, suggesting that, during LPS-mediated inflammation, IL-22 may play a minor role in mediating the hypoferremic response. These results may have implications for the treatment and management of the ACD.

Anti-mouse CD52 monoclonal antibody ameliorates intestinal epithelial barrier function in interleukin-10 knockout mice with spontaneous chronic colitis.

Intestinal inflammation causes tight junction changes and death of epithelial cells, and plays an important role in the development of Crohn's disease (CD). CD52 monoclonal antibody (CD52 mAb) directly targets the cell surface CD52 and is effective in depleting mature lymphocytes by cytolytic effects in vivo, leading to long-lasting changes in adaptive immunity. The aim of this study was to investigate the therapeutic effect of CD52 mAb on epithelial barrier function in animal models of IBD. Interleukin-10 knockout mice (IL-10(-/-) ) of 16 weeks with established colitis were treated with CD52 mAb once a week for 2 weeks. Severity of colitis, CD4(+) lymphocytes and cytokines in the lamina propria, epithelial expression of tight junction proteins, morphology of tight junctions, tumour necrosis factor-α (TNF-α)/TNF receptor 2 (TNFR2) mRNA expression, myosin light chain kinase (MLCK) expression and activity, as well as epithelial apoptosis in proximal colon were measured at the end of the experiment. CD52 mAb treatment effectively attenuated colitis associated with decreased lamina propria CD4(+) lymphocytes and interferon-γ/IL-17 responses in colonic mucosa in IL-10(-/-) mice. After CD52 mAb treatment, attenuation of colonic permeability, increased epithelial expression and correct localization of tight junction proteins (occludin and zona occludens protein-1), as well as ameliorated tight junction morphology were observed in IL-10(-/-) mice. CD52 mAb treatment also effectively suppressed the epithelial apoptosis, mucosa TNF-α mRNA expression, epithelial expression of long MLCK, TNFR2 and phosphorylation of MLC. Our results indicated that anti-CD52 therapy may inhibit TNF-α/TNFR2-mediated epithelial apoptosis and MLCK-dependent tight junction permeability by depleting activated T cells in the gut mucosa.

Protection of intestinal injury during heat stroke in mice by interleukin-6 pretreatment.

Heat stroke afflicts thousands of humans each year, worldwide. The immune system responds to hyperthermia exposure resulting in heat stroke by producing an array of immunological proteins, such as interleukin-6 (IL-6). However, the physiological functions of IL-6 and other cytokines in hyperthermia are poorly understood. We hypothesized that IL-6 plays a protective role in conditions of heat stroke. To test this, we gave small IL-6 supplements to mice prior to exposing them to hot environments sufficient to induce conditions of heat stroke. Pretreatment with IL-6 resulted in improved ability to withstand heat exposure in anaesthetized mice, it protected the intestine from injury, reducing the permeability of the intestinal barrier, and it attenuated the release of other cytokines involved in inflammation. The results support the hypothesis that IL-6 is a 'physiological stress hormone' that plays an important role in survival during acute life-threatening conditions such as heat stroke. The role of interleukin-6 (IL-6) in hyperthermia and heat stroke is poorly understood. Plasma IL-6 is elevated following hyperthermia in animals and humans, and IL-6 knockout mice are more intolerant of severe hyperthermia. We evaluated the effect of IL-6 supplementation on organ injury following severe hyperthermia exposure in anaesthetized mice. Two hours prior to hyperthermia, mice were treated with 0.6 μg intraperitoneal IL-6, or identical volumes of saline in controls. Mice were anaesthetized, gavaged with FITC-dextran for measures of gastrointestinal permeability, and exposed to incremental (0.5°C every 30 min) increases in temperature. Heating stopped when maximum core temperature (Tc) of 42.4°C was attained (Tc,max). The mice recovered at room temperature (≈22°C) for 30 or 120 min, at which time plasma and tissues were collected. IL-6-treated mice, on average, required ≈25 min longer to attain Tc,max . Injury and swelling of the villi in the duodenum was present in untreated mice after 30 min of recovery. These changes were blocked by IL-6 treatment. IL-6 also reduced gastrointestinal permeability, assayed by the accumulation of FITC-dextran in plasma. Plasma cytokines were also attenuated in IL-6-treated animals, including significant reductions in TNFα, MCP-1 (CXCL2), RANTES (CCL5) and KC (CCL5). The results demonstrate that IL-6 has a protective influence on the pattern of physiological responses to severe hyperthermia, suggesting that early endogenous expression of IL-6 may provide a protection from the development of organ damage and inflammation.

Does Decreased c-KIT Expression in Myenteric Interstitial Cells of Cajal Cause Decreased Spontaneous Contraction in Murine Proximal Colon?

Does Decreased c-KIT Expression in Myenteric Interstitial Cells of Cajal Cause Decreased Spontaneous Contraction in Murine Proximal Colon?

Alterations of colonic contractility in an interleukin-10 knockout mouse model of inflammatory bowel disease.

Background/AimsInflammatory bowel disease is commonly accompanied by colonic dysmotility and causes changes in intestinal smooth muscle contractility. In this study, colonic smooth muscle contractility in a chronic inflammatory condition was investigated using smooth muscle tissues prepared from interleukin-10 knockout (IL-10−/−) mice.MethodsPrepared smooth muscle sections were placed in an organ bath system. Cholinergic and nitrergic neuronal responses were observed using carbachol and electrical field stimulation with L-NG-nitroarginine methyl ester (L-NAME). The expression of interstitial cells of Cajal (ICC) networks, muscarinic receptors, neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) was observed via immunofluorescent staining.ResultsThe spontaneous contractility and expression of ICC networks in the proximal and distal colon was significantly decreased in IL-10−/− mice compared to IL-10+/+ mice. The contractility in response to carbachol was significantly decreased in the proximal colon of IL-10−/− mice compared to IL-10+/+ mice, but no significant difference was found in the distal colon. In addition, the expression of muscarinic receptor type 2 was reduced in the proximal colon of IL-10−/− mice. The nictric oxide-mediated relaxation after electrical field stimulation was significantly decreased in the proximal and distal colon of IL-10−/− mice. In inflamed colon, the expression of nNOS decreased, whereas the expression of iNOS increased.ConclusionsThese results suggest that damage to the ICC network and NOS system in the proximal and distal colon, as well as damage to the smooth muscle cholinergic receptor in the proximal colon may play an important role in the dysmotility of the inflamed colon.

IL-17A expression in HIV-specific CD8 T cells is regulated by IL-4/IL-13 following HIV-1 prime-boost immunization.

Although Th1 and Th2 cytokines can inhibit interleukin (IL)-17-secreting T cells, how these cells are regulated under different infectious conditions is still debated. Our previous studies have shown that vaccination of IL-4 and IL-13 gene knockout (KO) mice can induce high-avidity HIV K(d)Gag197-205-specific CD8 T cells with better protective efficacy. In this study, when IL-13, IL-4, STAT6 KO, and wild-type BALB/c mice were prime-boost immunized with an HIV poxviral modality, elevated numbers of IL-17A(+) splenic K(d)Gag197-205-specific CD8 T cells were observed in all the KO mice compared with the wt BALB/c control. Similarly, when wt BALB/c mice were immunized with IL-13Rα2-adjuvanted HIV vaccines (that transiently inhibited IL-13 activity and induced high-avidity CD8 T cells with enhanced protective efficacy), elevated IL-17A(+) K(d)Gag197-205-specific CD8 T cells were detected both in the lung and the spleen. However, at the transcriptional level, elevated TGF-β, IL-6, ROR-γt, and IL-17A mRNA copy numbers were mainly detected in IL-4 KO, but not the IL-13 KO mice. These data suggested that TGF-β, IL-6, ROR-γt, but not IL-23a, played a role in IL-17A regulation in K(d)Gag197-205-specific CD8 T cells. Collectively, our findings suggest that IL-4 and IL-13 differentially regulate the expression of IL-17A in K(d)Gag197-205-specific CD8 T cells at the transcriptional and translational level, respectively, implicating IL-17A as an indirect modulator of CD8 T cell avidity and protective immunity.

Interleukin 4 Deficiency Reverses Development of Secondary Pseudomonas aeruginosa Pneumonia During Sepsis-Associated Immunosuppression.

Interleukin 4 (IL-4) is an important cytokine that may modulate development of secondary bacterial pneumonia during sepsis-induced immunosuppression. We established an experimental model of cecal ligation and puncture (CLP)-induced sublethal polymicrobial sepsis followed by secondary Pseudomonas aeruginosa pulmonary infection, IL-4-deficient mice that underwent CLP were resistant to secondary pulmonary P. aeruginosa infection. As compared to wild-type mice, IL-4 knockout (KO) mice displayed improved survival and better bacterial clearance. Furthermore, IL-4 KO mice exhibited enhanced lung inflammation, neutrophil recruitment to airspaces, and elevated pulmonary cytokine production, with significantly increased tumor necrosis factor α (TNF-α) production. Neutralization of TNF-α could reverse the enhanced protection against secondary P. aeruginosa pneumonia in septic IL-4 KO mice, indicating that the resistance of septic IL-4 KO mice to secondary bacterial pneumonia was partially mediated by TNF-α. In addition, IL-4 priming displayed marked impairment of the ability of alveolar macrophages to phagocytose and kill P. aeruginosa in vitro, and this defect was associated with decreased activation of Akt, JNK, p38MAPK, and ERK intracellular signaling pathways by IL-4. Finally, neutralization of IL-4 in septic mice could improve survival and clearance of bacteria from the lungs of septic mice infected with P. aeruginosa. Our findings provide new insight for immunopathologic mechanisms of sepsis-induced secondary bacterial pneumonia.

Proximity-Based Single Cell Analysis of the Bone Marrow Niche Identifies Interleukin-18 As a Quiescence Regulator of Early Hematopoietic Progenitors

Background. Niche-derived signals are essential for maintenance and expansion of stem/progenitor cells in the bone marrow. Recently published studies used elegant genetic tools to dissect predominant cellular sources of known HSPC regulators, such as CXCL12 and kit-ligand (Ding et al, Nature 2012, Greenbaum et al, Nature 2013). However, strategies enabling identification of novel niche-derived factors are lacking. Here, we describe an approach which utilizes spatial proximity between osteolineage cells (OLC) and HSPC in the post-transplant bone marrow niche as a guide to niche factor discovery and reveals the role of Interleukin-18 (IL18) as a quiescence regulator of early hematopoietic progenitors.Results. We established a neonatal bone marrow (BM) transplantation model, in which adult BM-derived LKS CD34-Flk2- HSPCs fluorescently labeled with DiI were transplanted into irradiated newborn col2.3GFP recipients (in this mouse strain, the majority of OLCs are labeled with GFP). Forty-eight hours later, we obtained sections of femoral trabecular bone from transplanted mice and located rare OLCs harboring DiI-positive HSPCs in their immediate proximity. We harvested individual OLCs located within two cell diameters (proximal OLCs) and greater than five cell diameters (distal OLCs) from DiI-labeled HSPCs and performed comparative transcriptome analysis by single cell RNA-Seq. Remarkably, we found that proximal OLCs were distinct from their distal counterparts and showed a higher expression levels of known niche-associated molecules, as well as secreted factors not previously linked to regulatory niche function, including a pro-inflammatory cytokine IL18.Quantification of primitive subsets in the BM of IL18 knock-out (IL18KO) mice showed no abnormalities. However, cell cycle studies revealed that while long-term HSCs (which did not express the IL18 receptor) were unaffected by IL18 deletion, early progenitors - short-term HSC, multi-potent progenitor and common lymphoid progenitor – were more actively cycling. Accordingly, following sublethal exposure to 5-fluorouracil (5FU), IL18KO animals displayed a 2-fold increase in frequency of lin-kit+Sca1+ (LKS) cells, lin-kit+ myeloid progenitors and CLPs, as compared wild-type (WT) controls (Figure 1). On the other hand, exogenous administration of recombinant IL18 to 5FU-treated WT animals was associated with 25% mortality, while no deaths were observed in the vehicle-treated group. Taken together, these data illustrate that IL18 constrains the ability of the progenitor pool to respond to a genotoxic stress.Next, we tested if IL18 acts in a non cell-autonomous fashion. We transplanted progenitor-enriched bone marrow fraction (LKS cells) into IL18KO or WT hosts and observed an approximately 2-fold increase in both myeloid and lymphoid cells in peripheral blood of IL18KO animals. This finding was recapitulated in a reciprocal experiment, when LKS cells from IL18 receptor knock-out animals were transplanted into WT hosts, supporting the possibility that the effect of IL18 on progenitor proliferation is direct.Finally, we investigated if the loss of IL18-mediated quiescence can be therapeutically exploited in the transplant setting. To this end, we assessed survival of lethally irradiated WT and IL18KO mice following transplantation with a limiting BM dose, i.e. predicted to confer survival to 50% or less of WT animals. Notably, we observed reduced 30-day post-transplant mortality (33% versus 72%, p value = 0.05) in IL18KO animals, which likely resulted from accelerated progenitor expansion in the absence of IL18 in the host microenvironment (Figure 2).Conclusions.Our study demonstrates the capability of proximity-based single cell analysis of the post-transplant bone marrow microenvironment to identify a novel niche factor – IL 18. We show that IL18 specifically regulates quiescence of early hematopoietic progenitors, particularly under the conditions of stress hematopoiesis, such as exposure to 5FU or post-transplant hematopoietic expansion. The results presented here provide an insight into poorly understood molecular mechanisms of progenitor regulation by the niche. Moreover, improved post-transplant survival of animals in the absence of IL18 suggests that pharmacological inhibition of IL18 has the potential of improving clinical outcomes in transplant recipients. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Human adipose-derived stem cells attenuate inflammatory bowel disease in IL-10 knockout mice.

Inflammatory bowel disease (IBD) is a complex immunological disorder characterized by chronic inflammation caused mainly by unknown factors. The interleukin-10 knockout (IL-10 KO) mouse is a well-established murine model of IBD which develops spontaneous intestinal inflammation that resembles Crohn's disease. In the present study, human adipose-derived mesenchymal stem cells (hAMSCs) were administrated to IL-10 KO mice to evaluate the anti-inflammatory effects of hAMSCs that may attenuate the progress of or treat IBD. After IBD was induced by feeding the IL-10 KO mouse a 125-250 ppm piroxicam mixed diet for 1 week, 2×10(6) hAMSCs were injected into the peritoneum and the mice were switched to a normal diet. After 1 week, the mice were sacrificed and tissue samples were harvested. Tissue scores for inflammation and inflammation-related genes expression were determined. The hAMSC-treated group showed significantly reduced inflammatory changes in histological analysis. Reverse transcription-PCR analysis showed that RANTES, Toll-like receptor 9, and IL-4 expression levels were not significantly different between the groups while IL-12, INF-γ, and TNF-α levels were significantly decreased in the hAMSC treated group. hAMSC attenuated IBD in the IL-10 KO mice by suppressing inflammatory cytokine expression, was mediated by the type 1 helper T cell pathway. Even though only a single injection of hAMSCs was delivered, the effect influenced chronic events associated with inflammatory changes and demonstrated that hAMSCs are a powerful candidate for IBD therapy.

P-199 Lipocalin2 Affecting Bacterial Clearance in Activated Macrophages Contributes the Onset of Intestinal Inflammation

Lipocalin2 (Lcn2) is an extracellular transport protein expressed in neutrophil granules, activated macrophages, and epithelial cells of the respiratory and gastrointestinal tract in response to inflammatory signals. It is well known that Lcn2 exerts bacteriostatic effect by sequestrating iron-containing siderophores because iron is essential for the growth and virulence of most bacterial species. In this regards, Lcn2 is considered to be involved in innate immune response. However, little is known about the exact role of Lcn2 in the intestinal inflammation. The aim of this study is to investigate the role of Lcn2 in the intestinal inflammation using IL-10 knock out (KO) mice. (1) Time-dependent expression of Lcn2 in the colonic tissues of IL-10 KO mice was compared with that of C57BL/6 wild type (WT) mice by quantitative PCR (qPCR) and immunohistochemistry (IHC). (2) We compared intestinal inflammation between IL-10 KO and Lcn2/IL-10 double knock out (DKO) mice. Inflammation was quantified by histologic scores, TNF-alpha and IL-12p40 secretion from cultured colonic explants, and IFN-gamma and IL-17A secretion from unseparated mesenteric lymph node (MLN) cells stimulated with cecal bacterial lysate. (3) Thioglycollate-elicited peritoneal macrophages were obtained from IL-10 KO (IL-10 KO-derived TEPM) and Lcn2/IL-10 DKO mice (DKO-derived TEPM), and infected with Escherichia coli. We examined the bacteria-killing function of macrophages by gentamicin protection assay. Also, TNF-alpha secretion from infected macrophages was determined by enzyme-linked immune-sorbent assay (ELISA). (1) qPCR and IHC demonstrated that Lcn2 expression in the colonic tissues of IL-10 KO mice was apparently up-regulated compared to that of WT mice from early 4 weeks after birth, and further increased over time. (2) Histologic scores in DKO mice were significantly higher than those in IL-10 KO mice at 4 weeks after birth (DKO versus IL-10 KO mice, 15.0 ± 0.0 versus 0.6 ± 0.5). Gene expressions of IL-1beta, TNF-alpha, IFN-gamma, and IL-17A in the colonic tissues of DKO mice were significantly up-regulated compared to those in IL-10 KO mice. TNF-alpha and IL-12p40 secretion from colonic explants of DKO mice were significantly higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, TNF-alpha; 6.5 ± 0.7 versus 3.6 ± 1.2 ng/mL, IL-12p40; 30.6 ± 9.2 versus 5.3 ± 1.9 ng/mL). IFN-gamma and IL-17A production from MLN cells of DKO mice were higher than those of IL-10 KO mice (DKO versus IL-10 KO mice, IFN-gamma; 28.6 ± 33.7 versus 21.3 ± 32.7, P = 0.29, IL-17A; 2.5 ± 3.8 versus 0.6 ± 0.8 ng/mL, P < 0.05). (3) Uptake of bacteria was significantly enhanced in DKO-derived TEPM compared to that in IL-10 KO-derived TEPM (colony forming unit (CFU) at 2 hours after infection; DKO versus IL-10 KO-derived TEPM, 1461.7 ± 201.0 versus 868.3 ± 300.0 cfu/mL). In addition, intracellular survival of phagocytosed bacteria in DKO-derived TEPM persisted compared to that in IL-10 KO-derived TEPM (CFU at 8 hours after infection; DKO versus IL-10 KO-derived TEPM, 30.0 ± 17.9 versus 8.3 ± 13.3 cfu/mL, P < 0.05). Consequently, TNF-alpha production from DKO-derived TEPM significantly increased compared to IL-10 KO-derived TEPM (DKO versus IL-10 KO-derived TEPM, 14.6 ± 1.2 versus 4.0 ± 0.9 pg/mL at 16 hours after infection). Lcn2 affecting bacterial clearance in activated macrophages contributes to the onset of intestinal inflammation.

P-176 Epigenetic Regulation of Inflammation in IL-10-/- Mouse

Epigenetic modifications, exemplified by DNA methylation and histone methylation and acetylation, contribute to the regulation of gene expression. Abnormalities in these patterns of epigenetic modifications have been associated with disease processes, particularly cancer. However, the contribution of epigenetics to inflammatory bowel disease (IBD) is less known. Further, recent next-generation sequencing analyses uncovered mutations in interleukin-10 (IL-10) and the IL-10 receptor genes that associate with early onset of ulcerative colitis (UC) in children. Taken together with the IL-10 knockout (IL-10-/-) mouse model that develops chronic intestinal inflammation, it is clear that IL-10 signaling is essential to maintaining mucosal homeostasis. In concert with these observations, our previous studies identified Rc3h1 (Roquin-1), a RING finger ubiquitin ligase with immune modulatory and RNA stability functions, as a potential player in the IL-10 regulatory pathway. Given this complex etiology of IBD, we postulated that distinct epigenetic markers may demarcate individuals that are predisposed to developing IBD. To examine the epigenetic markers associated with defective IL-10 signaling, we investigated the epigenetic profile of Rc3h1 in mucosal biopsies from IL-10-/- mice. Using the IL-10-/- mouse model, we examined the epigenetic markers associated with intestinal inflammation. In these studies, chromatin immunoprecipitation (ChIP) followed by qPCR was used to obtain a detailed profile of methylation and acetylation at the Rc3h1 locus in the mucosal tissue and cells of IL-10-/- mice. ChIP analysis revealed that the Rc3h1 locus had increased methylation and reduced ubiquitylation in the IL-10-/- mice. In contrast, the Rc3h1 locus is poorly methylated and highly ubiquitylated in BALB/c mouse mucosal biopsies. Our data indicate that the Rc3h1 locus is transcriptionally silenced during inflammation and suggest a fundamental role for Rc3h1 in mucosal immune homeostasis. They further indicate that a lack of IL-10 signaling, as evidenced in the IL-10-/- mice, has significant epigenetic effects that may contribute to colitis.

Amylin-induced central IL-6 production enhances ventromedial hypothalamic leptin signaling.

Amylin acts acutely via the area postrema to reduce food intake and body weight, but it also interacts with leptin over longer periods of time, possibly via the ventromedial hypothalamus (VMH), to increase leptin signaling and phosphorylation of STAT3. We postulated that amylin enhances VMH leptin signaling by inducing interleukin (IL)-6, which then interacts with its gp130 receptor to activate STAT3 signaling and gene transcription downstream of the leptin receptor. We found that components of the amylin receptor (RAMPs1–3, CTR1a,b) are expressed in cultured VMH astrocytes, neurons, and microglia, as well as in micropunches of arcuate and ventromedial hypothalamic nuclei (VMN). Amylin exposure for 5 days increased IL-6 mRNA expression in VMH explants and microglia by two- to threefold, respectively, as well as protein abundance in culture supernatants by five- and twofold, respectively. Amylin had no similar effects on cultured astrocytes or neurons. In rats, 5 days of amylin treatment decreased body weight gain and/or food intake and increased IL-6 mRNA expression in the VMN. Similar 5-day amylin treatment increased VMN leptin-induced phosphorylation of STAT3 expression in wild-type mice and rats infused with lateral ventricular IgG but not in IL-6 knockout mice or rats infused with ventricular IL-6 antibody. Lateral ventricular infusion of IL-6 antibody also prevented the amylin-induced decrease of body weight gain. These results show that amylin-induced VMH microglial IL-6 production is the likely mechanism by which amylin treatment interacts with VMH leptin signaling to increase its effect on weight loss.

Transient low-dose methotrexate generates B regulatory cells that mediate antigen-specific tolerance to alglucosidase alfa.

Biologic drugs, including enzyme-replacement therapies, can elicit anti-drug Abs (ADA) that may interfere with drug efficacy and impact patient safety. In an effort to control ADA, we focused on identifying regimens of immune tolerance induction that may be readily available for clinical use. Data generated in both wild-type mice and a Pompe disease mouse model demonstrate that single-cycle, low-dose methotrexate can be as effective as three cycles of methotrexate in providing a long-lived reduction in alglucosidase alfa-specific ADA. In addition, we show that methotrexate induces Ag-specific tolerance as mice generate similar Ab responses to an irrelevant Ag regardless of prior methotrexate treatment. Methotrexate-induced immune tolerance does not seem to involve cell depletion, but rather a specific expansion of IL-10- and TGF-β-secreting B cells that express Foxp3, suggesting an induction of regulatory B cells. The mechanism of immune tolerance induction appears to be IL-10 dependent, as methotrexate does not induce immune tolerance in IL-10 knockout mice. Splenic B cells from animals that have been tolerized to alglucosidase alfa with methotrexate can transfer tolerance to naive hosts. We hypothesize that methotrexate induction treatment concomitant with initial exposure to the biotherapeutic can induce Ag-specific immune tolerance in mice through a mechanism that appears to involve the induction of regulatory B cells.

Abstract 3668: Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression

Abstract Background: The concept of tumor-promoting inflammation is a recognized enabling characteristic of cancers. Our group has demonstrated the significance of inflammation-related genes in poor outcome of children diagnosed with metastatic MYCN non-amplified neuroblastoma (NBL-NA). We investigated the effect of TAMs on neuroblastoma growth using a novel murine model. Methods: We have established a novel 100% penetrant NBL-NA murine model driven by SV40's Large T-antigen (NBL-Tag) and characterized expression of immune-related genes in tumors at various developmental stages. Single cell suspensions of murine tumors were analyzed using flow cytometry for determination of immune cells frequency and IL6 levels. NBL-Tag mice were crossed with IL6 knockout mice (NBL-Tag/IL6-/-) to investigate the role of IL6 in tumor pathogenesis. Results: NBL-Tag tumor growth coincided with IL6 levels becoming detectable and increasing in blood. Immune-related gene expression analysis of NBL-Tag adrenal glands in mice at pre-tumor development age (4, 8 and 12 week old) showed age-dependent increase in expression of IL6 and CCL2 (MCP1) compared to wild-type adrenal glands demonstrating early monocyte chemotaxis and establishment of a pro-inflammatory niche. FACS of tumors showed high expression of IL6, and infiltration by IL6-producing TAMs. In vitro co-culture of peritoneal macrophages from wild type mice increased proliferation of a NBL-Tag mice driven cell line (NBT2) by average of 50% over its basal rate, as measured by Brdu incorporation. This effect coincided with two-fold increase in IL6 protein level in the co-cultured media. However, the proliferative changes were only partially reversed by blocking IL6, suggesting other soluble factors also contribute to the observed effects. IL6 genetic ablation studies were conducted to generate NBL-Tag/IL6-/- mice, which convincingly demonstrated IL6 was not required for tumor growth and development. Macrophages from IL6-/- mice increased in vitro proliferation of tumor to similar levels as those from wild-type mice, while IHC staining of NBL-Tag/IL6-/- tumors revealed similar level of TAM infiltration and positivity for phospo-STAT3, indicating an IL6-independent pathway for STAT3 activation. Lastly, gene expression analyses revealed upregulation of MYC in tumor cells during co-culture and was confirmed by western analysis. Conclusions: This study demonstrates the recruitment and growth-promoting effects of TAMs in early pathogenesis of neuroblastoma in a novel MYCN non-amplified murine model. The macrophage and neuroblastoma cell cross-talk lead to tumor cell proliferation with phosphorylation of STAT3 and MYC upregulation in tumor cells. These effects were found to be independent of IL6 activity. Citation Format: Michael D. Hadjidaniel, Soheila Shirinbak, Sakunthala Muthugounder, Long Hung, Michael Sheard, Janahan Gnanachandran, Jin Kim, Richard Sposto, Hiroyuki Shimada, Shahab Asgharzadeh. Tumor-associated macrophages (TAMs) promote murine neuroblastoma tumor growth through upregulation of MYC and independent of IL6 expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3668. doi:10.1158/1538-7445.AM2014-3668

Long-term vitamin D3 supplementation does not prevent colonic inflammation or modulate bone health in IL-10 knockout mice at young adulthood.

Inflammatory bowel disease (IBD) is an idiopathic disease that can impair bone metabolism. Low vitamin D status has been implicated in its progress. This study used interleukin (IL)-10 knockout (KO) mice, that develop an intestinal inflammation when housed in a non-sterile environment, to determine if supplementation with vitamin D3 throughout life could mitigate inflammation and attenuate the lower bone mineral content (BMC) and density (BMD), and bone strength. Female IL-10 KO mice were randomized 25 or 5000 IU vitamin D3/kg diet throughout pregnancy and lactation. At weaning, offspring received the same or opposite diet as their mother until age three months. Body weight growth was similar among groups within a sex. At three months of age, there were no differences in inflammation and gene expression in the colon of offspring. Male offspring exposed to continuous 25 IU vitamin D3/kg diet had lower (p < 0.001) colonic VDR expression and those exposed only to low vitamin D3 until weaning had higher serum IL-6. There were no differences in femur or vertebral BMC, BMD or bone strength. In summary, long-term exposure to vitamin D3 did not attenuate intestinal inflammation or preserve bone mineral or bone strength. Thus, supplementation with vitamin D3 does not exert anti-inflammatory effects in this mouse model that mimics human inflammatory bowel disease.

Effects of ciprofloxacin on the enteritis of interleukin-10 knockout mice after ileocaecal resection

Objective To investigate the effects of ciprofloxacin on anastomotic stoma,small intestinal inflammation and fibrosis in interleukin-10 knock-out mice （mice model of Crohn′s disease （CD）） after ileocaecal resection.Methods Twelve interleukin-10 knockout male C3H/HeJBir mice were evenly divided into control group and ciprofloxacin group.The mice model was established with ileocaecal resection and ileocolostomy.There was no intervention in control group.Ciprofloxacin （50 mg/kg per day）was orally administrated in ciprofloxacin group for four weeks.All the mice were executed.The intestine around anastomotic stoma was taken for hematoxylin-eosin （HE）and Sirius red staining and histological scoring.The level of interferon （IFN）-γand tumor growth factor （TGF）-βin intestine around anastomotic stoma and small intestine was detected by enzyme-linked immunosorbent assay （ELISA） method.The content of procollagen α1 was determined by real-time polymerase chain reaction.The data were analyzed by t test.Results The inflammatory reaction level of anastomotic stoma and small intestine in mice of ciprofloxacin group （2.50±0.55 and 1 .67±0.52）was significantly lower than those of control group （3.67±0.63 and 2.83±0.75 ,t=-8.273,-5 .188,both P 〈0.05 ）.The IFN-γof anastomotic stoma and small intestine also significantly decreased （（0.72 ±0.22 ）pg/mg vs （1 .76 ±0.43 ）pg/mg, （0.23±0.12）pg/mg vs （0.78 ± 0.52 ）pg/mg;t = - 7.354,- 5 .268,both P 〈 0.05）.However, ciprofloxacin could not significantly decrease fibrosis in anastomotic stoma and small intestine of CD mice. There was no significant difference in the level of TGF-βcompared with control group.Conclusions The role of ciprofloxacin in improving inflammatory reation in anastomotic stoma of CD mice was limited. Maintenance therapy may be necessary to reduce the relapse of anastomotic stoma diseases. Key words: Ciprofloxacin; Inflammatory bowel diseases; Mice