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Intestinal fibrosis is a significant complication of Crohn's disease (CD). Gut microbiota reactive Th17 cells are crucial in the pathogenesis of CD; however, how Th17 cells induce intestinal fibrosis is still not completely understood.In this study, T-cell transfer model with wild-type (WT) and AregAlthough Th1 and Th17 cells induced intestinal inflammation at similar levels when transferred into TcrβxδThese findings reveal that Th17-derived AREG promotes intestinal fibrotic responses in experimental colitis and human patients with CD. Thereby, AREG might serve as a potential therapeutic target for fibrosis in CD.

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn's disease. CBir1 TCR transgenic naϊve CD4

Background & Aims Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. Methods We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn’s disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l -alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography–tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. Results Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. Conclusions Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

G protein-coupled receptor (GPR) 120 has been implicated in regulating metabolic syndromes with anti-inflammatory function. However, the role of GPR120 in intestinal inflammation is unknown. Here, we investigated whether and how GPR120 regulates CD4Dextran sodium sulfate (DSS)-induced colitis model, Citrobacter rodentium infection model, and CD4Deficiency of GPR120 in CD4Our findings show the role of GPR120 in regulating intestinal CD4

Innate lymphoid cells (ILCs) and CD4+ T cells produce IL-22, which is critical for intestinal immunity. The microbiota is central to IL-22 production in the intestines; however, the factors that regulate IL-22 production by CD4+ T cells and ILCs are not clear. Here, we show that microbiota-derived short-chain fatty acids (SCFAs) promote IL-22 production by CD4+ T cells and ILCs through G-protein receptor 41 (GPR41) and inhibiting histone deacetylase (HDAC). SCFAs upregulate IL-22 production by promoting aryl hydrocarbon receptor (AhR) and hypoxia-inducible factor 1α (HIF1α) expression, which are differentially regulated by mTOR and Stat3. HIF1α binds directly to the Il22 promoter, and SCFAs increase HIF1α binding to the Il22 promoter through histone modification. SCFA supplementation enhances IL-22 production, which protects intestines from inflammation. SCFAs promote human CD4+ T cell IL-22 production. These findings establish the roles of SCFAs in inducing IL-22 production in CD4+ T cells and ILCs to maintain intestinal homeostasis., Intestinal IL-22 has important regulatory effects on the barrier and intestinal diseases and its production is controlled by the intestinal microbiome. Here the authors show that intestinal immune cell production of IL-22 is regulated by short chain fatty acids via an aryl hydrocarbon receptor and HIF1α-mediated mechanism that protects mice from intestinal inflammation.

A case-control study of animal model of Modic changes (MCs) on rabbits.To evaluate the feasibility of inducing of MCs by injection of Propionibacterium acne (P. acnes) into the lumbar intervertebral discs of rabbits.MCs have been widely observed, and assume to be closely associated with low back pain and P. acnes, but there are few animal models showing the progression of MCs.Ten rabbits were used for the study. The L3-4 and L4-5 discs of all rabbits were injected with 100 μL P. acnes (1.6 × 10 CFU/mL) as P. acnes group, L2-3 disc were injected with 100 μL normal saline as vehicle, and L5-6 disc was untreated (blank). MCs were investigated by magnetic resonance imaging before operation and at 2 weeks, 1, 3, 4.5, 6, and 9 months postoperatively. Following sacrifice, histological analysis, blood test and micro-computed tomography were performed. Cytokine expression in nucleus and endplate tissues was quantified using real-time polymerase chain reaction.From 3 months postoperatively, the P. acnes group showed significantly decreased T1-weighted signal intensity, whereas the T2-weighted signal was significantly higher at 3 and 4.5 months, and then decreased remarkably at 6 and 9 months. Eleven of 20 inferior endplates were identified as type I MCs at 4.5 months, and 9 of 20 were identified as type II MCs at 9 months. Real-time polymerase chain reaction showed that expression of interleukin-1β, tumor necrosis factor α, interferon-γ, matrix metalloproteinase-9, and thrombospondin motifs-5 in the nucleus pulposus, and interleukin-1β, tumor necrosis factor α, and thrombospondin motifs-5 in the endplates, were significantly upregulated after injection of P. acnes. Histological slices of discs injected with P. acnes showed disc degeneration, endplate abnormalities, and inflammatory response, with micro-computed tomography confirming bone resorption.P. acnes infection of the disc can induce degeneration of the disc and an inflammatory response in the endplate region, presenting as MCs type I and II time dependently.N/A.

// Zhi Shan 1, * , Xianfeng Lin 1, * , Shengyu Wang 1, * , Xuyang Zhang 1, * , Yichuan Pang 2 , Shengyun Li 1 , Tianming Yu 1 , Shunwu Fan 1 and Fengdong Zhao 1 1 Department of Orthopaedic Surgery, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, 310016, China 2 MOE Key Laboratory of Macromolecular synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310027, China * These authors are contributed equally to this work Correspondence to: Fengdong Zhao, email: zhaodong68@hotmail.com Shunwu Fan, email: shunwu_fan@126.com Keywords: intervertebral disc degeneration, small intestinal submucosa, nucleus pulposus cell, decellularization, extracellular matrix Received: July 13, 2016 Accepted: February 27, 2017 Published: April 04, 2017 ABSTRACT We developed a nucleus pulposus cell (NPC)-modulated decellularized small intestinal submucosa (SIS) scaffold, and assessed the ability of this material to prevent Intervertebral disc degeneration (IVD) degeneration. Decellularized porcine SIS was squashed into particles and the biological safety and efficiency of these particles were evaluated. Next, SIS particles were seeded with rabbit NPCs, cultured for two months in vitro , decellularized again and suspended for intervertebral injection. We demonstrated that use of the decellularization protocol resulted in the removal of cellular components with maximal retention of extracellular matrix. The xenogeneic decellularized SIS did not display cytotoxicity in vitro and its application prevented NPC degradation. Furthermore, the xenogeneic SIS microparticles were effective in preventing IVD progression in vivo in a rabbit disc degeneration model. In conclusion, our study describes an optimized method for decellularized SIS preparation and demonstrated that the material is safe and effective for treating IVD degeneration.

Background Modic changes (inflammatory-like changes visible on magnetic resonance imaging [MRI] scans of a vertebral end plate) are common and are associated with low back pain, but their origin is unclear. To our knowledge, there have been no previous in vivo animal models of Modic changes. We hypothesized that Modic changes may be related to Propionibacterium acnes. Methods Ten New Zealand White rabbits were injected percutaneously with 1 mL of P. acnes (1.6 × 10 colony forming units/mL) into the subchondral bone superior to the L4-L5 and L5-L6 discs; 10 other control rabbits received sham injections at L4-L5 and 1 mL of normal saline solution (vehicle) at L5-L6. The subchondral bone superior to L3-L4 discs was untreated (blank). Development of Modic changes was investigated with MRI studies before the operation and at 2 weeks and 1, 2, 3, and 6 months postoperatively. Following sacrifice of the rabbits, histological analysis and microcomputed tomography (micro-CT) were performed, and blood samples were analyzed. Cytokine expression in end-plate tissues was quantified using real-time polymerase chain reaction (PCR). Results The group that received P. acnes showed significantly increased T1-weighted signal intensity at 6 months (mean and standard deviation, 3.43 ± 0.41 [range, 2.42 to 4.44] compared with 2.43 ± 0.66 [range, 1.98 to 2.87] before the injection) and higher T2-weighted signal intensity at 6 months. Positive culture results were obtained from 9 of 20 samples injected with P. acnes. Specimens with positive cultures had a higher prevalence of Modic changes (4 of 9 samples positive for P. acnes compared with 3 of 11 samples negative for P. acnes). Real-time PCR showed significantly increased expression of tumor necrosis factor-α, interleukin-1β, and interferon-γ following injection of P. acnes, but no changes were seen on histological analysis, micro-CT, or blood analysis. Conclusions P. acnes can survive within the end-plate region and can initiate mild inflammatory-like responses from host cells, leading to signal intensity changes in MRI scans, which potentially resemble Modic changes. Clinical relevance Disc degeneration and low back pain are associated with Modic changes. Our results indicate that Modic changes can be associated with P. acnes in the conjunction area of the disc and subchondral bone. These results may be useful for understanding the underlying mechanisms of Modic changes and related pain.

// Yingfeng Wei 1 , Chong Lu 1 , Jianing Chen 1 , Guangying Cui 1 , Lin Wang 1 , Tianming Yu 2 , Yue Yang 3 , Wei Wu 1 , Yulong Ding 1 , Lanjuan Li 1 , Toshimitsu Uede 4 , Zhi Chen 1 and Hongyan Diao 1 1 State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 2 Department of Orthopaedics, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China 3 Department of Biochemistry, University of Washington, Seattle, Washington, USA 4 Molecular Immunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan Correspondence to: Hongyan Diao, email: // Zhi Chen, email: // Keywords : high salt diet, Th17 cells, regulatory T cells, inflammatory bowel diseases, intestinal immunity, Immunology and Microbiology Section, Immune response, Immunity Received : August 01, 2016 Accepted : November 24, 2016 Published : December 01, 2016 Abstract This study focuses on characterizing the effect of a high salt diet (HSD) on intestinal immunity and the risk of inflammatory bowel diseases (IBD). We found that mice on a HSD had an increased frequency of IL-17A producing cells in the intestinal lamina propria (LP) compared to mice on a normal diet (ND). Furthermore, most intestinal IL-17A producing cells were CD4 + TCRβ + cells. A HSD increased the LP T helper 17 (Th17) responses in both the small and large intestines but did not increase the Th17 response of other gut-associated lymphoid organ. Although, HSD did not change the percentage of regulatory T (Treg) cells, HSD significantly inhibit secretion of IL-10 and the suppressive function of Treg cells. Moreover, we found that HSD exacerbates trinitrobenzenesulfonic acid (TNBS) induced colitis, and Th17 response was significantly increased in the colonic LP of HSD TNBS-treated mice compared with the ND TNBS-treated mice. This study demonstrates that HSD stimulates the intestinal Th17 response but inhibits the function of Treg cells. Moreover, HSD exacerbates TNBS induced mice colitis, suggesting that HSD disrupts the intestinal immunity and increases the risk of IBD.

Rho-associated kinase (ROCK) has been found to be involved in the pathogenesis of a variety of autoimmune diseases, but the role of ROCK in inflammatory bowel disease (IBD) is still elusive. In this study, we demonstrated that the levels of ROCK2, but not ROCK1, activity were significantly upregulated in peripheral blood mononuclear cells (PBMC) and inflamed mucosa from IBD patients using a ROCK activity assay, and that ROCK2 activity in intestinal mucosa was positively correlated with disease severity. Stimulation with TNF markedly upregulated ROCK2 activity in IBD CD4

Neutrophils are found to be infiltrated in tumour tissues of patients with colitis-associated cancer (CAC) and colorectal cancer (CRC), and CD177 is mainly expressed in neutrophils. In our study, expression of CD177 in tumour tissues from patients with CAC or CRC was analysed byquantitative real-time polymerase chain reaction, flow cytometry and immunohistochemistry. We recruited 378 patients with CRC, determined CD177 expression in tumours and examined its correlation with clinicopathological features. Moreover, CAC model was induced in wild-type and CD177-/- mice by azoxymethane/dextran sodium sulphate. CD177+ neutrophils were significantly increased in colon tumour tissues from patients with CRC or CAC compared with controls. Expression of CD177 mRNA and percentages of CD177+ neutrophils were also markedly increased in tumour tissues from CRC patients compared with controls. Patients with high density of CD177+ neutrophils had better overall survival and disease-free survival compared with controls. Multivariate analyses revealed that the density of CD177+ neutrophils was an independent factor in predicting overall survival and disease-free survival. Consistently, CD177 depletion aggravated azoxymethane/dextran sodium sulphate-induced CAC in mice. Expression of Ki67 and proliferating cell nuclear antigen was increased in tumour tissues from CD177-/- mice compared with wild-type counterparts. Moreover, CD177-/- neutrophils failed to migrate in response to fMLP[AU: Please expand fMLP, DN, TNM and HIF-1α.] stimulation compared with wild-type controls. Our data indicate that CD177+ neutrophils suppress epithelial cell tumourigenesis and act as an independent factor in predicting the prognosis in patients with CRC. CD177+ neutrophils may serve as a novel therapeutic target in the treatment and predict the prognosis of CAC and CRC.

Tripartite motif-containing (TRIM) 21 has been implicated in the pathogenesis of several types of autoimmune diseases.We sought to elucidate TRIM21 expression in patients with inflammatory bowel diseases (IBDs) and its role in regulating intestinal mucosal inflammation.TRIM21 expression was analyzed in the inflamed mucosa of patients with IBDs by means of quantitative RT-PCR and immunohistochemistry. Peripheral blood CD4TRIM21 was expressed predominantly in CD4TRIM21 plays a protective role in mucosal inflammation through inhibiting T

Neutrophils are accumulated in inflamed mucosa of IBD and play an important role in the pathogenesis. CD177 is expressed in neutrophils specifically and upregulated during inflammation. However, the role of CD177Expression of CD177 was analysed in peripheral blood and intestinal mucosa from patients with IBD using quantitative RT-PCR, flow cytometry and immunohistochemistry. CD177CD177CD177

Background & Aims Intestinal tissues from patients with inflammatory bowel disease (IBD) and colorectal cancer have increased expression of microRNA-301a (MIR301A) compared with tissues from patients without IBD. We studied the mechanisms of MIR301A in the progression of IBD in human tissues and mice. Methods We isolated intestinal epithelial cells (IECs) from biopsy samples of the colon from 153 patients with different stages of IBD activity, 6 patients with colitis-associated cancer (CAC), and 35 healthy individuals (controls), enrolled in the study in Shanghai, China. We measured expression of MIR301A and BTG anti-proliferation factor 1 (BTG1) by IECs using quantitative reverse-transcription polymerase chain reaction. Human colon cancer cell lines (HCT-116 and SW480) were transfected with a lentivirus that expresses MIR301A; expression of cytokines and tight junction proteins were measured by quantitative reverse transcription polymerase chain reaction, flow cytometry, and immunofluorescence staining. We generated mice with disruption of the microRNA-301A gene (MIR301A-knockout mice), and also studied mice that express a transgene-encoding BTG1. Colitis was induced in knockout, transgenic, and control (C57BL/B6) mice by administration of dextran sulfate sodium (DSS), and mice were given azoxymethane to induce colorectal carcinogenesis. Colons were collected and analyzed histologically and by immunohistochemistry; tumor nodules were counted and tumor size was measured. SW480 cells expressing the MIR301A transgene were grown as xenograft tumors in nude mice. Results Expression of MIR301A increased in IECs from patients with IBD and CAC compared with controls. MIR301A-knockout mice were resistant to the development of colitis following administration of DSS; their colon tissues expressed lower levels of interleukin 1β (IL1β), IL6, IL8, and tumor necrosis factor than colons of control mice. Colon tissues from MIR301A-knockout mice had increased epithelial barrier integrity and formed fewer tumors following administration of azoxymethane than control mice. Human IECs expressing transgenic MIR301A down-regulated expression of cadherin 1 (also called E-cadherin or CDH1). We identified BTG1 mRNA as a target of MIR301A; levels of BTG1 mRNA were reduced in inflamed mucosa from patients with active IBD compared with controls. There was an inverse correlation between levels of BTG1 mRNA and levels of MIR301A in inflamed mucosal tissues from patients with active IBD. Human colon cancer cell lines that expressed a MIR301A transgene increased proliferation; they had increased permeability and decreased expression of CDH1 compared with cells transfected with a control vector, indicating reduced intestinal barrier function. BTG1 transgenic mice developed less severe colitis than control mice following administration of DSS. SW480 cells expressing anti-MIR301A formed fewer xenograft tumors in nude mice than cells expressing a control vector. Conclusions Levels of MIR301A are increased in IECs from patients with active IBD. MIR301A reduces expression of BTG1 to reduce epithelial integrity and promote inflammation in mouse colon and promotes tumorigenesis. Strategies to decrease levels of MIR301A in colon tissues might be developed to treat patients with IBD and CAC.