Early Life Stress Triggers Persistent Colonic Barrier Dysfunction and Exacerbates Colitis in Adult IL-10−/− Mice:

It has become increasingly evident that disease flares in the human inflammatory bowel diseases (IBD) are influenced by life stress. It is known that life stress can trigger disturbances in intestinal barrier function and activate proinflammatory signaling pathways, which are important contributors to intestinal inflammation and clinical disease; however, the exact mechanisms of stress-induced IBD exacerbations remain to be elucidated. Here we present a model of early life stress-induced exacerbation of colitis in IL-10-/- mice

Combined Serological, Genetic, and Inflammatory Markers Differentiate Non-IBD, Crohnʼs Disease, and Ulcerative Colitis Patients:

Previous studies have demonstrated that serological markers can assist in diagnosing inflammatory bowel disease (IBD). In this study, we aim to build a diagnostic tool incorporating serological markers, genetic variants, and markers of inflammation into a computational algorithm to examine patterns of combinations of markers to (1) identify patients with IBD and (2) differentiate patients with Crohn’s disease (CD) from ulcerative colitis (UC)

Enteroendocrine cells express functional Toll-like receptors

Intestinal epithelial cells (IECs) provide a physical and immunological barrier against enteric microbial flora. Toll-like receptors (TLRs), through interactions with conserved microbial patterns, activate inflammatory gene expression in cells of the innate immune system. Previous studies of the expression and function of TLRs in IECs have reported varying results. Therefore, TLR expression was characterized in human and murine intestinal sections, and TLR function was tested in an IEC line. TLR1, TLR2, and TLR4 are coexpressed on a subpopulation of human and murine IECs that reside predominantly in the intestinal crypt and belong to the enteroendocrine lineage. An enteroendocrine cell (EEC) line demonstrated a similar expression pattern of TLRs as primary cells. The murine EEC line STC-1 was activated with specific TLR ligands: LPS or synthetic bacterial lipoprotein. In STC-1 cells stimulated with bacterial ligands, NF-κB and MAPK activation was demonstrated. Furthermore, the expression of TNF and macrophage inhibitory protein-2 were induced. Additionally, bacterial ligands induced the expression of the anti-inflammatory gene transforming growth factor-β. LPS triggered a calcium flux in STC-1 cells, resulting in a rapid increase in CCK secretion. Finally, conditioned media from STC-1 cells inhibited the production of nitric oxide and IL-12 p40 by activated macrophages. In conclusion, human and murine IECs that express TLRs belong to the enteroendocrine lineage. Using a murine EEC model, a broad range of functional effects of TLR activation was demonstrated. This study suggests a potential role for EECs in innate immune responses

A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

Nuclear factor of activated T cells (NFAT) plays a critical role in the development and function of immune and non-immune cells. Although NFAT is a central transcriptional regulator of T cell cytokines, its role in macrophage specific gene expression is less defined. Previous work from our group demonstrated that NFAT regulates Il12b gene expression in macrophages. Here, we further investigate NFAT function in murine macrophages and determined the effects of a cell permeable NFAT inhibitor peptide 11R-VIVIT on experimental colitis in mice. Treatment of bone marrow derived macrophages (BMDMs) with tacrolimus or 11R-VIVIT significantly inhibited LPS and LPS plus IFN-γ induced IL-12 p40 mRNA and protein expression. IL-12 p70 and IL-23 secretion were also decreased. NFAT nuclear translocation and binding to the IL-12 p40 promoter was reduced by NFAT inhibition. Experiments in BMDMs from IL-10 deficient (Il10−/−) mice demonstrate that inhibition of IL-12 expression by 11R-VIVIT was independent of IL-10 expression. To test its therapeutic potential, 11R-VIVIT was administered systemically to Il10−/− mice with piroxicam-induced colitis. 11R-VIVIT treated mice demonstrated significant improvement in colitis compared to mice treated with an inactive peptide. Moreover, decreased spontaneous secretion of IL-12 p40 and TNF in supernatants from colon explant cultures was demonstrated. In summary, NFAT, widely recognized for its role in T cell biology, also regulates important innate inflammatory pathways in macrophages. Selective blocking of NFAT via a cell permeable inhibitory peptide is a promising therapeutic strategy for the treatment of inflammatory bowel diseases

An Antibiotic-Responsive Mouse Model of Fulminant Ulcerative Colitis

Paul Allen and colleagues describe the development of a mouse model of fulminant ulcerative colitis with multiple genetic hits in immune regulation which can be moderated by anti-cytokine therapy and broad-spectrum antibiotics

Functional Characterization of N297A, A Murine Surrogate for low-Fc Binding Anti-Human CD3 Antibodies

Several low- or non-FcR binding anti-human CD3 monoclonal antibodies have been under investigation for the treatment of autoimmune diseases. To model the mechanism of action of these anti-human CD3 mAbs in the murine system, an Fc-modified anti-mouse CD3 antibody (N297A) was generated. N297A exhibited similar biological effects as Fc-modified anti-human CD3 antibodies including rapid, reversible reduction in peripheral leukocyte numbers, differential modulation of activated versus resting T cells, and reduced levels of induced cytokine release compared to the non-Fc-modified parent antibody. In an in vivo model of colitis induced by adoptive transfer of IL–10-deficient cells, administration of N297A significantly reduced body weight loss. As N297A shared many functional characteristics of non-FcR binding anti-human CD3 mAbs both in vitro and in vivo, it provides a means to model the mechanisms of action of Fc-modified anti-human CD3 antibodies in mouse

Genetic Variation in the Familial Mediterranean Fever Gene (MEFV) and Risk for Crohn's Disease and Ulcerative Colitis

BACKGROUND AND AIMS: The familial Mediterranean fever (FMF) gene (MEFV) encodes pyrin, a major regulator of the inflammasome platform controlling caspase-1 activation and IL-1beta processing. Pyrin has been shown to interact with the gene product of NLRP3, NALP3/cryopyrin, also an important active member of the inflammasome. The NLRP3 region was recently reported to be associated with Crohn's disease (CD) susceptibility. We therefore sought to evaluate MEFV as an inflammatory bowel disease (IBD) susceptibility gene. METHODOLOGY AND RESULTS: MEFV colonic mucosal gene expression was significantly increased in experimental colitis mice models (TNBS p<0.0003; DSS p<0.006), in biopsies from CD (p<0.02) and severe ulcerative colitis (UC) patients (p<0.008). Comprehensive genetic screening of the MEFV region in the Belgian exploratory sample set (440 CD trios, 137 UC trios, 239 CD cases, 96 UC cases, and 107 healthy controls) identified SNPs located in the MEFV 5' haplotype block that were significantly associated with UC (rs224217; p = 0.003; A allele frequency: 56% cases, 45% controls), while no CD associations were observed. Sequencing and subsequent genotyping of variants located in this associated haplotype block identified three synonymous variants (D102D/rs224225, G138G/rs224224, A165A/rs224223) and one non-synonymous variant (R202Q/rs224222) located in MEFV exon 2 that were significantly associated with UC (rs224222: p = 0.0005; A allele frequency: 32% in cases, 23% in controls). No consistent associations were observed in additional Canadian (256 CD trios, 91 UC trios) and Scottish (495 UC, 370 controls) sample sets. We note that rs224222 showed marginal association (p = 0.012; G allele frequency: 82% in cases, 70% in controls) in the Canadian sample, but with a different risk allele. None of the NLRP3 common variants were associated with UC in the Belgian-Canadian UC samples and no significant interactions were observed between NLRP3 and MEFV that could explain the observed flip-flop of the rs224222 risk allele. CONCLUSION: The differences in association levels observed between the sample sets may be a consequence of distinct founder effects or of the relative small sample size of the cohorts evaluated in this study. However, the results suggest that common variants in the MEFV region do not contribute to CD and UC susceptibility.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

NLRP12 attenuates colon inflammation by maintaining colonic microbial diversity and promoting protective commensal bacterial growth

Inflammatory bowel diseases involve the dynamic interplay of host genetics, microbiome and inflammatory response. Here, we report that NLRP12, a negative regulator of innate immunity, is reduced in human ulcerative colitis by comparing monozygotic twins and other patient cohorts. In parallel, Nlrp12-deficiency in mice caused increased colonic basal inflammation, leading to a less-diverse microbiome, loss of protective gut commensal strains (Lachnospiraceae) and increased colitogenic strains (Erysipelotrichaceae). Dysbiosis and colitis susceptibility associated with Nlrp12-deficency were reversed equally by treatment with antibodies targeting inflammatory cytokines or by administration of beneficial commensal Lachnospiraceae isolates. Fecal transplants from specific pathogen free reared mice into germ-free Nlrp12-deficient mice showed that NLRP12 and the microbiome each contribute to immune signaling that culminates in colon inflammation. These findings reveal a feed-forward loop where NLRP12 promotes specific commensals that can reverse gut inflammation, while cytokine blockade during NLRP12-deficiency can reverse dysbiosis

Restoration of IFNγR Subunit Assembly, IFNγ Signaling and Parasite Clearance in Leishmania donovani Infected Macrophages: Role of Membrane Cholesterol

Despite the presence of significant levels of systemic Interferon gamma (IFNγ), the host protective cytokine, Kala-azar patients display high parasite load with downregulated IFNγ signaling in Leishmania donovani (LD) infected macrophages (LD-MØs); the cause of such aberrant phenomenon is unknown. Here we reveal for the first time the mechanistic basis of impaired IFNγ signaling in parasitized murine macrophages. Our study clearly shows that in LD-MØs IFNγ receptor (IFNγR) expression and their ligand-affinity remained unaltered. The intracellular parasites did not pose any generalized defect in LD-MØs as IL-10 mediated signal transducer and activator of transcription 3 (STAT3) phosphorylation remained unaltered with respect to normal. Previously, we showed that LD-MØs are more fluid than normal MØs due to quenching of membrane cholesterol. The decreased rigidity in LD-MØs was not due to parasite derived lipophosphoglycan (LPG) because purified LPG failed to alter fluidity in normal MØs. IFNγR subunit 1 (IFNγR1) and subunit 2 (IFNγR2) colocalize in raft upon IFNγ stimulation of normal MØs, but this was absent in LD-MØs. Oddly enough, such association of IFNγR1 and IFNγR2 could be restored upon liposomal delivery of cholesterol as evident from the fluorescence resonance energy transfer (FRET) experiment and co-immunoprecipitation studies. Furthermore, liposomal cholesterol treatment together with IFNγ allowed reassociation of signaling assembly (phospho-JAK1, JAK2 and STAT1) in LD-MØs, appropriate signaling, and subsequent parasite killing. This effect was cholesterol specific because cholesterol analogue 4-cholestene-3-one failed to restore the response. The presence of cholesterol binding motifs [(L/V)-X1–5-Y-X1–5-(R/K)] in the transmembrane domain of IFNγR1 was also noted. The interaction of peptides representing this motif of IFNγR1 was studied with cholesterol-liposome and analogue-liposome with difference of two orders of magnitude in respective affinity (KD: 4.27×10−9 M versus 2.69×10−7 M). These observations reinforce the importance of cholesterol in the regulation of function of IFNγR1 proteins. This study clearly demonstrates that during its intracellular life-cycle LD perturbs IFNγR1 and IFNγR2 assembly and subsequent ligand driven signaling by quenching MØ membrane cholesterol