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Type 1 interferon (IFN-1) promotes regulatory T-cell function to suppress inflammation in the mouse intestine, but little is known about IFN-1 in the human gut. We therefore assessed the influence of IFN-1 on CD4+ T-cells isolated from human colon tissue obtained from healthy controls or patients with inflammatory bowel disease (IBD). Immunofluorescent imaging revealed constitutive expression of IFNβ in human intestinal tissue, and colonic T-cells were responsive to exogenous IFN-1 as assessed by phosphorylation of signal transduction and activator of transcription 1 (pSTAT1) and induction of interferon stimulated genes (ISGs). Unlike their blood counterparts, intestinal T-cells from non-inflamed regions of IBD colon displayed enhanced responsiveness to IFN-1, increased frequency of pSTAT1+ cells, and greater induction of ISGs upon IFN-1 exposure in vitro. In healthy tissue, antibody neutralization of IFNβ selectively reduced T-cell production of the pro-regulatory cytokine interleukin-10 (IL-10) and increased IFNγ synthesis. In contrast, neutralization of IFNβ in IBD tissue cultures increased the frequency of T-cells producing inflammatory cytokines but did not alter IL-10 expression. These data support a role for endogenous IFN-1 as a context-dependent modulator of T-cell function that promotes regulatory activity in healthy human intestine, but indicate that the IFN-1/STAT1 pathway is dysregulated in inflammatory bowel disease.

Background & Aims T cells mediate the development of inflammation in inflammatory bowel disease (IBD). We investigated the effects of an antibody against CD3 called otelixizumab, which induces immune tolerance, in intestinal mucosa samples from patients. Methods Intestinal tissues were isolated from patients undergoing routine endoscopy or from patients undergoing intestinal surgery for colon cancer or IBD; healthy surrounding tissues were collected as controls. Isolated lamina propria mononuclear cells (LPMCs) and mucosal tissue explants were incubated with otelixizumab for 24 or 48 hours. Production of inflammatory cytokines was determined by enzyme-linked immunosorbent assay. Levels of 36 cytokines and chemokines and phosphorylation of 39 receptor tyrosine kinases and signaling molecules were measured using protein arrays. Immunoblot analysis was used to analyze T-cell transcription factors. Results Incubation of intestinal tissues or LPMCs with otelixizumab reduced production of interferon gamma, interleukin (IL)-17A, and other inflammatory cytokines and chemokines, simultaneously increasing production of IL-10. Mucosal biopsy specimens from patients with IBD retained inflammation-associated tyrosine phosphoprotein profiles ex vivo. Incubation of the inflamed tissue with otelixizumab reduced phosphorylation of these proteins to levels observed in control tissues. Otelixizumab also markedly reduced phosphorylation of proteins associated with T-cell receptor activation. Neutralization of IL-10 blocked the anti-inflammatory effects of otelixizumab. Conclusions We observed anti-inflammatory effects of anti-CD3 in inflamed intestinal tissues from patients with IBD. The antibody appears to down-regulate T-cell activation via IL-10.

Dendritic cells (DCs) are immunoregulatory antigen-presenting cells. DCs can be potent activators of naive T cells and influence the generation and homing of effector lymphocytes; they can also induce regulatory mechanisms and maintain non-responsiveness. In part, these different outcomes are influenced by exposure of the DC to microbial products. The regulatory role of DCs is of particular importance at mucosal surfaces such as the intestine, where the immune system exists in intimate association with the external antigenic environment. Much of what we know about DCs has come from studies on the cells outside the gastrointestinal tract but information about gut DCs and their contribution to the specialized immune environment of the gut is now emerging. Here, we review current knowledge on gut DCs, suggest models for interactions between DCs and the commensal microflora in health and disease, and discuss gut DCs as targets for probiotic therapies.

Different antigen-presenting cells elicit responses in different T-cell populations for primary activation, secondary stimulation and cytotoxic effector functions. Maturing bone marrow derived dendritic cells may acquire and process antigens, transport them to lymph nodes and activate naive T cells located there. By contrast, follicular dendritic cells, acquiring antigen-antibody complexes, maintain 'memory' via B-cell activation. Effector memory T cells recognize various tissue cells bearing antigen and we speculate that they may also target specialized antigen-presenting dendritic populations.

The life history of dendritic cells (DC) is now established from their origins from bone marrow stem cells, their distribution through blood to the tissues, and their movement via afferent lymph to lymph nodes for the initiation of immune responses. Bone-marrow stem cells, and occasional stem cells in peripheral blood (about 1 per 10(5) mononuclear cells), can give rise both to DC and macrophages (MO). In addition to stem cells in blood, after short-term culture of mononuclear cells, three major morphologic types of DC can be separated (types I-III), which probably represent the maturational pathway of this cell type; type II cells resemble tissue DC such as Langerhans cells and type III have a veiled morphology similar to that seen in cells of afferent lymph and in the interdigitating cells of the paracortex of lymph nodes. Functionally, DC cultured from peripheral blood are able to acquire large antigens and process them like Langerhans cells of the skin. They can also present antigens to stimulate primary T-cell responses, a property associated with lymph node DC. In tissues, DC appear to act as outposts of the immune system, acquire antigens, and, particularly in primary responses, carry the antigens to lymph nodes where they initiate T-cell responses. In secondary responses, activation of memory T cells in the periphery and the acquisition of antigen/antibody complexes by follicular dendritic cells of the lymph node follicles, which stimulate B cell memory, may be more important pathways for immune activation. DC may play a role in the development of many immunologic diseases including cancer, autoimmunity, and acquired immunodeficiency syndrome (AIDS).

Background Reduced mucosal concentrations of Faecalibacterium prausnitzii predict disease recurrence in patients with Crohn's disease (CD). Siblings of CD patients have elevated risk of developing CD and share aspects of disease phenotype compared with healthy controls (HC), including dysbiosis in the faecal microbiota.[1] No study has compared the mucosal microbiota of CD siblings with unrelated healthy controls. Aim: to determine whether dysbiosis is present in the mucosal microbiota of siblings of CD patients with reference to HC, and to apply 16S rRNA gene pyrosequencing in order to accomplish a more comprehensive characterisation of that dysbiosis. Methods Rectal biopsies were taken from 21 patients with quiescent CD, 17 of their healthy siblings and 19 unrelated HC. Total DNA was extracted using phenol/chloroform based method. The V1 to V3 region of the bacterial 16S ribosomal RNA gene was amplified using PCR, and microbiota composition resolved by 454 pyrosequencing. Sequence processing and analyses were performed using the open source Mothur software package (www.mothur.org). Results For each group the resulting species in the microbiota were classified into core (common and abundant among similar subjects) versus infrequent and rare.[2] In terms of both microbial diversity (measured by both the ShannonWiener and Simpson's indexes of diversity) and species richness, the core mucosal microbiota of both siblings and CD patients were significantly less diverse than HC. Although the diversity of the rare microbiota was lower in CD compared with HC, there was no difference in diversity of rare microbiota between siblings and HC. Metacommunity profiling using the Bray-Curtis (SBC) index of similarity with unweighted pair group averages showed that the core microbial metacommunity of siblings was more similar to CD (SBC=0.70) than to HC, whereas the rare microbial metacommunity of siblings was more similar to HC (SBC= 0.42). As in CD patients, the species that contributed most to the dissimilarity between healthy siblings and HC was F. prausnitzii, Table 1. Conclusions This is the first in depth case-control study of the mucosal microbiota in the siblings of CD patients. We report a dysbiosis characterised by reduced diversity of core microbiota and lower abundance of F. prausnitzii. Given that siblings of CD patients have elevated risk of developing CD, this dysbiosis in otherwise healthy people implicates microbiological processes in CD pathogenesis and risk.

Introduction The intestinal microbiota drive inflammation associated with Crohn9s disease.1 The composition of the intestinal microbiota can be influenced by prebiotic9s such as fructo-oligosaccharides (FOS). Preliminary data suggest that FOS can increase colonic bifidobacteria and induce immunoregulatory dendritic cell (DC) epithelial responses.2 Methods To assess the impact of a diet supplemented with FOS in patients with active Crohn9s disease using an appropriately powered randomised, double-blind, placebo-controlled trial. Patients with active Crohn9s disease (CDAI ≥ 220, plus one marker of inflammation) were randomised to receive 15 g/day FOS or placebo for 4 weeks. The primary endpoint was clinical response at week 4 (change in CDAI of −70 in the intention-to-treat (ITT) population). Multiple pre-specified secondary endpoints were analysed, including intracellular cytokine staining assessed by flow cytometry (n=27). Results In total, 103 patients were randomised to receive FOS (n=54) or placebo (n=49). The mean (SD) baseline CDAI was 283 (61.1) and 286 (61.5) in the FOS and placebo groups, respectively (p=0.79). Significantly more patients receiving FOS (n=14.26%) than placebo (n=4.8%) withdrew before the 4-week endpoint (p=0.018). There was no significant clinical benefit of FOS compared to placebo (Abstract 003). Patients receiving FOS, but not placebo, had a reduced proportion of IL-6+ intestinal DC, and increased DC staining of IL-10 (both p Conclusion An adequately powered trial of a diet supplemented with FOS in a well defined population of patients with active Crohn9s disease showed no clinical benefit despite impacting on DC immunology. Patient withdrawal was greater in patients receiving FOS.

INTRODUCTION: The immunopathology of ulcerative colitis (UC) relates to an inappropriate mucosal immune response to constituents of the intestinal microbiota in genetically susceptible individuals. HLA-DR+ lin-/dim (lin = anti-CD3,14,16,19,34) cells extracted from human intestinal tissue contain CD11c+ myeloid dendritic cells (DC) that contribute to the immunoregulatory events that normally limit inflammatory responses to commensal bacteria. Also present within the HLA-DR+ lin-/dim population are hitherto poorly characterized CD11ccells. We hypothesized that this population in the gut may also contribute to intestinal inflammation in active UC AIMS & METHODS: HLA-DR+ lin-/dim cells were identified in freshly isolated lamina propria mononuclear cells by multicolour flow cytometry, from patients with UC (n=48) and controls (n=22). The proportion and number of CD11c+ and CD11ccells within this population were determined. Surface expression of the activation/maturation markers CD40, CD86, Toll-like receptors (TLR), TLR-2, TLR-4, and the natural killer cell marker, CD56 was assessed on each cell population. Morphology was assessed by electron microscopy and T cell stimulatory capacity measured in allogenic mixed leucocyte reaction (MLR). RESULTS: Lamina propria colonic HLA-DR+ lin-/dim cells, of the CD11c-, but not the CD11c+ subset, were significantly increased in inflamed tissue of patients with UC compared with control tissue (UC 447±94 versus Control 104±17 per mg; p

expression of TNBS colitis in mice. Methods: Samples were obtained from healthy and inflamed colonic mucosa from 28 IBD patients (14 CD, 14 UC) not receiving therapy, and from 19 non-IBD controls. TNBS colitis was induced in 57 Balb/c mice and compared with 20 control mice. P38 and JNK expression and activity were quantified in colonic specimens by western blot and kinase assays. A pilot investigation with 36 mice was used to determine optimal dose and mode of SB203580 administration. Subsequendy, a controlled study was performed using SB203580 given 30 minutes before and 18 hours after induction of TNBScolitis or a saline/ethanol control. Mice were sacrificed 2 days after induction. Colitis severity was quantified by survival rates, macroscopic and microscopic colitis scoring systems, and quantification of myeloperoxidase (MPO), TNF-a mRNA and IL-1 iB mRNA, and p38 and JNK activity. Results: p38 and JNK expression and activity were similar in all specimens from humans. In mice, p38 and JNK expression or activity did not increase following TNBS colitis induction. Treatment with 5B203580 had no therapeutic effect on macroscopic or microscopic inflammation, nor did it modify the levels of MPO, TNF-Ix mRNA or IL-I~ mRNA. SB203580 treatment was associated with a 30% reduction in p38 activity, but JNK activity was unchanged. Conclusion: We were unable to demonstrate any consistent role in IBD for p38 and JNK MAPKs in either humans or in animal models of IBD. These results suggest that the inflammatory cascades in IBD act independently of p38 and JNK MAPK signaling pathways.

To design effective mucosal vaccines against pathogens such as HIV the relative importance of different immune mechanisms in mediating protection needs to be determined and strategies to optimize their induction should be developed.In a model system, Belyakov et al.1xThe importance of local mucosal HIV-specific CD8+ cytotoxic T lymphocytes for resistance to mucosal viral transmission in mice and enhancement of resistance by local administration of IL-12. Belyakov, I.M. et al. J. Clin. Invest. 1998; 102: 2072–2081Crossref | PubMedSee all References1 have dissected the mechanisms by which mucosal immunization with an HIV peptide protects mice against subsequent intrarectal challenge with recombinant vaccinia virus expressing HIV-1 gp160. The peptide immunogen stimulates both antibody and cytotoxic T lymphocyte (CTL) responses. However, the construction of the challenge virus was such that gp160 was expressed in infected host cells but was not incorporated into the virus particle itself, making a protective role for neutralizing antibody unlikely. Instead, long-lasting resistance to mucosal infection was mediated by CD8+ CTLs. Protective mucosal immunization induced CTLs in both local tissues and the spleen; subcutaneous immunization only induced CTLs in the spleen and was not protective, suggesting that local responses are necessary for protection.Recombinant interleukin 12 (rIL-12), mixed with the cationic lipofection agent DOTAP and administered with the peptide vaccine, increased CTL responses and protection. When given systemically, IL-12 did not enhance CTL generation. Locally applied low doses of cytokine might be an effective way of manipulating immune responses in other contexts; it might also avoid problems, such as toxicity, that are associated with systemic administration.

Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells Gong, J. et al . (1998) Proc. Natl. Acad. Sci. U. S. A . 95, 6279–6283