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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.

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b+Ly6G+Ly6Clow with granulocytic phenotype (G-MDSCs) and CD11b+Ly6G-Ly6Chigh with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H2S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H2S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus (Hh), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh-infected mice. Following, we observed that chronic oral administration of the H2S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh-infected mice. Thus, we identify the metabolic pathway l-cysteine/H2S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

The intestine is the tissue of the body with the highest constitutive exposure to foreign antigen and is also a common entry portal for many local and systemic pathogens. Therefore, the local immune system has the unenviable task of balancing efficient responses to dangerous pathogens with tolerance toward beneficial microbiota and food antigens. As in most tissues, the decision between tolerance and immunity is critically governed by the activity of local myeloid cells. However, the unique challenges posed by the intestinal environment have necessitated the development of several specialized mononuclear phagocyte populations with distinct phenotypic and functional characteristics that have vital roles in maintaining barrier function and immune homeostasis in the intestine. Intestinal mononuclear phagocyte populations, comprising dendritic cells and macrophages, are crucial for raising appropriate active immune responses against ingested pathogens. Recent technical advances, including microsurgical approaches allowing collection of cells migrating in intestinal lymph, intravital microscopy, and novel gene-targeting approaches, have led to clearer distinctions between mononuclear phagocyte populations in intestinal tissue. In this review, we present an overview of the various subpopulations of intestinal mononuclear phagocytes and discuss their phenotypic and functional characteristics. We also outline their roles in host protection from infection and their regulatory functions in maintaining immune tolerance toward beneficial intestinal antigens.

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.

In nonhuman primates, Vγ9Vδ2+ (Vδ2)T cells proliferate and accumulate in mucosal tissues following microbial activation. Human Vδ2T cells produce proinflammatory cytokines in response to bacterial species that colonize the gut, but the role played by Vδ2T cells in intestinal immunity is unknown. We hypothesized that circulating Vδ2T cells can populate the human intestine and contribute to mucosal inflammation. Cell suspensions prepared from peripheral blood and intestinal biopsies were stimulated with microbial phosphoantigen (1-hydroxy-2-methyl-2-buten-4-yl 4-diphosphate [HDMAPP]) and analyzed by flow cytometry to determine Vδ2T cell phenotype, cytokine production, and proliferative potential. Circulating Vδ2T cells expressed gut-homing integrin α4β7 (>70%), which was coexpressed with skin-associated cutaneous leukocyte Ag by up to 15% of the total population. However, Vδ2T cell activation with HDMAPP and exposure to retinoic acid (signaling via retinoic acid receptor α) increased α4β7 expression and enhanced binding to mucosal addressin cell adhesion molecule-1 in vitro while simultaneously suppressing cutaneous leukocyte Ag, thereby generating a committed gut-tropic phenotype. Confocal microscopy and flow cytometry identified frequent Vδ2T cells that migrated out of human intestinal biopsies and comprised both CD103+ and CD103− subsets that produced TNF-α and IFN-γ upon phosphoantigen exposure, with more frequent cytokine-producing cells in the CD103− population. Activated intestinal Vδ2T cells expressed CD70 and HLA-DR but were unable to drive the proliferation of allogeneic naive CD4+ T cells. Instead, phosphoantigen-activated CD103− Vδ2T cells increased T-bet expression and enhanced IFN-γ production by autologous colonic αβ T cells via an IFN-γ–dependent mechanism. These data demonstrate that circulating Vδ2T cells display enhanced gut-homing potential upon microbial activation and populate the human intestinal mucosa, generating functionally distinct CD103+ and CD103− subsets that can promote inflammation by colonic αβ T cells.

Tumor-derived and bacterial phosphoantigens are recognized by unconventional lymphocytes that express a Vγ9Vδ2 T cell receptor (Vδ2 T cells) and mediate host protection against microbial infections and malignancies. Vδ2 T cells are absent in rodents but readily populate the human intestine, where their function is largely unknown. Here, we assessed Vδ2 T cell phenotype and function by flow cytometry in blood and intestinal tissue from Crohn’s disease patients (CD patients) and healthy controls. Blood from CD patients included an increased percentage of gut-tropic integrin β7–expressing Vδ2 T cells, while “Th1-committed” CD27-expressing Vδ2 T cells were selectively depleted. A corresponding population of CD27+ Vδ2 T cells was present in mucosal biopsies from CD patients and produced elevated levels of TNFα compared with controls. In colonic mucosa from CD patients, Vδ2 T cell production of TNFα was reduced by pharmacological blockade of retinoic acid receptor-α (RARα) signaling, indicating that dietary vitamin metabolites can influence Vδ2 T cell function in inflamed intestine. Vδ2 T cells were ablated in blood and tissue from CD patients receiving azathioprine (AZA) therapy, and posttreatment Vδ2 T cell recovery correlated with time since drug withdrawal and inversely correlated with patient age. These results indicate that human Vδ2 T cells exert proinflammatory effects in CD that are modified by dietary vitamin metabolites and ablated by AZA therapy, which may help resolve intestinal inflammation but could increase malignancy risk by impairing systemic tumor surveillance.

Transitional 2 B cells home to gut-associated lymphoid tissue and present an activated phenotype in healthy subjects, but gut immune compartments are depleted in SLE., We have tracked the fate of immature human B cells at a critical stage in their development when the mature B cell repertoire is shaped. We show that a major subset of bone marrow emigrant immature human B cells, the transitional 2 (T2) B cells, homes to gut-associated lymphoid tissue (GALT) and that most T2 B cells isolated from human GALT are activated. Activation in GALT is a previously unknown potential fate for immature human B cells. The process of maturation from immature transitional B cell through to mature naive B cell includes the removal of autoreactive cells from the developing repertoire, a process which is known to fail in systemic lupus erythematosus (SLE). We observe that immature B cells in SLE are poorly equipped to access the gut and that gut immune compartments are depleted in SLE. Thus, activation of immature B cells in GALT may function as a checkpoint that protects against autoimmunity. In healthy individuals, this pathway may be involved in generating the vast population of IgA plasma cells and also the enigmatic marginal zone B cell subset that is poorly understood in humans.

Background & Aims Reduced generation of all-trans retinoic acid (RA) by CD103+ intestinal dendritic cells (DCs) is linked to intestinal inflammation in mice. However, the role of RA in intestinal inflammation in humans is unclear. We investigated which antigen-presenting cells (APCs) produce RA in the human intestine and whether generation of RA is reduced in patients with Crohn's disease (CD). Methods Ileal and colonic tissues were collected from patients with CD during endoscopy or surgery, and healthy tissues were collected from subjects who were undergoing follow-up because of rectal bleeding, altered bowel habits, or cancer (controls). Cells were isolated from the tissue samples, and APCs were isolated by flow cytometry. Retinaldehyde dehydrogenase (RALDH) activity was assessed by Aldefluor assay, and ALDH1A expression was measured by quantitative real-time polymerase chain reaction. Macrophages were derived by incubation of human blood monocytes with granulocyte-macrophage colony–stimulating factor (GM-CSF). Results CD103+ and CD103− DCs and CD14+ macrophages from healthy human intestine had RALDH activity. Although ALDH1A1 was not expressed by DCs, it was the predominant RALDH enzyme isoform expressed by intestinal CD14+ macrophages and their putative precursors, CD14+ monocytes. RALDH activity was up-regulated in all 3 populations of APCs from patients with CD; in CD14+ macrophages, it was associated with local induction of ALDH1A1 expression. Blocking of RA receptor signaling during GM-CSF–mediated differentiation of monocytes into macrophages down-regulated CD14 and HLA-DR expression and reduced the development of tumor necrosis factor α–producing inflammatory macrophages. Conclusions RA receptor signaling promotes differentiation of human tumor necrosis factor α–producing inflammatory macrophages in vitro. In vivo, more CD14+ macrophages from the intestinal mucosa of patients with CD than from controls are capable of generating RA, which might increase the inflammatory phenotype of these cells. Strategies to reduce the generation of RA by CD14+ macrophages could provide new therapeutic options for patients with CD.

Background & Aims It is a challenge to determine the dynamics of stem cells within human epithelial tissues such as colonic crypts. By tracking methylation patterns of nonexpressed genes, we have been able to determine how rapidly individual stem cells became dominant within a human colonic crypt. We also analyzed methylation patterns to study clonal expansion of entire crypts via crypt fission. Methods Colonic mucosa was obtained from 9 patients who received surgery for colorectal cancer. The methylation patterns of Cardiac-specific homeobox, Myoblast determination protein 1, and Biglycan were examined within clonal cell populations, comprising either part of, or multiple adjacent, normal human colonic crypts. Clonality was demonstrated by following cytochrome c oxidase-deficient (CCO − ) cells that shared an identical somatic point mutation in mitochondrial DNA. Results Methylation pattern diversity among CCO − clones that occupied only part of a crypt was proportional to clone size; this allowed us to determine rates of clonal expansion. Analysis indicated a slow rate of niche succession within the crypt. The 2 arms of bifurcating crypts had distinct methylation patterns, indicating that fission can disrupt epigenetic records of crypt ancestry. Adjacent clonal CCO − crypts usually had methylation patterns as dissimilar to one another as methylation patterns of 2 unrelated crypts. Mathematical models indicated that stem cell dynamics and epigenetic drift could account for observed dissimilarities in methylation patterns. Conclusions Methylation patterns can be analyzed to determine the rates of recent clonal expansion of stem cells, but determination of clonality over many decades is restricted by epigenetic drift. We developed a technique to follow changes in intestinal stem cell dynamics in human epithelial tissues that might be used to study premalignant disease.

Introduction Vγ9Vδ2+ “unconventional” (Vδ2) T-cells are a population of circulating anti-microbial lymphocytes found only in higher primates and whose role in human intestinal immunity is unknown. In macaques, microbe-activated Vδ2T-cells expand and accumulate in mucosal tissues, and human Vδ2T-cells can produce key mediators of intestinal inflammation such as IFNγ, TNFα and IL-17A in response to bacterial species present among the gut microbiota. We therefore hypothesised that Vδ2T-cells might contribute to the pathogenesis of Crohn9s disease (CD). Methods Disaggregated intestinal biopsies and peripheral blood were analysed by flow-cytometry in CD patients (n=22), and healthy controls (n=36). Blood and biopsy-derived cell suspensions were stimulated with microbial phosphoantigen (HDMAPP) and IL-2 in vitro to determine Vδ2T-cell phenotype, cytokine production and proliferative potential in the presence or absence of azathioprine. Results Blood Vδ2T-cells proliferated, expressed “gut-homing” integrin β7, and produced IFNγ and TNFα upon activation with HDMAPP and IL-2 in vitro. Vδ2T-cells were also identified by confocal microscopy in both healthy and inflamed colonic lamina propria. In contrast to their blood counterparts, mucosal Vδ2T-cells expressed high levels of CD103 integrin, which is implicated in interactions with the intestinal epithelium. Although the frequency of mucosal Vδ2T-cells was low, these cells proliferated rapidly and up-regulated CD70 co-stimulatory molecule upon exposure to HDMAPP and IL-2 in vitro, consistent with responsiveness to the gut microbiota. In CD patients receiving azathioprine therapy, Vδ2T-cells were selectively lost from the blood and were markedly depleted from the lamina propria. Accordingly, physiological concentrations of azathioprine were sufficient to block HDMAPP activation of Vδ2T-cells in vitro. Conclusion Human Vδ2T-cells primarily reside in the blood but display gut-homing potential upon microbial activation and can be detected in the intestinal mucosa. Intestinal Vδ2T-cells may contribute to local pro-inflammatory responses against the gut microbiota but are depleted by azathioprine therapy in CD. Competing interests None declared.

Introduction All-trans retinoic acid (RA) has emerged as an important immunoregulatory molecule with specific functions in the intestine. RA is generated enzymatically by the sequential action of alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH) enzymes. In the mouse, RALDH expression is confined to CD103+ intestinal dendritic cells (DC) giving this subset the unique ability to mediate RA-dependent functions such as the imprinting of gut tropism on T cells. RALDH activity is downregulated in mouse models of inflammatory bowel disease (IBD) but little is known about its role in human intestine. The aim of this study was to assess RA-dependent immune regulation by human intestinal antigen presenting cells (APC). Methods Lamina propria mononuclear cells (LPMC) were extracted from intestinal biopsies by enzymatic digestion and analysed by multicolour flow cytometry. Purified subsets of intestinal APC were obtained by immunomagnetic or FACS sorting. Expression of ADH and RALDH enzymes was quantified by quantitative RT-PCR and RALDH activity assessed using the Aldefluor assay. The ability to induce the gut homing receptor α4β7 on T cells was assessed by flow cytometry following stimulation of CFSE-labelled naive CD4+ T cells. Results Induction of the gut homing receptor α4β7 on human T cells was RA dependent. Intestinal myeloid APC were potent stimulators of naive CD4+ T cells and induced high levels of α4β7. Both of these activities were attributed to the CD103+ APC fraction. However, analysis of ex vivo intestinal populations revealed RALDH activity in all myeloid APC populations studied: CD103+ and CD103− DC identified as HLA−DR+CD11c+Lin− (Lin=anti-CD3,14,16,19,34) as well as HLA−DR+CD11c+Lin+ monocyte-like cells. Lymphocytes had little or no Aldefluor activity. Comparison of intestinal DC from healthy controls and IBD patients showed similar RALDH activity in inflamed and non-inflamed tissue. RALDH activity was equivalent in CD103+ and CD103− DC. In contrast, the ADH RDH10 was expressed at levels 4.5-fold higher in CD103+ DC. Conclusion As in the mouse, RA-mediated induction of α4β7 is a property of the CD103+ subset of intestinal DC. However in divergence from murine data, this property is associated with restricted expression of the ADH enzyme RDH10 rather than “downstream” RALDH enzymes. These data imply that RA availability is regulated differently in mice and man, with expression of RDH10 providing an important control point in humans. Competing interests None declared.