Edward M. Giles

Increased production of retinoic acid by intestinal macrophages contributes to their inflammatory phenotype in patients with Crohn's disease.

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 Crohns 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.

Proinflammatory Vδ2+ T Cells Populate the Human Intestinal Mucosa and Enhance IFN-γ Production by Colonic αβ T Cells

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.

Systemic inflammatory response syndrome after major abdominal surgery predicted by early upregulation of TLR4 and TLR5

Objectives:To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. Background:Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. Methods:Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. Results:Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-&kgr;B/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon &agr;-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14++CD16+ monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). Conclusions:These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Regulation of human intestinal T-cell responses by type 1 interferon-STAT1 signaling is disrupted in inflammatory bowel disease

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.

EWTD: incompatible with subspecialty training?

Subspecialty paediatric training in the UK is organised through a national grid, with trainees centrally appointed for their final 3 years of training. Historically, the Royal College of Paediatrics and Child Health (RCPCH) College Specialty Advisory Committee has recommended that a trainee in paediatric gastroenterology, hepatology and nutrition (PGHAN) should spend at least 70% of working hours in that subspecialty. As a result of changes due to the European Working Time Directive (EWTD), there has been an increasing perception from both trainees and trainers that there is insufficient time spent within their subspecialty. To attempt to quantify the effects of the EWTD on subspecialty training in PGHAN, we surveyed current UK trainees. For comparison, 14 consultants …

OC-009 Human anti-microbial vδ2+ t-cells are novel intestinal lymphocytes with functional relevance in crohn's 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 Crohns 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.

Sa1778 Constitutive Type I Interferon, via STAT1 Activation, Selectively Promotes Regulatory T Cell Function in the Healthy Human Intestine, but Not in Inflammatory Bowel Disease

WT Balb/C mice (p<0.001 for each cytokine btw uninfected control and helminth-infected; N=at least 3 independent experiments), while no regulation of IFNγ and modest induction of IL4 or IL10 was seen in STAT6-/mice. Helminths did not induce TGFβ production in STAT6-/animals. In an acute GVHD and colitis model, helminths regulated WT donor T cell Th1 cytokine generation, serum Th1 cytokines, colitis and survival in WT and not STAT6-/recipients (p<0.05 for each parameter btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Helminth infection was associated with the induction of donor and recipient FoxP3+ regulatory T cells (Tregs) in WT and not STAT6-/bone marrow transplanted mice (p<0.05 btw. helminth infected WT and helminthinfected STAT6-/recipients, multiple experiments). Addition of TGFβ to STAT6-/T cell cultures restored T cell IL10 production and Treg generation that regulate Th1 inflammation. Conclusions: STAT6 is a critical transcription factor directing mucosal TGFβ producing Th3 cell generation. Helminths utilize STAT6 pathway to induce mucosal Tregs, trigger mucosal T cell IL10 secretion and regulate alloreactive colitis in a TGFβ dependent manner.

PWE-256 Intestinal inflammation regulates retinoic acid dependent imprinting of gut tropism by dendritic cells independently of RALDH expression

Introduction In the mouse, tissue-specific expression of retinaldehyde dehydrogenase (RALDH) enzymes by CD103+ intestinal dendritic cells (DC) enables them to generate all-trans retinoic acid (RA) and thereby imprint a gut-tropic phenotype on T cells via induction of homing receptors including α4β7 integrin. In health, RA from CD103+ also enhances their generation of Treg, contributing to intestinal homeostasis. In murine models of inflammatory bowel disease (IBD) RALDH expression by CD103+ DC is reduced but little is known about the function of RA in the human intestine. The aim of this study was to determine whether factors present in the healthy and inflamed human intestine regulate RA generation and activity. Methods Conditioned media (CM) were generated by culture of intestinal biopsies from healthy individuals and IBD patients (inflamed/non-inflamed regions). DC were differentiated from monocytes using GM-CSF and IL-4 in the presence or absence of CM. Expression of RA-generating enzymes was assessed by qRT-PCR and RALDH activity determined using the Aldefluor assay. Induction of α4β7 following activation of naïve allogeneic CD4+ T cells was determined by flow cytometry. Results Activation of naïve CD4+ T cells by human monocyte-derived DC resulted in RA-dependent upregulation of α4β7. These DC possessed retinal-inhibitable Aldefluor activity and expressed both alcohol dehydrogenase (RDH10) and RALDH (RALDH1,2,3) enzymes required for the generation of RA from retinol via retinal. Aldefluor activity was regulated by GM-CSF and RA, and reflected predominately the activity of RALDH2 as suggested by qRT-PCR analysis of sorted Aldefluor+ DC. CM significantly suppressed Aldefluor activity (p<0.0001) irrespective of whether generated from healthy or IBD tissue (inflamed or non-inflamed). The inhibitory effect of CM generated from healthy tissue could be partially reversed with the prostaglandin E2 (PGE2) EP-2 receptor antagonist AH6809 but this effect was less marked with CM from IBD tissue suggesting the involvement of distinct RALDH regulators. Although the effects of inflamed and non-inflamed CM on Aldefluor activity were similar, DC differentiated in the presence of inflamed CM induced significantly higher (p<0.05) levels of CD4 T cell α4β7 expression. Conclusion Factors generated in the human intestinal mucosa limit RALDH activity in DC and may thereby impact upon their generation of RA. Factors other than PGE2 are involved particularly in inflamed tissue. Intestinal mediators influence the imprinting of gut tropism independently of effects on RA-generating enzymes. Manipulation of RA availability may offer new therapeutic options in IBD. Competing interests None declared.

PWE-257 The role of RDH10 and RALDH enzymes in retinoic acid-mediated immune regulation by antigen presenting cells in the human intestine

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 naïve 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 naïve 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.