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Monocytes are mononuclear phagocytes that can differentiate to a variety of cell fates under the influence of their microenvironment and hardwired commitment. We found that inhibition of TRPM8 in human blood CD14+ monocytes during a critical 3-h window at the beginning of their differentiation into macrophages led to enhanced survival and LPS-driven TNFα production after 24 h. TRPM8 antagonism also promoted LPS-driven TNFα production in CD14+ monocytes derived from the intestinal mucosa. Macrophages that had been derived for 6 days under blockade of TRPM8 had impaired phagocytic capacity and were transcriptionally distinct. Most of the affected genes were altered in a way that opposed normal monocyte to macrophage differentiation indicating that TRPM8 activity promotes aspects of this differentiation programme. Thus, we reveal a novel role for TRPM8 in regulating human CD14+ monocyte fate and function.

Targeting leukocyte trafficking in the management of inflammatory bowel disease [IBD] has been a significant therapeutic advance over the past 15 years. However, as with other advanced therapies, phase III clinical trials report response to trafficking inhibitors in only a proportion of patients, with fewer achieving clinical remission or mucosal healing. Additionally, there have been significant side effects, most notably progressive multifocal leukoencephalopathy in association with the α4 inhibitor natalizumab. This article reviews the mechanisms underpinning T cell recruitment and residence, to provide a background from which the strength and limitations of agents that disrupt leukocyte trafficking can be further explored. The therapeutic impact of trafficking inhibitors is underpinned by the complexity and plasticity of the intestinal immune response. Pathways essential for gut homing in health may be bypassed in the inflamed gut, thus providing alternative routes of entry when conventional homing molecules are targeted. Furthermore, there is conservation of trafficking architecture between proinflammatory and regulatory T cells. The persistence of resident memory cells within the gut gives rise to local established pro-inflammatory populations, uninfluenced by inhibition of trafficking. Finally, trafficking inhibitors may give rise to effects beyond the intended response, such as the impact of vedolizumab on innate immunity, as well as on target side effects. With significant research efforts into predictive biomarkers already underway, it is ultimately hoped that a better understanding of trafficking and residence will help us predict which patients are most likely to respond to inhibition of leukocyte trafficking, and how best to combine therapies.

Background & aims: Siblings of people with Crohn's disease (CD) share aspects of the disease phenotype (raised faecal calprotectin, altered microbiota), which are markers of risk for their own development of CD. The aim was to determine whether supplementation with prebiotic oligofructose/inulin induces a prebiotic response and impacts the risk phenotype in CD patients and siblings. Methods: Patients with inactive CD (n = 19, CD activity index 50 μg/g) ingested oligofructose/inulin (15 g/day) for three weeks. Faecal microbiota (qPCR), intestinal permeability (lactulose-rhamnose test), blood T cells (flow-cytometry) and calprotectin (ELISA) were measured at baseline and follow-up. Results: Following oligofructose/inulin, calprotectin did not significantly change in patients (baseline mean 537 SD 535 μg/g; follow-up mean 974 SD 1318 μg/g, p = 0.08) or siblings (baseline mean 73 SD 90 μg/g: follow up mean 58 SD 72 μg/g, p = 0.62). Faecal Bifidobacteria and Bifidobacterium longum increased in patients and siblings; Bifidobacterium adolescentis and Roseburia spp. increased only in siblings. Compared with patients, siblings had a greater magnitude change in Bifidobacteria (+14.6% vs +0.4%, p = 0.028), B. adolescentis (+1.1% vs 0.0% p = 0.006) and Roseburia spp. (+1.5% vs −0.1% p = 0.004). Intestinal permeability decreased significantly in patients after oligofructose/inulin to a level that was similar to siblings. Blood T cell abundance reduced in siblings but not patients following oligofructose/inulin. Conclusions: Oligofructose/inulin supplementation did not significantly impact calprotectin, but the prebiotic effect was more marked in healthy siblings compared with patients with inactive CD and was associated with alterations in other CD risk markers. Future research should focus on dietary intervention, including with prebiotics, in the primary prevention of CD.

Cytokine storm can result from cancer immunotherapy or certain infections, including COVID-19. Though short-term immune-related adverse events are routinely described, longer-term immune consequences and sequential immune monitoring are not as well defined. In 2006, six healthy volunteers received TGN1412, a CD28 superagonist antibody, in a first-in-man clinical trial and suffered from cytokine storm. After the initial cytokine release, antibody effect-specific immune monitoring started on Day + 10 and consisted mainly of evaluation of dendritic cell and T-cell subsets and 15 serum cytokines at 21 time-points over 2 years. All patients developed problems with concentration and memory; three patients were diagnosed with mild-to-moderate depression. Mild neutropenia and autoantibody production was observed intermittently. One patient suffered from peripheral dry gangrene, required amputations, and had persistent Raynaud’s phenomenon. Gastrointestinal irritability was noted in three patients and coincided with elevated γδT-cells. One had pruritus associated with elevated IgE levels, also found in three other asymptomatic patients. Dendritic cells, initially undetectable, rose to normal within a month. Naïve CD8+ T-cells were maintained at high levels, whereas naïve CD4+ and memory CD4+ and CD8+ T-cells started high but declined over 2 years. T-regulatory cells cycled circannually and were normal in number. Cytokine dysregulation was especially noted in one patient with systemic symptoms. Over a 2-year follow-up, cognitive deficits were observed in all patients following TGN1412 infusion. Some also had signs or symptoms of psychological, mucosal or immune dysregulation. These observations may discern immunopathology, treatment targets, and long-term monitoring strategies for other patients undergoing immunotherapy or with cytokine storm. Electronic supplementary material The online version of this article (10.1007/s00262-020-02725-2) contains supplementary material, which is available to authorized users.

Following infusion of the anti-CD28 superagonist monoclonal antibody TGN1412, three of six previously healthy, young male recipients developed gastrointestinal irritability associated with increased expression of ‘gut-homing’ integrin β7 on peripheral blood αβT cells. This subset of patients with intestinal symptoms also displayed a striking and persistent expansion of putative Vδ2+ γδT cells in the circulation which declined over a 2-year period following drug infusion, concordant with subsiding gut symptoms. These data demonstrate that TGN1412-induced gastrointestinal symptoms were associated with dysregulation of the ‘gut-homing’ pool of blood αβ and γδT cells, induced directly by the antibody and/or arising from the subsequent cytokine storm. Electronic supplementary material The online version of this article (10.1007/s00262-020-02723-4) contains supplementary material, which is available to authorized users.

Gastrointestinal (GI) graft-versus-host disease (GVHD) is a major barrier in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The metabolite retinoic acid (RA) potentiates GI-GVHD in mice via alloreactive T cells expressing the RA receptor-α (RARα), but the role of RA-responsive cells in human GI-GVHD remains undefined. Therefore, we used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T cells in tissues and blood of patients who had received allo-HSCT and to characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after exposure to RA. RARαhi mononuclear cells were increased in GI-GVHD tissue, contained more cellular RA-binding proteins, localized with tissue damage, and correlated with GVHD severity and mortality. By using a targeted candidate protein approach, we predicted the phenotype of RA-responsive T cells in the context of increased microenvironmental interleukin-23 (IL-23). Sequential immunostaining confirmed the presence of a population of RARαhi CD8 T cells with the predicted phenotype that coexpressed the effector T-cell transcription factor T-bet and the IL-23–specific receptor (IL-23R). These cells were increased in GI- but not skin-GVHD tissues and were also selectively expanded in the blood of patients with GI-GVHD. Finally, functional approaches demonstrated that RA predominantly increased alloreactive GI-tropic RARαhi CD8 effector T cells, including cells with the phenotype identified in vivo. IL-23–rich conditions potentiated this effect by selectively increasing β7 integrin expression on CD8 effector T cells and reducing CD4 T cells with a regulatory cell phenotype. In summary, we have identified a population of RA-responsive effector T cells with a distinctive phenotype that is selectively expanded in human GI-GVHD and that represents a potential new therapeutic target.

Urban particulate matter (PM) enhances airway dendritic cell (DC) maturation in vitro. However, to date, there are no data on the association between exposure to urban PM and DC maturation in vivo. We sought to determine whether exposure of school-age children (8 to 14 y) to PM was associated with expression of CD86, a marker of maturation of airway conventional DCs (cDC). Healthy London school children underwent spirometry and sputum induction. Flow cytometry was used to identify CD86 and CCR7 expression on cDC subsets (CD1c+ cDC2 and CD141+ cDC1). Tertiles of mean annual exposure to PM ≤ 10 microns (PM10) at the school address were determined using the London Air Quality Toolkit model. Tertiles of exposure from the 409 children from 19 schools recruited were; lower (23.1 to 25.6 μg/m3, n = 138), middle (25.6 to 26.8 μg/m3, n = 126), and upper (26.8 to 31.0 μg/m3, n = 145). DC expression was assessed in 164/370 (44%) children who completed sputum induction. The proportion (%) of cDC expressing CD86 in the lower exposure tertile (n = 47) was lower compared with the upper exposure tertile (n = 49); (52% (44 to 70%) vs 66% (51 to 82%), p

Background & Aims There is limited evidence that a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) reduces gut symptoms in quiescent inflammatory bowel disease (IBD). We performed a randomized, controlled trial to investigate the effects of a low FODMAP diet on persistent gut symptoms, the intestinal microbiome, and circulating markers of inflammation in patients with quiescent IBD. Methods We performed a single-blind trial of 52 patients with quiescent Crohn's disease or ulcerative colitis and persistent gut symptoms at 2 large gastroenterology clinics in the United Kingdom. Patients were randomly assigned to groups that followed a diet low in FODMAPs (n = 27) or a control diet (n = 25), with dietary advice, for 4 weeks. Gut symptoms and health-related quality of life were measured using validated questionnaires. Stool and blood samples were collected at baseline and end of trial. We assessed fecal microbiome composition and function using shotgun metagenomic sequencing and phenotypes of T cells in blood using flow cytometry. Results A higher proportion of patients reported adequate relief of gut symptoms following the low FODMAP diet (14/27, 52%) than the control diet (4/25, 16%, P=.007). Patients had a greater reduction in irritable bowel syndrome severity scores following the low FODMAP diet (mean reduction of 67; standard error, 78) than the control diet (mean reduction of 34; standard error, 50), although this difference was not statistically significant (P = .075). Following the low FODMAP diet, patients had higher health-related quality of life scores (81.9 ± 1.2) than patients on the control diet (78.3 ± 1.2, P = .042). A targeted analysis revealed that in stool samples collected at the end of the study period, patients on the low FODMAP diet had significantly lower abundance of Bifidobacterium adolescentis, Bifidobacterium longum, and Faecalibacterium prausnitzii than patients on control diet. However, microbiome diversity and markers of inflammation did not differ significantly between groups. Conclusions In a trial of the low FODMAP diet vs a control diet in patients with quiescent IBD, we found no significant difference after 4 weeks in change in irritable bowel syndrome severity scores, but significant improvements in specific symptom scores and numbers reporting adequate symptom relief. The low FODMAP diet reduced fecal abundance of microbes believed to regulate the immune response, compared with the control diet, but had no significant effect on markers of inflammation. We conclude that a 4-week diet low in FODMAPs is safe and effective for managing persistent gut symptoms in patients with quiescent IBD. www.isrctn.com no.: ISRCTN17061468

Siblings of patients with Crohn's disease (CD) have elevated risk of developing CD and display aspects of disease phenotype, including faecal dysbiosis. In our recent article we have used 16S rRNA gene targeted high-throughput sequencing to comprehensively characterize the mucosal microbiota in healthy siblings of CD patients, and determine the influence of genotypic and phenotypic factors on the gut microbiota (dysbiosis). We have demonstrated that the core microbiota of both patients with CD and healthy siblings is significantly less diverse than controls. Faecalibacterium prausnitzii contributed most to core metacommunity dissimilarity between both patients and controls and between siblings and controls. Phenotype/genotype markers of CD risk significantly influenced microbiota variation between and within groups, of which genotype had the largest effect. Individuals with elevated CD-risk display mucosal dysbiosis characterized by reduced diversity of core microbiota and lower abundance of F. prausnitzii. The presence of this dysbiosis in healthy people at-risk of CD implicates microbiological processes in CD pathogenesis.

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.

Impaired immune responses and increased susceptibility to infection characterize acute inflammatory conditions such as pancreatitis and alcoholic hepatitis and are major causes of morbidity and mortality. However, the mechanisms that drive this apparent immune paresis remain poorly understood. Monocytes mediate host responses to damage and pathogens in health and disease, and three subsets of monocytes have been defined based on CD14 and CD16 expression. We sought to determine the changes in monocyte subsets in acute pancreatitis (AP) and acute alcoholic hepatitis (AAH), together with functional consequences and mechanisms that underlie this change. Peripheral blood mononuclear cells (PBMCs) from patients with AP or AAH were compared with healthy controls. Monocyte subsets were defined by HLA-DR, CD14, and CD16 expression. Changes in surface and intracellular protein expression and phosphorylation were determined by flow cytometry. Phenotype and function were assessed following stimulation with lipopolysaccharide (LPS) or other agonists in the presence of specific inhibitors of TNFα and a disintegrin and metalloproteinase 17 (ADAM17). Patients with AP and AAH had reduced CD14++CD16+ intermediate monocytes compared to controls. Reduction of intermediate monocytes was recapitulated ex vivo by stimulating healthy control PBMCs with Toll-like receptor (TLR) agonists LPS, flagellin or polyinosilic:polycytidylic acid (poly I:C). Stimulation caused shedding of CD14 and CD16, which could be reversed using the ADAM17 inhibitor, TMI005 but not direct inhibitors of TNFα, a known ADAM17-target. Culturing PBMCs from healthy controls resulted in expansion of intermediate monocytes, which did not occur when LPS was in the culture medium. Cultured intermediate monocytes showed reduced expression of CX3CR1, CCR2, TLR4, and TLR5. We found reduced migratory responses, intracellular signaling and pro-inflammatory cytokine production, and increased expression of IL-10. Stimulation with TLR agonists results in ADAM17-mediated shedding of phenotypic markers from CD16+ monocytes, leading to apparent "loss" of intermediate monocytes. Reduction in CD14++CD16- monocytes and increased CD14++CD16+ is associated with altered responses in functional assays ex vivo. Patients with AP and AAH had reduced proportions of CD14++CD16+ monocytes and reduced phosphorylation of NFκB and IL-6 production in response to bacterial LPS. Together, these processes may contribute to the susceptibility to infection observed in AP and AAH.

Background T cell trafficking contributes to inflammation and is a therapeutic target in IBD. Exposure of T cells to retinoic acid during activation induces expression of the gut trafficking integrin α4β7 and suppresses expression of cutaneous leukocyte antigen (CLA) which is required for entry into the skin. Although α4β7+ (gut homing) and CLA+ (skin homing) T cells are generally non-overlapping populations, we have recently observed a low frequency of α4β7+CLA+ (dual tropic) T cells in human blood; these cells may provide an immunological link between gut and skin. Here we investigate the generation of these population by DC in vitro, analyse the function of cells with different tissue tropism and examine the impact of intestinal and skin inflammation. Methods Using flow cytometry, expression of α4β7 and CLA was assessed in whole blood, gut tissues, and on proliferating cells generated by stimulation of naïve CD4+ T cells with monoclonal antibodies (anti-CD3/28/2), or with allogeneic colonic or monocyte-derived DC (moDC). Production of TNFα and IFNγ was measured by intracellular cytokine staining. Results α4β7+CLA+ T cells were found at low frequency in ileal lamina propria tissue as well as peripheral blood. Naïve CD4 T cells activated in vitro with antibodies expressed α4β7 but not CLA. In contrast, activation with DC resulted in the expression of both α4β7 and CLA and generated a dual tropic a4b7+CLA+ population. Colonic DC generated significantly more α4β7+ cells (p Conclusion CLA expression is induced by gut DC even though they potently upregulate α4β7. The dual tropic population generated is enriched for cells that produce inflammatory cytokines and it may provide a link between gut and skin inflammation. Monocyte-derived cells may contribute to the imprinting of T cell homing to the intestine and upregulation of this activity, specifically in active IBD, supports the concept that intestinal inflammation reprogrammes circulating monocytes.

Background Dysregulated Toll-like receptor (TLR)-mediated responses in intestinal monocyte-derived cells contribute to pathology in inflammatory bowel disease (IBD). JAK inhibitors regulate the production of inflammatory mediators and are an emerging therapy in IBD. Recent data suggest that combination therapies may be more effective than single agents, and therapies targeting aspects of TLR signalling pathways are being developed. We assessed the impact of inhibitors of IRAK4, TPL2 and JAK1 (IRAK4i, TPL2i and JAK1i, respectively) either alone or in combination on TLR-mediated cytokine responses and associated signalling pathways in classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14lowCD16+) human blood monocyte subsets. Methods Peripheral Blood Mononuclear Cells (PBMCs) or CD14+ monocytes from healthy volunteers were pre-incubated with TPL2i, IRAK4i or JAK1i individually or with JAK1i in combination with TPL2i or IRAK4i for 1 hour at 37°C. Cells were then stimulated at 37°C with agonists of TLR4 (E.coli lipopolysaccharide; LPS) or TLR2 (Pam3CYSK4). Intra-cellular staining and flow cytometry were used to measure phosphorylation of signalling molecules (NF-kappaB p65, p38 MAPK and ERK) or cytokines (TNF-alpha and IL-1-beta) in the three monocyte subsets after stimulation for 15 minutes or 3 hours respectively. Results Pre-incubation of PBMCs or CD14+ monocytes with IRAK4i, TPL2i or JAK1i individually led to a significant dose-dependent reduction in TLR-stimulated cytokine production (the frequency of TNF-alpha+ cells and the per cell level of IL-1-beta) within the classical, intermediate and non-classical monocyte subsets. The combination of JAK1i with IRAK4i or TPL2i had an additive effect. TPL2i reduced LPS-stimulated ERK phosphorylation, but p65 and p38 phosphorylation in response to LPS was not affected by any of the inhibitors. Conclusion Small molecule inhibitors of TPL2, IRAK4 and JAK1 dampen TLR4- and TLR2- mediated inflammatory responses in human monocyte subsets, by affecting pathways independent of p65 or p38. This is due to a cell intrinsic effect on CD14+ monocytes. TPL2 inhibition may in part act to dampen cytokine production in monocytes through the reduction of ERK phosphorylation. Combining JAK1i with IRAK4i or TPL2i may be more effective in reducing inflammatory responses than single agents. Further work is required to elucidate the mechanisms by which IRAK4i and JAK1i dampen TLR-mediated inflammation in monocyte subsets.

Background Tissue resident memory T cells (Trm) persist in peripheral tissues where they protect against pathogens but can also contribute to inflammatory disease. Recent work shows that Trm can re-enter the circulation and give rise to new effector T cell and Trm populations in secondary tissue sites. Such ‘ex -Trm’ derived from the skin co-express the residency marker CD103 with cutaneous leukocyte antigen (CLA), a marker associated with skin tropism. Many T cells in the human intestine are Trm but it is unknown whether these cells re-enter the circulation; the existence of gut-derived ex-Trm would have important implications for IBD treatment targeting the recruitment of circulating gut-homing cells. Here, we identify a population of blood cells that co-express CD103 and the gut-homing integrin a4b7 and determine how they are changed in IBD. Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteers and patients with active IBD (Crohn’s disease or ulcerative colitis). Cell surface staining and multi-colour flow cytometry were used to identify CD4+ and CD8+ subsets of antigen experienced (CD45RA-) conventional T cells (abTCR+) and determine expression of markers associated with tissue tropism and residency. Results Staining with antibodies to CD103 and b7 integrin were used to define CD103b7+a4b7+ putative gut ex-Trm based on the excess per cell expression of b7 resulting from its contribution to both integrins. A separate CD103b7+a4b7- population defined by 1:1 expression of CD103 and b7 contained CLA+ skin ex-Trm. Gut ex-Trm comprised 0.3% total circulating CD8+ T cells (range 0.02–1.4%), and 1.2% CD4+ T cells (range 0.3–3%). Gut and skin ex-Trm were phenotypically similar; both expressed the residency associated markers CD101 and CD9 but lacked expression of CD69. Gut ex-Trm were phenotypically distinct from both traditional CD103-a4b7+ gut tropic CD45RA- antigen-experienced T cells and naïve T cells; significantly more gut ex-Trm expressed CD101 and CD9 and fewer expressed CD27. The proportion of gut ex-Trm did not differ between heath and IBD. However, the ratio of gut:skin ex Trm was significantly reduced in active Crohn’s disease but not ulcerative colitis indicating a selective reduction in the population derived from the intestine. Conclusion A putative population of gut-derived ex-Trm can be identified in the blood of healthy controls and IBD patients. This population has a distinctive phenotype similar to that of previously described skin-derived ex-Trm. Circulating ex-Trm could link discreet areas of intestinal inflammation in Crohn’s disease and there is a selective loss of the gut ex-Trm population from the blood of these patients. The role of ex-Trm in IBD merits further study.

OBJECTIVES\ud \ud 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.\ud \ud BACKGROUND\ud \ud 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.\ud \ud METHODS\ud \ud 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.\ud \ud RESULTS\ud \ud 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-κB/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P

This article reports on the sixth scientific workshop of the European Crohn's and Colitis Organisation [ECCO] on the pathogenesis of extraintestinal manifestations [EIMs] in inflammatory bowel disease [IBD]. This paper has been drafted by 15 ECCO members and 6 external experts [in rheumatology, dermatology, ophthalmology, and immunology] from 10 European countries and the USA. Within the workshop, contributors formed subgroups to address specific areas. Following a comprehensive literature search, the supporting text was finalized under the leadership of the heads of the working groups before being integrated by the group consensus leaders.

IntroductionDiet may modulate immune responses in health and disease. We hypothesised a role for nutrient sensing pathways. Nutrient sensing enteroendocrine cells (EEC) within intestinal epithelium release local neuroendocrine peptides.1 We examined their effects on immune responses in intestinal mu

Introduction Many patients with quiescent inflammatory bowel disease (IBD) also experience functional-like GI symptoms. The low FODMAP diet improves GI symptoms in quiescent IBD in uncontrolled trials but there are no placebo-controlled trials to confirm this. We performed a randomised, placebo-controlled trial to assess the effects of a low FODMAP diet on GI symptoms, microbiota, inflammatory markers and circulating gut-tropic (α4β7+) T-cells in patients with quiescent IBD. Methods Patients with Crohn’s disease (CD) or ulcerative colitis (UC) were included. Quiescent IBD was defined as: 1) Physician Global Assessment, 2) faecal calprotectin [FC] Results Fifty two patients were randomised (27 low FODMAP diet, 25 sham diet). At the end of trial, more patients reported adequate relief of GI symptoms following the low FODMAP diet (14/27, 52%) than the sham diet (4/25, 16%) (p=0.007). Total IBS Severity Scoring System score decreased by 67 points (SD 78) during the low FODMAP diet and 34 points (SD 50) during the sham diet (p=0.075). Daily stool frequency was lower following low FODMAP diet (1.7 SD 0.5) than sham diet (2.1 SD 0.5) (p=0.012). Bacterial gene richness was not different between the groups at end of trial (p=0.620). Relative abundance of Bifidobacterium longum (1.24–7 vs 6.95–7, p=0.003) and B. adolescentis (1.99–7 vs 2.55–6, p=0.015) was lower following low FODMAP diet compared to sham diet. Between baseline and end of trial, Faecalibacterium prausnitzii SL3/3 M21/2 (2.30–6 vs 1.52–6, p=0.029) and F. prausnitzii KLE1255 (4.49–6 vs. 2.68–6,p=0.006) declined in the low FODMAP diet group. There was no difference in proportions of α4β7+T cells between groups at end of trial. Conclusions The low FODMAP diet improved functional-like GI symptoms in patients with quiescent IBD but reduces immunoregulatory species of the intestinal microbiota, though does not impact on inflammatory markers or α4β7+blood T-cell numbers.

SummaryBackground Airway dendritic cells (DC) are critical mediators of lung inflammation in asthma, but the characteristics of DC in the airways of healthy children, and children with asthma, are currently unknown. Objective We sought to identify changes in DC subset distribution and activation profile in paediatric asthma using flow cytometry to analyse induced sputum samples obtained from healthy and asthmatic children. Methods Lung function and atopic status were determined by spirometry and skin prick testing. Induced sputum samples were analysed using 7-colour flow cytometry to identify airway DC populations (lineage− HLA-DR+ sputum cells expressing either CD11c as conventional DC or CD123 as plasmacytoid DC). Results Sputum samples containing lower airway plugs were obtained from 10 healthy children and 8 children with asthma. Lineage− HLA-DR+ DC were successfully identified in all samples, and DC comprised a significantly higher proportion of sputum cells in children with asthma compared with age-matched healthy controls (1.29% vs. 0.67%, P = 0.02). DC expression of the costimulatory marker CD86 was significantly reduced in asthmatic children (73.4% vs. 59.7%, P = 0.04). Sputum DC also included numerous CD1c+ cells (mean 57% of the total DC population) and low frequencies of cells expressing the subset markers CD141 or CD123, although the proportions of these did not differ between groups. Conclusions Airway DC can be identified and characterized non-invasively using flow cytometry to analyse paediatric sputum samples. Our data reveal that children with steroid-treated asthma exhibit increased frequency of airway DC with reduced expression of the costimulatory marker CD86, suggesting altered trafficking and/or maturation of these cells either due to asthma or steroid therapies.

The pathogenesis of inflammatory bowel disease is incompletely understood but results from a dysregulated intestinal immune response to the luminal microbiota. CD4 T cells mediate tissue injury in the inflammatory bowel disease-associated immune response. Dendritic cells (DC) generate primary T-cell responses and mediate intestinal immune tolerance to prevent overt inflammation in response to the gut microbiota. However, most information regarding function of intestinal DC has come from mouse models, and information in humans is scarce. We show here that intestinal DC subsets are skewed in ulcerative colitis (UC) in humans, with a loss of CD103 lymph-node homing DC; this intestinal DC subset preferentially generates regulatory T cells in mice. We show infiltrates of DC negative for myeloid marker CD11c, with enhanced expression of Toll-like receptors for bacterial recognition. After mixed leukocyte reaction, DC from the inflamed UC colon had an enhanced ability to generate gut-specific CD4 T cells with enhanced production of interleukin-4 but a loss of interferon γ and interleukin-22 production. Conditioning intestinal DC with probiotic strain Lactobacillus casei Shirota in UC partially restored their normal function indicated by reduced Toll-like receptor 2/4 expression and restoration of their ability to imprint homing molecules on T cells and to generate interleukin-22 production by stimulated T cells. This study suggests that T-cell dysfunction in UC is driven by DC. T-cell responses can be manipulated indirectly through effects of bacterial conditioning on gut DC with implications for immunomodulatory effects of the commensal microbiota in vivo. Manipulation of DC to allow generation of DC-specific therapy may be beneficial 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.

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.

The mechanism of CD4 T-cell decline in HIV-1 infection is unclear, but the association with plasma viral RNA load suggests viral replication is involved. Indeed, viremic controller patients with low viral RNA loads typically maintain high CD4 T-cell counts. Within a local cohort of 86 viremic controllers, we identify a subgroup (18 "discord controllers") with low CD4 T-cell counts that present clinical uncertainty. The underlying mechanism accounting for CD4 T-cell decline in the face of low or undetectable plasma (RNA) viral load remains unresolved. The objective of this study was to investigate the viral and host immune system dynamics in discord controllers by measuring cellular HIV-1 DNA load, T-cell populations, and T-cell activation markers.We compared discord controllers (viral RNA load2000 copies/mL,450 CD4 T-cells/mm) with typical controllers (viral RNA load2000 copies/mL,450 CD4 T-cells/mm) and progressors (viral RNA load10,000 copies/mL,450 CD4 T-cells/mm). We quantified CD4/CD8 naive/central memory/effector memory subsets (CD45RA/RO ± CD62L), activation levels (CD38HLA-DR), and HIV-1 DNA load.Discord controllers resembled progressors showing high viral DNA load, depletion of naive CD4 T-cells, and higher activation in all CD4 T-cell subsets, compared with typical controllers. They were similar to typical controllers with lower CD8 T-cell activation compared with progressors.Our data are consistent with a relationship between CD4 T-cell activation and disease progression. HIV-1 DNA load may be a better marker of viral replication and disease progression than viral RNA load. Lower level CD8 T-cell activation correlates with low viral RNA load but not with disease progression or viral DNA load.

Background: Dendritic cells (DC) dictate not only the type of T-cell immunity, but also homing patterns of T cells in mice. In humans, we characterized normal human gut DC and tested whether gut-specific homeostatic DC could be generated from blood precursors by factors in the gut microenvironment. Methods: We characterized the phenotype and function of healthy human gut DC compared with blood and skin DC, and studied whether conditioning of blood DC in the presence of colonic biopsy supernatants (Bx-SN) induced gut-like phenotype and functions. Results: Blood DC mostly expressed both gut and skin homing markers, indicating potential to migrate to both major immune surface organs, and induced multi-homing T cells. However, DC within gut or skin did not demonstrate this multi-homing phenotype, were tissue-specific, and induced tissue-specific T cells. Human gut DC were less stimulatory for allogeneic T cells than their dermal and blood counterparts. Human blood DC cultured in vitro lost homing marker expression. Conditioning of human enriched blood DC with colonic Bx-SN from healthy controls induced a gut-homing phenotype and a homeostatic profile. Moreover, Bx-SN-conditioned DC demonstrated a restricted T-cell stimulatory capacity and preferentially induced gut-specific T cells. Retinoic acid and transforming growth factor beta (TGF-β) mediated the acquisition of the gut-homing and homeostatic properties, respectively, induced by colonic Bx-SN on blood enriched DC. Conclusions: Tissue-specific factors manipulate immunity via modulating characteristics of DC and may provide tools to generate tissue-specific immunotherapy. (Inflamm Bowel Dis 2011;)

Patients with Crohn's disease (CD) have an intestinal dysbiosis with components of the microbiota exerting differential immune effects. Smoking is associated with an increased incidence of CD, more frequent relapse, and greater burden of surgery. This study aimed to investigate the association between smoking and the intestinal microbiota in patients with active CD.Patients with active CD (n = 103) and healthy controls (n = 66) were recruited and demographic and clinical data recorded including current smoking behavior. Fecal samples were collected and analyzed by fluorescent in situ hybridization using probes targeting 16S rRNA of bacteria previously shown to be altered in active CD (bifidobacteria, bacteroides, Clostridium coccoides-Eubacterium rectale, Escherichia coli, and Faecalibacterium prausnitzii).In total, 29/101 (29%) patients with CD and 8/58 (14%) controls were current smokers (P = 0.032). Following multivariate analysis, smoking was found to have a significant and independent effect on the microbiota of patients with CD, with higher Bacteroides-Prevotella in smokers (38.4%) compared with nonsmokers (28.1%) (F((1,93)) = 12.6, P = 0.001). Healthy controls who smoked also had higher Bacteroides-Prevotella (34.8%) than nonsmokers (24.1%) (F((1,55)) = 4.5, P = 0.038). In the pooled multivariate analysis, patients with CD had higher bifidobacteria (F((1,156)) = 30.5, P0.001), higher Bacteroides-Prevotella (F((1,156)) = 6.5, P = 0.012), and lower F. prausnitzii (F((1,156)) = 3.8, P = 0.052) compared with healthy controls.Smokers have luminal microbiota that consist of significantly higher bacteroides. Investigation of whether this is one mechanism through which the negative effects of smoking on CD are mediated is warranted.

Altered intestinal dendritic cell (DC) function underlies dysregulated T-cell responses to bacteria in Crohn's disease (CD) but it is unclear whether composition of the intestinal microbiota impacts local DC function. We assessed the relationship between DC function with disease activity and intestinal microbiota in patients with CD.Surface expression of Toll-like receptor (TLR)-2, TLR-4, and spontaneous intracellular interleukin (IL)-10, IL-12p40, IL-6 production by freshly isolated DC were analyzed by multicolor flow cytometry of cells extracted from rectal tissue of 10 controls and 28 CD patients. Myeloid DC were identified as CD11c(+) HLA-DR(+lin-/dim) cells (lin = anti-CD3, CD14, CD16, CD19, CD34). Intestinal microbiota were analyzed by fluorescent in situ hybridization of fecal samples with oligonucleotide probes targeting 16S rRNA of bifidobacteria, bacteroides-prevotella, C. coccoides-E. rectale, and Faecalibacterium prausnitzii.DC from CD produced higher amounts of IL-12p40 and IL-6 than control DC. IL-6(+) DC were associated with the CD Activity Index (r = 0.425; P = 0.024) and serum C-reactive protein (CRP) (r = 0.643; P = 0.004). DC expression of TLR-4 correlated with disease activity. IL-12p40(+) DC correlated with ratio of bacteroides: bifidobacteria (r = 0.535, P = 0.003). IL-10(+) DC correlated with bifidobacteria, and IL-6(+) DC correlated negatively with F. prausnitzii (r = -0.50; P = 0.008). The amount of TLR-4 on DC correlated negatively with the concentration of F. prausnitzii.IL-6 production by intestinal DC is increased in CD and correlates with disease activity and CRP. Bacterially driven local IL-6 production by intestinal DC may overcome regulatory activity, resulting in unopposed effector function and tissue damage. Intestinal DC function may be influenced by the composition of the commensal microbiota.

Crohn's disease (CD) is an incurable intestinal disorder in which the loss of immune tolerance to the commensal gut microbiota leads to chronic inflammation. The reason this occurs in specific individuals is unclear; however, a genetic predisposition is fundamental and relatives of patients with CD are at significant risk of developing the disease. Knowledge relating to the genetic loci that predispose to CD is accumulating, which raises the possibility of disease prediction and prevention in susceptible populations. However, the genetic basis of CD is complex and genotyping alone is likely to be insufficient to predict disease risk accurately. Specific physiological abnormalities associated with CD, such as increased intestinal permeability and raised faecal calprotectin, are also abnormal in some relatives of patients with CD. The combination of genotypic factors and biomarkers of risk makes the development of models of disease prediction a realistic possibility. Furthermore, enhanced understanding of the genotype and phenotype of the at-risk state in relatives of patients with CD allows the earliest stages in the pathogenesis of CD to be investigated and may allow intervention to prevent disease onset. This article reviews current knowledge of the at-risk phenotype in relatives of patients with CD and focuses on the implications for the design of future studies.

The commensal intestinal microbiota drive the inflammation associated with Crohn's disease. However, bacteria such as bifidobacteria and Faecalibacterium prausnitzii appear to be immunoregulatory. In healthy subjects the intestinal microbiota are influenced by prebiotic carbohydrates such as fructo-oligosaccharides (FOS). Preliminary data suggest that FOS increase faecal bifidobacteria, induce immunoregulatory dendritic cell (DC) responses and reduce disease activity in patients with Crohn's disease.To assess the impact of FOS in patients with active Crohn's disease using an adequately powered randomised double-blind placebo-controlled trial with predefined clinical, microbiological and immunological end points. Patients with active Crohn's disease were randomised to 15 g/day FOS or non-prebiotic placebo for 4 weeks. The primary end point was clinical response at week 4 (fall in Crohn's Disease Activity Index of ≥ 70 points) in the intention-to-treat (ITT) population.103 patients were randomised to receive FOS (n = 54) or placebo (n = 49). More patients receiving FOS (14 (26%) vs 4 (8%); p = 0.018) withdrew before the 4-week end point. There was no significant difference in the number of patients achieving a clinical response between the FOS and placebo groups in the ITT analysis (12 (22%) vs 19 (39%), p = 0.067). Patients receiving FOS had reduced proportions of interleukin (IL)-6-positive lamina propria DC and increased DC staining of IL-10 (p0.05) but no change in IL-12p40 production. There were no significant differences in the faecal concentration of bifidobacteria and F prausnitzii between the groups at baseline or after the 4-week intervention.An adequately powered placebo-controlled trial of FOS showed no clinical benefit in patients with active Crohn's disease, despite impacting on DC function. ISRCTN50422530.

Dendritic cells (DCs) play a key role in discriminating between commensal microorganisms and potentially harmful pathogens and in maintaining the balance between tolerance and active immunity. The regulatory role of DC is of particular importance in the gut where the immune system lies in intimate contact with the highly antigenic external environment. Intestinal DC constantly survey the luminal microenvironment. They act as sentinels, acquiring antigens in peripheral tissues before migrating to secondary lymphoid organs to activate naive T cells. They are also sensors, responding to a spectrum of environmental cues by extensive differentiation or maturation. Recent studies have begun to elucidate mechanisms for functional specializations of DC in the intestine that may include the involvement of retinoic acid and transforming growth factor-β. Specialized CD103(+) intestinal DC can promote the differentiation of Foxp3(+) regulatory T cells via a retinoic acid-dependent process. Different DC outcomes are, in part, influenced by their exposure to microbial stimuli. Evidence is also emerging of the close interaction between bacteria, epithelial cells, and DC in the maintenance of intestinal immune homeostasis. Here we review recent advances of functionally specialized intestinal DC and their mechanisms of antigen uptake and recognition. We also discuss the interaction of DC with intestinal microbiota and their ability to orchestrate protective immunity and immune tolerance in the host. Lastly, we describe how DC functions are altered in intestinal inflammation and their emerging potential as a therapeutic target in inflammatory bowel disease.

Background: In ulcerative colitis (UC) gut bacteria drive inflammation. Bacterial recognition and T-cell responses are shaped by intestinal dendritic cells (DCs); therapeutic effects of probiotic bacteria may relate to modulation of intestinal DC. The probiotic mixture, VSL#3, increases interleukin (IL)-10 and downregulates IL-12p40 production by DC in vitro. We evaluated in vivo effects of oral VSL#3 and steroids on colonic DC in patients with acute UC. Methods: Rectal biopsies were obtained from patients with active UC before and after treatment with VSL#3, corticosteroids, or placebo, and from healthy controls. Myeloid colonic DC were studied from freshly isolated lamina propria cells using multicolor flow cytometry. Surface expression of activation markers, CD40, CD86, pattern recognition receptors, Toll-like receptor (TLR)-2 and TLR-4 were assessed. Changed function was measured from ongoing intracellular IL-10, IL-12p40, IL-6, and IL-13 production. Results: Acute UC colonic myeloid DC were producing more IL-10 and IL-12p40 than control DC (P = 0.01). In VSL#3-treated patients DC TLR-2 expression decreased (P < 0.05), IL-10 production increased and IL-12p40 production decreased (P < 0.005); 10/14 patients on VSL#3 showed a clinical response. Corticosteroids also resulted in increased IL-10 and reduced IL-12p40 production by DC. Conversely, in patients on placebo, TLR-2 expression and intensity of staining for IL-12p40 and IL-6 increased (all P < 0.05); 5/14 patients on placebo showed a clinical response (P = NS). Conclusions: Despite small numbers of human colonic DC available, we showed that treatment of UC patients with probiotic VSL#3 and corticosteroids induced “favorable” intestinal DC function in vivo, increasing regulatory cytokines and lowering proinflammatory cytokines and TLR expression. These effects may contribute to therapeutic benefit. (Inflamm Bowel Dis 2010)

Background: Gastro-intestinal (GI) graft-versus-host disease (GvHD) results in significant morbidity and mortality after allogeneic hematopoietic stem cell transplantation (AHSCT). Retinoic acid (RA), a metabolite of vitamin A, has diverse effects on immune cells. RA signaling promotes immune tolerance under steady state conditions. However in pro-inflammatory conditions RA increases Th1, Th17 CD4+ and Tc1 CD8+ T cell responses and is implicated in the pathogenesis of autoimmune inflammatory bowel disease. Furthermore, RA exposure and RA-receptor alpha (RARα) signaling in allogeneic T cells potentiates GI-GvHD in mice. Thus RA and cytokines that influence the effect of RA on T cells may represent potential therapeutic targets for GI-GvHD. However, the impact of RA at a tissue level in human GI-GvHD is unknown. We therefore investigated the role of RA-responsive T cells in human GvHD in vivo and human allogeneic T-cell responses in vitro . Methods: We scrutinized GI biopsies from 47 patients after AHSCT (36 with and 11 without GI-GvHD confirmed by conventional histological criteria). We also analysed skin biopsies from 11 AHSCT patients. We used conventional immunohistochemistry and a novel deep phenotyping method using sequential staining, stripping and re-probing on the same fixed embedded biopsy to determine cellular co-expression of multiple markers to identify RA-responsive T cells in human GvHD biopsies at sites of GvHD damage. Finally, we used an HLA-mismatched allogeneic mixed lymphocyte co-culture (MLRs) system, combining CFSE dye dilution and multi-parameter flow cytometry to enable the phenotyping of alloproliferative T cells after manipulation of RA signalling. Results: Firstly we observed thatthe number of cells expressing high levels of cytoplasmic RA binding proteins I/II (a surrogate marker of RA) was increased in gut biopsies in patients with GI-GvHD versus controls (median 155 vs 43 cells/mm2, p=0.056). Importantly the number of cells expressing RARα was also significantly increased (median 1315 vs 544 cells/mm2, p=0.001) consistent with RA-responsiveness. Crucially the number of RARα+ cells correlated with clinical stage of acute GI-GvHD with higher numbers in patients with severe (stage 3-4) versus less severe (stage 1-2) disease. Having demonstrated that RA-responsive cells were associated with both occurrence and severity of GI-GvHD we went on to identify potential RA-responsive T cell subsets. Additionally, as IL-23 and IL-33 direct RA-programming of T cells to either pro-inflammatory or tolerogenic phenotypes we also measured cellular expression of these cytokines and their receptors. CD8+ T cells co-localized with RARα+ cells and were present in significantly increased numbers in gut biopsies from patients with GI-GvHD, as were Tbet+ Th1/Tc1 cells. In contrast, numbers of RORγ+ Th17 cells were unchanged and CD4+ cells were decreased. IL-23p19+ and IL-23R+ cell numbers were significantly increased in GI-GvHD biopsies, whereas IL33R (ST2+)cells were unchanged. In order to determine if the RA-responsive, CD8+ and IL-23R+ cells represented a single effector subset, we used sequential stripping/re-probing to deep phenotype the T cells. We identified a unique single CD8+ T-cell population that co-expressed RARα+, T-bet and IL23R, present in significantly increased numbers in gut biopsies from patients with GI-GvHD versus controls. This is consistent with a RA-responsive, IL-23-dependent, inflammatory Tc1 effector cell mediating tissue damage in human gut GvHD. Importantly, there was no increase in this T cell subset in skin biopsies of skin GvHD patients demonstrating tissue-specificity. Finally we determined the impact of exogenous RA exposure on T cell alloresponses in MLRs. RA significantly increased the proportion of alloproliferative effector CD8+ T cells co-expressing Tbet and gut-homing molecules, confirming that RA can directly potentiate human alloreactive CD8 T cell responses with capacity to home to the GI tract. Conclusions: This is the first data to demonstrate a role for RA at a tissue level in human GI-GvHD. Furthermore we have identified a gut specific CD8+ T cell subset which co-expresses RARα, T-bet, and IL-23R localised in areas of GI-damage likely to represent the RA-responsive effector cell. Therapeutic blockade of IL-23R could target this cellular response to prevent or treat GI-GvHD. Disclosures Gribben: Acerta: Honoraria; Janssen: Honoraria; Kite: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Karyopharm: Honoraria; Pharmacyclics: Honoraria; Genentech/Roche: Honoraria; Abbvie: Honoraria.

Background Dendritic cells (DC) mediate inflammation in rodent models of allergic airway disease, but the role played by human respiratory-tract DC (hRTDC) in atopic asthma remains poorly defined. Recent data suggest that CD 1 antigen presentation by hRTDC may contribute to asthma pathogenesis. Objective To investigate the influence of hRTDC on the balance between atopy and allergic asthma in human subjects and to determine whether CD1 expression by hRTDC is modulated during asthmatic inflammation. Methods Sputum cells were induced from steroid-naive, allergen-challenged and allergen-naive subjects (atopic asthmatics, atopic non-asthmatics and non-atopic controls). hRTDC were identified using monoclonal antibody labelling and analysis by flow cytometry. Results hRTDC stained HLA-DR + (negative for markers of other cell lineages) were predominantly myeloid and comprised ∼0.5% of viable sputum cells. Sputum cells were potent stimulators of allogeneic CD4 + naive T cells and enrichment/depletion experiments correlated stimulatory potency with DC numbers. Sputum contained cells that exhibited typical dendritic morphology when analysed by electron microscopy. Myeloid hRTDC were endocytically active, but uptake of FITC-dextran was enhanced in cells from asthmatics (P < 0.001). Despite their increased endocytic capacity, asthmatic myeloid hRTDC appeared mature and expressed increased levels of maturation markers (P < 0.05-P < 0.001), CD1c, CD1d and langerin (P < 0.05). CD1c expression by asthmatic myeloid hRTDC was enhanced upon in vivo allergen challenge (three to ninefold within 24h; P < 0.05). CD11c - CD123 high hRTDC were only detected in asthmatic sputum and were increased in number following allergen challenge. Conclusion Despite limited cell numbers, it proved possible to analyse human RTDC in induced sputum, providing evidence that increased antigen uptake and enhanced CD 1 presentation by activated hRTDC may contribute to allergic airway disease. CD1 presentation by hRTDC in atopic asthma may therefore constitute a novel target for future intervention strategies.

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.

Patients who develop colorectal cancer or chronic colitis often require surgical gut resection. To facilitate healing a temporary redirection of luminal contents, via stoma formation upstream of th...

Background and Aims: Prebiotic inulin-type fructans are widely consumed in the diet and may have contrasting effects in Crohn’s disease by stimulating gut microbiota and/or by generating functional gastrointestinal symptoms. The aim of this study was to measure fructan and oligofructose intakes in patients with active and inactive Crohn’s disease compared with healthy controls. Methods: Patients with active Crohn’s disease ( n = 98), inactive Crohn’s ( n = 99) and healthy controls ( n = 106) were recruited to a case–control study. Dietary intake of inulin-type fructans was measured using a specific food frequency questionnaire and was compared between the three groups and between patients with different disease phenotypes (Montreal classification). Associations between intakes and disease activity (Harvey–Bradshaw Index, HBI) were also undertaken. Results: Patients with active Crohn’s disease had lower fructan intakes (median 2.9 g/d, interquartile range [IQR] 1.8) than those with inactive Crohn’s (3.6 g/d, 2.1, p = 0.036) or controls (3.9 g/d, 2.1, p = 0.003) and lower oligofructose intakes (2.8 g/d, 1.8) than those with inactive Crohn’s (3.5 g/d, 2.2, p = 0.048) or controls (3.8 g/d, 2.1, p = 0.003). There were no differences in intakes related to disease site or behaviour. There were negative correlations between HBI well-being score and fructan intake ( ρ = –0.154, p = 0.03) and oligofructose intake ( ρ = –0.156, p = 0.028) and for the HBI abdominal pain score and fructan ( ρ = –0.164, p = 0.021) and oligofructose intake ( ρ = –0.157, p = 0.027). Conclusions: Patients with active Crohn’s disease consume lower quantities of fructans and oligofructose than their inactive counterparts and healthy controls. The impact of lower intakes of prebiotic fructans on gut microbiota is unknown and warrants further research.