Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

BACKGROUND & AIMS: Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103+ DC specialize in generating immune tolerance with the functionality of CD11b+/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. METHODS: Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. RESULTS: Colonic DC identified were myeloid (mDC, CD11c+CD123-) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103-SIRPα+ DC were the major population and with CD103+SIRPα+ DC were CD1c+ILT3+CCR2+ (although CCR2 was not expressed on all CD103+SIRPα+ DC). CD103+SIRPα- DC constituted a minor subset that were CD141+ILT3-CCR2-. Proximal colon samples had higher total DC counts and fewer CD103+SIRPα+ cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4+CD45RA+ T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (ß7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c+, but not CD141+ mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. CONCLUSIONS: Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

Compartment-specific immunity in the human gut: properties and functions of dendritic cells in the colon versus the ileum.

OBJECTIVE: Dendritic cells (DC) mediate intestinal immune tolerance. Despite striking differences between the colon and the ileum both in function and bacterial load, few studies distinguish between properties of immune cells in these compartments. Furthermore, information of gut DC in humans is scarce. We aimed to characterise human colonic versus ileal DC. DESIGN: Human DC from paired colonic and ileal samples were characterised by flow cytometry, electron microscopy or used to stimulate T cell responses in a mixed leucocyte reaction. RESULTS: A lower proportion of colonic DC produced pro-inflammatory cytokines (tumour necrosis factor-α and interleukin (IL)-1ß) compared with their ileal counterparts and exhibited an enhanced ability to generate CD4(+)FoxP3(+)IL-10(+) (regulatory) T cells. There were enhanced proportions of CD103(+)Sirpα(-) DC in the colon, with increased proportions of CD103(+)Sirpα(+) DC in the ileum. A greater proportion of colonic DC subsets analysed expressed the lymph-node-homing marker CCR7, alongside enhanced endocytic capacity, which was most striking in CD103(+)Sirpα(+) DC. Expression of the inhibitory receptor ILT3 was enhanced on colonic DC. Interestingly, endocytic capacity was associated with CD103(+) DC, in particular CD103(+)Sirpα(+) DC. However, expression of ILT3 was associated with CD103(-) DC. Colonic and ileal DC differentially expressed skin-homing marker CCR4 and small-bowel-homing marker CCR9, respectively, and this corresponded to their ability to imprint these homing markers on T cells. CONCLUSIONS: The regulatory properties of colonic DC may represent an evolutionary adaptation to the greater bacterial load in the colon. The colon and the ileum should be regarded as separate entities, each comprising DC with distinct roles in mucosal immunity and imprinting.

Human gut dendritic cells drive aberrant gut-specific t-cell responses in ulcerative colitis, characterized by increased IL-4 production and loss of IL-22 and IFNγ.

: 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.

Altered human gut dendritic cell properties in ulcerative colitis are reversed by Lactobacillus plantarum extracellular encrypted peptide STp.

SCOPE: The human/microbiota cross-talk is partially mediated by bacteria-derived peptides like Serine-Threonine peptide (STp), which is resistant to gut proteolysis, is found in the human healthy colon and induces regulatory properties on gut dendritic cells (DCs); here we characterized human gut DC in ulcerative colitis (UC) patients and studied the effect of STp on their properties. METHODS AND RESULTS: Human colonic DC from healthy controls and UC patients were isolated, conditioned for 24 h +/- STp and characterized by flow cytometry, immunohistochemistry, and electron microscopy. Expression of immature DC markers DC-SIGN and ILT3, and Toll-like receptors were increased on gut UC-DC. Langerin (involved in phagocytosis), lymph node homing marker CCR7, and activation markers CD40/CD80/CD86 were decreased in UC. Gut DC had restricted stimulatory capacity for T-cells in UC. Conditioning of DC with STp in vitro reduced Toll-like receptor expression, increased CD40 and CD80 expression, and restored their stimulatory capacity. CONCLUSION: Colonic DCs display an abnormal immature phenotype in UC, which was partially restored following STp treatment. Bacteria-derived metabolites, like STp, seem to have a role in gut homeostasis that is missing in UC so they might lead a new era of probiotic products setting the basis for nondrug dietary therapy in inflammatory bowel disease.

Oral tacrolimus as maintenance therapy for refractory ulcerative colitis--an analysis of outcomes in two London tertiary centres.

BACKGROUND: The medical management of refractory ulcerative colitis (UC) remains a significant challenge. Two randomised controlled studies have demonstrated tacrolimus therapy is effective for the induction of remission of moderate to severe UC. However, the long term outcomes of UC patients treated with tacrolimus as maintenance therapy are not certain. AIMS: This study aims to assess the efficacy of tacrolimus maintenance therapy for refractory UC. METHODS: A retrospective review of patients with UC treated with tacrolimus at two London tertiary centres was performed. Clinical outcomes were assessed at six months, at the end of tacrolimus treatment, or at the last follow-up for patients continuing tacrolimus treatment. Modified Truelove-Witts score (mTW) and Mayo endoscopy subscores were calculated. RESULTS: 25 patients with UC, treated with oral tacrolimus between 2005 and 2011, were identified. The median duration of tacrolimus treatment was 9 months (IQR 3.7-18.2 months). The median duration of follow-up was 27 months (range 3-66 months). At six months thirteen (52%) patients had achieved and maintained clinical response and eleven (44%) were in clinical remission. The mean mTW score decreased from 10+/-0.5 before therapy, to 5.8+/-0.8 (p≤0.001 95% CI 2.7-5.8) at cessation of treatment or last follow-up. Mayo endoscopy subscore decreased from 2.6+/-0.1 to 1.2+/-0.2 (p≤0.001 mean reduction 1.4, 95% CI 0.8-1.9). Eight patients (32%) subsequently underwent a colectomy within a mean time of 17 months (range 2-45 months). CONCLUSION: Tacrolimus is effective for the maintenance of refractory UC and can deliver sustained improvement in mucosal inflammation.

Mechanisms of action of anti-tumor necrosis factor α agents in Crohn's disease.

Crohn's disease (CD) is characterized by inflammation that can affect any part of the gastrointestinal tract. It is a chronic destructive condition that follows a relapsing-remitting course and can lead to disability and a poor quality of life. Lifelong pharmacotherapy with systemic immunomodulator therapies remains the cornerstone of CD management. Advances in understanding of the immunopathogenic mechanisms underlying chronic gut inflammation in CD have led to the development of effective biological therapies for patients with CD. Tumor necrosis factor α (TNF-α) is a potent proinflammatory cytokine that plays a pivotal role in the development of Crohn's inflammation. Therapies designed to target this cytokine have revolutionized treatment of CD since their introduction in the late 1990s, thanks to their ability to induce and maintain remission, heal mucosa, reduce hospital admissions and surgical procedures, and restore quality of life. Despite widespread use of these therapies in CD, their precise mechanism of action remains unclear, although several different mechanisms have been proposed. This review summarizes the biology of the TNF-α cytokine and the development of biological therapies targeting TNF-α, and updates our current understanding of mechanisms of action of the commercially available anti-TNF-α therapies used in the treatment of CD.

Intestinal dendritic cells: their role in intestinal inflammation, manipulation by the gut microbiota and differences between mice and men.

The intestinal immune system maintains a delicate balance between immunogenicity against invading pathogens and tolerance of the commensal microbiota and food antigens. Dendritic cells (DC) generate primary T-cell responses, and determine whether these responses are immunogenic or tolerogenic. The regulatory role of DC is of particular importance in the gut due to the high antigenic load. Intestinal DC act as sentinels, sampling potentially pathogenic antigens but also harmless antigens including the commensal microbiota. Following antigen acquisition, intestinal DC migrate to secondary lymphoid organs to activate naive T-cells. DC also imprint specific homing properties on T-cells that they stimulate; gut DC specifically induce gut-homing properties on T-cells upon activation, enabling T-cell migration back to intestinal sites. Data regarding properties on gut DC in humans is scarce, although evidence now supports the role of DC as important players in intestinal immunity in humans. Here, we review the role of intestinal DC in shaping mucosal immune responses and directing tissue-specific T-cell responses, with a special focus on the importance of distinguishing DC subsets from macrophages at intestinal sites. We compare and contrast human DC with their murine counterparts, and discuss the ability of the gut microbiota to shape intestinal DC function, and how this may be dysregulated in inflammatory bowel disease (IBD). Lastly, we describe recent advances in the study of probiotics on intestinal DC function, including the use of soluble secreted bacterial products.

IL-6 promotes immune responses in human ulcerative colitis and induces a skin-homing phenotype in the dendritic cells and Tcells they stimulate.

Dendritic cells (DCs) control the type and location of immune responses. Ulcerative colitis (UC) is considered a Th2 disease mediated by IL-13 where up to one third of patients can develop extraintestinal manifestations. Colonic biopsies from inflamed and noninflamed areas of UC patients were cultured in vitro and their supernatants were used to condition human blood enriched DCs from healthy controls. Levels of IL-13 in the culture supernatants were below the detection limit in most cases and the cytokine profile suggested a mixed profile rather than a Th2 cytokine profile. IL-6 was the predominant cytokine found in inflamed areas from UC patients and its concentration correlated with the Mayo endoscopic score for severity of disease. DCs conditioned with noninflamed culture supernatants acquired a regulatory phenotype with decreased stimulatory capacity. However, DCs conditioned with inflamed culture supernatants acquired a proinflammatory phenotype with increased expression of the skin-homing chemokine CCR8. These DCs did not have decreased T-cell stimulatory capacity and primed T cells with the skin-homing CLA molecule in an IL-6-dependent mechanism. Our results highlight the role of IL-6 in UC and question the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology.

T-cell proliferation and forkhead box P3 expression in human T cells are dependent on T-cell density: physics of a confined space?

T-cell proliferation rates in vitro depend on factors including initial T-cell number, dose of stimulus, culture time, and available physical space. The role of forkhead box P3 (FoxP3) in the identification of T cells with a regulatory phenotype remains controversial in humans. Through 5-carboxyfluorescein diacetate succinimidyl ester labeling of human T cells and subsequent culture of different numbers of T cells and antigen-presenting cells (APC), we studied proliferative T-cell responses and FoxP3 expression in divided T cells. T-cell proliferation rates depended on initial T-cell/APC numbers. Proliferation rates decreased when high initial T-cell numbers were increased. FoxP3 expression was expressed exclusively in virtually all divided T cells cultured at high T-cell densities, irrespective of their CD4 nature or cytokine content, and was coexpressed with T-bet. However, when T cells were cultured on larger surfaces or at lower initial numbers, FoxP3 expression was not induced in divided T cells, even when most of the cells had undergone cell division. FoxP3(+) T cells generated at high cell densities did not elicit a suppressive phenotype and FoxP3 expression was subsequently lost in time when the stimulus was removed. Therefore, caution should be observed in the use of FoxP3 expression to identify regulatory T cells in humans because its expression may be only a consequence of activation status in a restricted environment.

A dangerous combination of binge and purge.

We present a 36-year-old female diagnosed with Crohn's disease at the age of 11 years. In 2001, she underwent a total colectomy and further small bowel resection as a result of active Crohn's. Her residual anatomy consisted of 150 cm of small bowel to an end jejunostomy. Subsequently, she developed short bowel syndrome with recurrent episodes of hypomagnesaemia, hypocalcaemia, and hypokalaemia. Dietetic assessment revealed her to be severely underweight at 37 kg with a bodymass index (BMI) of 14.4 kg/m(2) . During her admission, our patient underwent psychiatric assessment and was established on home parenteral nutrition (HPN). At the time of discharge, 1 month later, her weight had increased to 44 kg (BMI = 17.7 kg/m(2) ). Over the following 12-month period, she lost weight (BMI, 15.4 mg/m(2) ; weight, 39.5 kg) and she described a high stoma output (up to 17 L) and dehydration. Assessment of her oral intake found she was consuming an estimated 14,000 kcal and 600 g protein per day. At this time, the possibility of a new form of eating disorder was discussed with the patient and she agreed that her behavior i.e., using her stoma as a purging device, fulfilled the criteria for a diagnosis of bulimia nervosa and she was referred to a specialist eating disorder unit.

H1N1 vaccines in a large observational cohort of patients with inflammatory bowel disease treated with immunomodulators and biological therapy.

BACKGROUND: Safety data are lacking on influenza vaccination in general and on A (H1N1)v vaccination in particular in patients with inflammatory bowel disease (IBD) receiving immmunomodulators and/or biological therapy. AIMS AND METHODS: The authors conducted a multicentre observational cohort study to evaluate symptoms associated with influenza H1N1 adjuvanted (Pandemrix, Focetria, FluvalP) and non-adjuvanted (Celvapan) vaccines and to assess the risk of flare of IBD after vaccination. Patients with stable IBD treated with immunomodulators and/or biological therapy were recruited from November 2009 until March 2010 in 12 European countries. Harvey-Bradshaw Index and Partial Mayo Score were used to assess disease activity before and 4 weeks after vaccination in Crohn's disease (CD) and ulcerative colitis (UC). Vaccination-related events up to 7 days after vaccination were recorded. RESULTS: Of 575 patients enrolled (407 CD, 159 UC and nine indeterminate colitis; 53.9% female; mean age 40.3 years, SD 13.9), local and systemic symptoms were reported by 34.6% and 15.5% of patients, respectively. The most common local and systemic reactions were pain in 32.8% and fatigue in 6.1% of subjects. Local symptoms were more common with adjuvanted (39.3%) than non-adjuvanted (3.9%) vaccines (p < 0.0001), whereas rates of systemic symptoms were similar with both types (15.0% vs 18.4%, p = 0.44). Among the adjuvanted group, Pandemrix more often induced local reactions than FluvalP and Focetria (51.2% vs 27.6% and 15.4%, p < 0.0001). Solicited adverse events were not associated with any patient characteristics, specific immunomodulatory treatment, or biological therapy. Four weeks after vaccination, absence of flare was observed in 377 patients with CD (96.7%) and 151 with UC (95.6%). CONCLUSION: Influenza A (H1N1)v vaccines are well tolerated in patients with IBD. Non-adjuvanted vaccines are associated with fewer local reactions. The risk of IBD flare is probably not increased after H1N1 vaccination.