Defective IL-10 production in severe phenotypes of Crohn’s disease

et al.Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohn's disease. IL-10 is a key anti-inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL-10 production in response to bacterial components in Crohn's disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohn's disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4(+) cells, and monocyte-derived dendritic cells (MDDCs). Under unstimulated conditions, IL-10, but not IL-12, was down-regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohn's disease patients. In response to LPS, IL-10 was up-regulated more significantly in patients with no fistulae or fibrosis. Study of IL-10 production by isolated cell subsets showed that DCs, but not CD4(+) T cells, from penetrating Crohn's disease produced significantly less IL-10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL-10 gene promoter. We show a defect in IL-10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohn's disease. This defect in IL-10 production by a group of Crohn's disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL-10 or IL-10-inducing therapies could be of particular benefit to these group of patientsThis work was supported by Grant SAF2005-03755 from the Ministerio de Educación y Ciencia, Spain.Peer reviewe

Btla signaling in conventional and regulatory lymphocytes coordinately tempers humoral immunity in the intestinal mucosa

The Btla inhibitory receptor limits innate and adaptive immune responses, both preventing the development of autoimmune disease and restraining anti-viral and anti-tumor responses. It remains unclear how the functions of Btla in diverse lymphocytes contribute to immunoregulation. Here, we show that Btla inhibits activation of genes regulating metabolism and cytokine signaling, including Il6 and Hif1a, indicating a regulatory role in humoral immunity. Within mucosal Peyer\u27s patches, we find T-cell-expressed Btla-regulated Tfh cells, while Btla in T or B cells regulates GC B cell numbers. Treg-expressed Btla is required for cell-intrinsic Treg homeostasis that subsequently controls GC B cells. Loss of Btla in lymphocytes results in increased IgA bound to intestinal bacteria, correlating with altered microbial homeostasis and elevations in commensal and pathogenic bacteria. Together our studies provide important insights into how Btla functions as a checkpoint in diverse conventional and regulatory lymphocyte subsets to influence systemic immune responses

Differences in peripheral and tissue immune cell populations following haematopoietic stem cell transplantation in Crohn's disease patients

Background and aims: recent studies have shown the efficacy of autologous haematopoietic stem cell transplantation [HSCT] in severely refractory Crohn's disease [CD] patients. HSCT is thought to eliminate auto-reactive cells; however, no specific studies of immune reconstitution in CD patients are available. Methods: we followed a group of CD patients [n = 18] receiving autologous HSCT, with 50% of them achieving endoscopic drug-free remission. To elucidate the mechanisms driving efficacy, we monitored changes after HSCT in blood and intestine immune-cell composition. CD patients [n = 22] receiving anti-tumour necrosis factor [TNF]-α were included for comparison. Results: severe immune ablation followed by HSCT induced dramatic changes in both peripheral blood T and B cells in all patients regardless of the efficacy of the treatment. Endoscopic remission at week 52 following HSCT was associated with significant intestinal transcriptional changes. A comparison of the remission signature with that of anti-TNFα identified both common and unique genes in the HSCT-induced response. Based on deconvolution analysis of intestinal biopsy transcriptome data, we show that response to HSCT, but not to anti-TNFα, is associated with an expansion of naïve B-cells, as seen in blood, and a decrease in the memory resting T-cell content. As expected, endoscopic remission, in response to both HSCT and anti-TNFα, led to a significant reduction in intestinal neutrophil and M1 macrophage content. Conclusions: peripheral blood immune remodelling after HSCT does not predict efficacy. In contrast, a profound intestinal T-cell depletion that is maintained long after transplant is associated with mucosal healing following HSCT, but not anti-TNFα

Nous mediadors de la resposta T efectora en la malaltia inflamatòria intestinal

La malaltia de Crohn és una malaltia inflamatòria intestinal de base immunitària. L’etiologia de la malaltia és desconeguda, encara que en general s’admet la següent definició: la malaltia és conseqüència d’una desregulació de la resposta immunitària en front als antígens comuns de la flora bacteriana intestinal en individus genèticament susceptibles. Un component important de la desregulació immunitària es la hiperactivació dels limfòcits T que es dóna en aquesta i altres malalties de tipus autoimmunitari, i en les que tradicionalment s’ha atribuït una major presència i producció de citocines per part de la població limfocitària Th1 en el teixit inflamat. Recentment, amb la descripció de la citocina IL-23 i la població Th17 se ha posat en dubte el paper principal de la població Th1 en les malalties inflamatòries de base immunitària. De fet, en algunes d’aquestes malalties s’ha descrit un augment de las cèl•lules Th17 així com de les citocines produïdes per aquesta població. L’objectiu d’aquesta tesi ha sigut caracteritzar el paper relatiu de les poblacions Th1 i Th17 en la mucosa intestinal inflamada i en la circulació perifèrica de pacients amb malaltia de Crohn activa o en remissió així com avaluar el paper i l’evolució d’aquestes poblacions limfocitàries en els estadis inicials (primers brots de la malaltia) i avançats de la malaltia. En aquest estudi observem que els períodes d’activitat inflamatòria en la malaltia de Crohn s’associen a una resposta sistèmica exacerbada de la població Th17, essent la producció de IL-17 en sobrenedant del cultiu de sang total, el percentatge de limfòcits CD4+IL-17+ i la producció de IL-17 per part d’aquestes cèl•lules més elevada que en els pacients amb malaltia inactiva i que en els controls sans. Paral•lelament hem descrit que la població Th1 circulant no presenta durant un brot d’activitat un augment tan generalitzat com la població Th17, sinó que només observem un augment en el percentatge de limfòcits CD4+IFN-γ+ circulants i en els dobles productors de IL-17 y IFN-γ. Els pacients que denominem debut (pateixen el primer brot de la malaltia) no presenten a nivell sistèmic un augment de la resposta Th17 ni Th1. A diferència del que observem en circulació perifèrica, en la mucosa intestinal inflamada trobem un augment dels trànscrits de les citocines característiques de la població Th17, tant en els pacients debut com en els que es troben en un estat més avançat de la malaltia. A més, en la mucosa inflamada d’ambdós grups de pacients trobem una major infiltració de cèl•lules IL-17+. A partir d’aquests resultats hipotetitzem un model per a la fisiopatologia de la malaltia de Crohn en el que la generació de limfòcits Th17 memòria podrien estar implicats en la cronicitat i recurrència de la malaltia. Així en les primeres fases de la malaltia només detectem un augment d’aquesta població en la mucosa intestinal, com seria d’esperar d’una resposta immunitària local. A conseqüència d’aquesta es generarà una població limfocitària de memòria immunitària que només detectem en circulació en aquells malalts que es troben ja en estadis més crònics de la malaltia. Amb aquesta hipòtesi podem explicar mitjançant mecanismes immunitaris diferents el fet que, en general, la malaltia de Crohn no es manifesta fins la segona o tercera dècada de vida d’un individu, mentre que un cop s’ha manifestat, la recurrència d’activitat inflamatòria és molt més freqüent (degut a la participació dels limfòcits memòria).One component of the immune deregulation observed in Crohn’s disease is the massive infiltration of T lymphocytes in the inflamed tissue. Classically these hyperreactive lymphocytes have been attributed to the Th1 subpopulation. Recently the description of IL-23 cytokine and Th17 population has questioned the main role given to Th1 population. In this regard, an increase in the frequency of Th17 cells and in the production of their signature cytokines has been already described in some inflammatory diseases. In this study we have observed that active inflammation in Crohn’s disease associates to an increased systemic response of Th17 cells described as overproduction of IL-17 in supernatants of whole blood cultures, increased percentage of CD4+IL-17+ lymphocytes and increased production of IL-17 from these cells related to remission periods of disease or healthy controls. Early patients (patients who suffer their first flare of the disease) do not present a systemic increase of Th1 or Th17 cells despite suffering of active inflammation. Otherwise, in inflamed intestinal mucosa there is an increase in the expression of Th17 cytokines both in early as well as late chronic patients. Moreover we found a significant higher number of Th17 cells infiltrating the mucosa of both groups of patients. Taking these results into account we hypothesize a model for the physiopathology of Crohn’s disease in which the generation of memory Th17 lymphocytes could be involved in the chronicity and recurrence of the disease. Therefore, in early phases of disease we only detect an increase of this population in the intestinal mucosa, as we could expect from a local immune response. As a consequence of this activation there would be a generation of memory lymphocytes that we can detect in peripheral circulation only in that patients that have already suffered repeated flares of the disease. This hypothesis allows us to explain the different immune mechanisms that could be acting during the evolution of Crohn’s disease, as it is the first appearance of disease, generally occurring during the second or third decade of the individual life, and the subsequent appearance of recurrences that are much more frequent and life-long lasting

Late Crohn's disease patients present an increase in peripheral Th17 cells and cytokine production compared with early patients

[Background]: Th1 and Th17 cells have been implicated in Crohn's disease (CD) pathophysiology and may play a role in disease persistence. [Aim]: To determine Th1 and Th17 responses in intestine and peripheral blood of early (2 years) CD patients. [Methods]: Cytokine mRNA in intestinal biopsies was determined by RT-PCR. Cytokine concentration in culture was measured by ELISA and cytokine-producing cells were identified by intracellular staining. [Results]: The inflamed mucosa showed significantly increased IL-17 mRNA levels compared with non-inflamed areas, both in early and late CD patients. However, only patients with late (n = 12), but not early (n = 9), active disease showed increased IL-17 production, as well as a significantly higher percentage of IL-17+CD4+ cells in blood, compared with controls (n = 12) or patients in remission (n = 13). Moreover, cultured peripheral CD4 + cells from late active CD patients presented significantly higher percentages of IL-17+, IL-22+ and IFN-γ+ and a significantly increased production of IL-17 and IL-22, but not IFN-γ+. [Conclusions]: Increased IL-17 gene transcription is common to early and late CD mucosa. However, exacerbated Th17 responses in the peripheral blood appear only in late disease. We propose that this population may constitute a mechanism of perpetuating the disease. © 2010 Blackwell Publishing Ltd.This work was funded by grants SAF2005⁄03755 and BFU2008-02683⁄BFI to A.S. and SAF2006⁄03074 to J.P. from the Ministerio de Ciencia e Innovación, Spain. M.V. is the recipient of a FPI fellowship (SAF2005⁄03755) from the Ministerio de Ciencia e Innovación, Spain.Peer Reviewe

Chemokine receptor CCR7 regulates the intestinal TH1/TH17/Treg balance during Crohn’s-like murine ileitis

The regulation of T cell and DC retention and lymphatic egress within and from the intestine is critical for intestinal immunosurveillance; however, the cellular processes that orchestrate this balance during IBD remain poorly defined. With the use of a mouse model of TNF-driven Crohn's-like ileitis (TNF(Δ) (ARE)), we examined the role of CCR7 in the control of intestinal T cell and DC retention/egress during experimental CD. We observed that the frequency of CCR7-expressing TH1/TH17 effector lymphocytes increased during active disease in TNF(Δ) (ARE) mice and that ΔARE/CCR7(-/-) mice developed exacerbated ileitis and multiorgan inflammation, with a marked polarization and ileal retention of TH1 effector CD4(+) T cells. Furthermore, adoptive transfer of ΔARE/CCR7(-/-) effector CD4(+) into lymphopenic hosts resulted in ileo-colitis, whereas those transferred with ΔARE/CCR7(+/+) CD4(+) T cells developed ileitis. ΔARE/CCR7(-/-) mice had an acellular draining MLN, decreased CD103(+) DC, and decreased expression of RALDH enzymes and of CD4(+)CD25(+)FoxP3(+) Tregs. Lastly, a mAb against CCR7 exacerbated ileitis in TNF(Δ) (ARE) mice, phenocopying the effects of congenital CCR7 deficiency. Our data underscore a critical role for the lymphoid chemokine receptor CCR7 in orchestrating immune cell traffic and TH1 versus TH17 bias during chronic murine ileitis

Expression Levels of 4 Genes in Colon Tissue Might Be Used to Predict Which Patients Will Enter Endoscopic Remission After Vedolizumab Therapy for Inflammatory Bowel Diseases

BACKGROUND & AIMS: We aimed to identify biomarkers that might be used to predict responses of patients with inflammatory bowel diseases (IBD) to vedolizumab therapy. METHODS: We obtained biopsies from inflamed colon of patients with IBD who began treatment with vedolizumab (n = 31) or tumor necrosis factor (TNF) antagonists (n = 20) and performed RNA-sequencing analyses. We compared gene expression patterns between patients who did and did not enter endoscopic remission (absence of ulcerations at month 6 for patients with Crohn's disease or Mayo endoscopic subscore ≤1 at week 14 for patients with ulcerative colitis) and performed pathway analysis and cell deconvolution for training (n = 20) and validation (n = 11) datasets. Colon biopsies were also analyzed by immunohistochemistry. We validated a baseline gene expression pattern associated with endoscopic remission after vedolizumab therapy using 3 independent datasets (n = 66). RESULTS: We identified significant differences in expression levels of 44 genes between patients who entered remission after vedolizumab and those who did not; we found significant increases in leukocyte migration in colon tissues from patients who did not enter remission (P < .006). Deconvolution methods identified a significant enrichment of monocytes (P = .005), M1-macrophages (P = .05), and CD4+ T cells (P = .008) in colon tissues from patients who did not enter remission, whereas colon tissues from patients in remission had higher numbers of naïve B cells before treatment (P = .05). Baseline expression levels of PIWIL1, MAATS1, RGS13, and DCHS2 identified patients who did vs did not enter remission with 80% accuracy in the training set and 100% accuracy in validation dataset 1. We validated these findings in the 3 independent datasets by microarray, RNA sequencing and quantitative PCR analysis (P = .003). Expression levels of these 4 genes did not associate with response to anti-TNF agents. We confirmed the presence of proteins encoded by mRNAs using immunohistochemistry. CONCLUSIONS: We identified 4 genes whose baseline expression levels in colon tissues of patients with IBD associate with endoscopic remission after vedolizumab, but not anti-TNF, treatment. We validated this signature in 4 independent datasets and also at the protein level. Studies of these genes might provide insights into the mechanisms of action of vedolizumab.status: publishe