The interplay between the microbiota, diet and T regulatory cells in the preservation of the gut barrier in inflammatory bowel disease

Inflammatory bowel disease (IBD) is becoming more common in the Western world due to changes in diet-related microbial dysbiosis, genetics and lifestyle. Incidences of gut permeability can predate IBD and continued gut barrier disruptions increase the exposure of bacterial antigens to the immune system thereby perpetuating chronic inflammation. Currently, most of the approved IBD therapies target individual pro-inflammatory cytokines and pathways. However, they fail in approximately 50% of patients due to their inability to overcome the redundant pro inflammatory immune responses. There is increasing interest in the therapeutic potential of T regulatory cells (Tregs) in inflammatory conditions due to their widespread capability to dampen inflammation, promote tolerance of intestinal bacteria, facilitate healing of the mucosal barrier and ability to be engineered for more targeted therapy. Intestinal Treg populations are inherently shaped by dietary molecules and gut microbiota-derived metabolites. Thus, understanding how these molecules influence Treg-mediated preservation of the intestinal barrier will provide insights into immune tolerance-mediated mucosal homeostasis. This review comprehensively explores the interplay between diet, gut microbiota, and immune system in influencing the intestinal barrier function to attenuate the progression of colitis

Interleukin 6 increases production of cytokines by colonic innate lymphoid cells in mice and patients with chronic intestinal inflammation

Background & Aims: Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods: ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results: IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions: IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells

Developing in vitro expanded CD45RA⁺ regulatory T cells as an adoptive cell therapy for Crohn's disease

BACKGROUND AND AIM: Thymus-derived regulatory T cells (T(regs)) mediate dominant peripheral tolerance and treat experimental colitis. T(regs) can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. T(reg) cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of T(regs) expanded from Crohn's blood is unknown. The potential for adoptively transferred T(regs) to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to T(reg)-mediated suppression in active CD. The capacity for expanded T(regs) to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for T(reg) cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(−) T(reg) subsets were isolated from patients’ blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: T(regs) can be expanded from the blood of patients with CD to potential target dose within 22–24 days. Expanded CD45RA(+) T(regs) have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(−) T(regs). CD45RA(+) T(regs) highly express α(4)β(7) integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) T(regs) also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) T(regs). These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) T(regs) may be the most appropriate population from which to expand T(regs) for autologous T(reg) therapy for CD, paving the way for future clinical trials

A Crohn’s Disease-associated IL2RA Enhancer Variant Determines the Balance of T Cell Immunity by Regulating Responsiveness to IL-2 Signalling

BACKGROUND AND AIMS: Differential responsiveness to interleukin [IL]-2 between effector CD4(+) T cells [T(eff)] and regulatory T cells [T(reg)] is a fundamental mechanism of immunoregulation. The single nucleotide polymorphism [SNP] rs61839660, located within IL2RA [CD25], has been associated with the development of Crohn’s disease [CD]. We sought to identify the T cell immune phenotype of IBD patients who carry this SNP. METHODS: T(eff) and T(reg) were isolated from individuals homozygous [TT], heterozygous [CT], or wild-type [CC] for the minor allele at rs61839660, and used for phenotyping [flow cytometry, Cytometry Time Of Flight] functional assays or T cell receptor [TCR] sequencing. Phosphorylation of signal transducer and activator of transcription 5 [STAT5] was assessed in response to IL-2, IL-7, and in the presence of basiliximab, a monoclonal antibody directed against CD25. T(eff) pro-inflammatory cytokine expression levels were assessed by reverse transcription quantitative polymerase chain reaction after IL-2 and/or TCR stimulation. RESULTS: Presence of the minor T allele enhances CD25 expression, leading to increased STAT5 phosphorylation and pro-inflammatory cytokine transcript expression by T(eff) in response to IL-2 stimulation in vitro. T(eff) from TT individuals demonstrate a more activated gut homing phenotype. TCR sequencing analysis suggests that TT patients may have a reduced clonal capacity to mount an optimal regulatory T cell response. CONCLUSIONS: rs61839660 regulates the responsiveness of T cells to IL-2 signalling by modulating CD25 expression. As low-dose IL-2 is being trialled as a selective T(reg) modulator in CD, these findings highlight the potential for adverse effects in patients with this genotype

SNPs associated with mucosal autoimmune diseases are enriched at T-bet binding sites.

<p>Scatter plots showing log-odds ratio against –log(10) p-value for the enrichment of disease-associated SNPs at different functional annotation datasets (DHS, histone modification, FAIRE-seq and transcription factor binding). Selected enriched functional annotation datasets are highlighted. GM12878 H3K4me1 indicates sites of H3K4me1 in the GM12878 lymphoblastoid cell line. Celiac disease, Crohn’s disease and UC-associated SNPs, but not RA, psoriasis or coronary artery disease-associated SNPs, are strongly enriched at T-bet binding sites (red dots with arrows).</p

Genetic variants alter T-bet binding <i>in vitro</i>.

<p><b>A.</b> Outline of the OligoFlow method. Double-stranded oligonucleotides are annealed to beads and incubated with cell lysate containing the transcription factor of interest. Fluorescently labelled antibody is added and MFI of the beads measured by flow cytometry. The histograms show the MFI of beads coated with oligonucleotides containing a T-bet binding motif (Motif +) or a mutated sequence (Motif -) after incubation with YT lysate, normalised for the number of beads acquired. <b>B.</b> Summary of OligoFlow results for the 11 SNPs tested. In each case, MFI for both alleles is normalised such that the negative control equals 1. Normalised MFI for the lowest binding allele was then subtracted from the value for the highest binding allele. Each cross represents one experiment, with the average difference between alleles represented by a horizontal line. * Significantly different binding between the two alleles (p < 0.05, paired t-test.) <b>C.</b> Representative experiment measuring the binding of T-bet to the A and G alleles of rs1465321. Data for the different oligonucleotide probes are separated according to the key on the right and the MFI is also shown. <b>D.</b> Bar chart showing all replicate experiments for rs1465321. The y-axis shows MFI for each allele normalised to the MFI of the negative control oligonucleotide (set to 1). Each pair of bars represents one experiment, performed with either YT cells (YT) or Th1-polarised primary CD4⁺ cells (Th1). <b>E.</b> As C but for rs1006353. <b>F.</b> As D but for rs1006353. <b>G.</b> As C but for rs11135484. <b>H.</b> As D but for rs11135484.</p

Motif analysis does not reliably predict impact on T-bet binding.

<p><b>A.</b> T-bet binding, IgG control and H3K27ac modification (ChIP-seq reads/million) at the genomic regions surrounding the SNPs rs1465321 (left), rs11135484 (center) and rs1006353 (right). The location of the SNPs are indicated by dashed vertical lines. The regions highlighted in grey are expanded in B. <b>B.</b> Expanded view of T-bet binding at the regions highlighted in grey in A. The locations of sequences matching the identified T-bet DNA binding motif (inset) are marked by red lines, together with their score (a negative value indicates a poor match). Only rs1006353 overlaps a T-bet DNA binding motif and the A allele is predicted to disrupt the motif and T-bet binding.</p