PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.Ian C. Nicholson, Christos Mavrangelos, Daniel R.G. Bird, Suzanne Bresatz-Atkins, Nicola G. Eastaff-Leung, Randall H. Grose, Batjargal Gundsambuu, Danika Hill, Debbrah J. Millard, Timothy J. Sadlon, Sarah To, Heddy Zola, Simon C. Barry, Doreen Krumbiege

Inhibition of activation induced CD154 on CD4+ CD25- cells: a valid surrogate for human Treg suppressor function

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3–6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations. Keywords: regulatory T cells; functional assays; iTreg; nTregDanika Hill, Nicola Eastaff-Leung, Suzanne Bresatz-Atkins, Noel Warner, Joyce Ruitenberg, Dorren Krumbiegel, Steve Pederson, Natasha McInnes, Cheryl Y. Brown, Timothy Sadlon and Simon C. Barr

Cytokine profile and induction of T helper type 17 and regulatory T cells by human peripheral mononuclear cells after microbial exposure

The immunomodulatory effects of probiotics were assessed following exposure of normal peripheral blood mononuclear cells (PBMC), cord blood cells and the spleen-derived monocyte/macrophage cell line CRL-9850 to Lactobacillus acidophilus LAVRI-A1, Lb. rhamnosus GG, exopolysaccharides (EPS)-producing Streptococcus thermophilus St1275, Bifidobacteriun longum BL536, B. lactis B94 and Escherichia coli TG1 strains. The production of a panel of pro- and anti-inflammatory cytokines by PBMC following bacterial stimulation was measured, using live, heat-killed or mock gastrointestinal tract (GIT)-exposed bacteria, and results show that (i) all bacterial strains investigated induced significant secretion of pro- and anti-inflammatory cytokines from PBMC-derived monocytes/macrophages; and (ii) cytokine levels increased relative to the expansion of bacterial cell numbers over time for cells exposed to live cultures. Bifidobacteria and S. thermophilus stimulated significant concentrations of transforming growth factor (TGF)-β, an interleukin necessary for the differentiation of regulatory T cells (Treg)/T helper type 17 (Th17) cells and, as such, the study further examined the induction of Th17 and Treg cells after PBMC exposure to selected bacteria for 96 h. Data show a significant increase in the numbers of both cell types in the exposed populations, measured by cell surface marker expression and by cytokine production. Probiotics have been shown to induce cytokines from a range of immune cells following ingestion of these organisms. These studies suggest that probiotics' interaction with immune-competent cells produces a cytokine milieu, exerting immunomodulatory effects on local effector cells, as well as potently inducing differentiation of Th17 and Treg cells

IL-17Aは赤痢アメーバの腸管感染においてIFN-γ/IL-4比の減少と持続感染に寄与する

Amebiasis is an infectious disease caused by Entamoeba histolytica, an anaerobic protozoan parasite, and is a major public health problem worldwide, particularly in areas with inadequate sanitation and poor hygiene. Th1 responses, represented by interferon gamma (IFN-γ), play a protective role by clearing the amebae from the gut, whereas Th2 responses are responsible for chronic infection. Th17 responses preconditioned by vaccination or by modulating the intestinal microbiome protect mice from the settlement of E. histolytica. However, the role of interleukin-17A (IL-17A), which is upregulated during the natural course of intestinal amebiasis, has not been clarified. The aim of this study was to investigate the role of IL-17A during intestinal amebiasis in a mouse model. IL-17A knockout and wild-type CBA/J mice were challenged intracecally with 2 × 106 E. histolytica trophozoites, and their infection, pathology, and immune responses were monitored. Neither the initial settlement of E. histolytica nor the inflammation of the cecum was affected by the absence of IL-17A for week 1, but the infection rate and parasite burden declined in a late stage of infection, accompanied by an increased IFN-γ/IL-4 ratio. Therefore, IL-17A contributes to the persistence of E. histolytica and modulates the immune response, including the IFN-γ/IL-4 ratio, which may be responsible for the reduction of the parasite burden in the IL-17A knockout mice during the chronic phase of intestinal amebiasis.長崎大学学位論文 学位記番号:博(医歯薬)甲第1005号 学位授与年月日:平成29年12月6日Author: Sharmina Deloer, Risa Nakamura, Mihoko Kikuchi, Taeko Moriyasu, Yombo Dan Justin Kalenda, Eman Sayed Mohammed, Masachika Senba, Yoichiro Iwakura, Hiroki Yoshida, Shinjiro HamanoCitation: Parasitology International, 66(6), pp.817-823; 2017Nagasaki University (長崎大学)課程博

Immune activation in irritable bowel syndrome: can neuroimmune interactions explain symptoms?

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.Patrick A. Hughes, Heddy Zola, Irmeli A. Penttila, L. Ashley Blackshaw, Jane M. Andrews, and Doreen Krumbiege

Foxp3(+) regulatory T cells, Th17 effector cells, and cytokine environment in inflammatory bowel disease

Background: Inflammatory bowel disease (IBD) is thought to result from an aberrant immune response. Inflammation in IBD may be caused by the loss of homeostasis between CD4+ CD25high Foxp3+ regulatory cells (T reg) and proinflammatory Th17 cells. The aim of this study was to investigate T reg and Th17 cells in the peripheral blood and intestinal mucosa of IBD patients and to assess the mucosal cytokine environment. Methods: T reg and Th17 cells were measured in peripheral blood of 63 IBD patients and 28 controls by flow cytometry. Forkhead box p3 (Foxp3), interleukin (IL)-17a, IL-1β, IL-6, IL-21, IL-23, and transforming growth factor (TGF)-β mRNA were analyzed using real-time reverse transcription polymerase chain reaction in intestinal biopsies of 24 IBD and 18 control subjects. Results: A decrease in T reg and increase in Th17 cells was observed in the peripheral blood of IBD patients. When measured in the same patient and expressed as a ratio, a significant decrease in T reg/Th17 ratio was observed in IBD. Elevated expression of Foxp3, IL-17a, IL-1β, and IL-6 was observed in the mucosa of IBD patients, while TGF-β was only elevated in ulcerative colitis. Conclusion: IBD is associated with a reduced ratio of T reg to Th17 cells in peripheral blood and is characterized by a proinflammatory cytokine microenvironment, which supports the continued generation of Th17 cells.Nicola Eastaff-Leung, Nicholas Mabarrack, Angela Barbour, Adrian Cummins and Simon Barr

Oral Serum-Derived Bovine Immunoglobulin/Protein Isolate Has Immunomodulatory Effects on the Colon of Mice that Spontaneously Develop Colitis

Dietary immunoglobulin concentrates prepared from animal plasma can modulate the immune response of gut-associated lymphoid tissue (GALT). Previous studies have revealed that supplementation with serum-derived bovine immunoglobulin/protein isolate (SBI) ameliorates colonic barrier alterations in the mdr1a-/- genetic mouse model of IBD. Here, we examine the effects of SBI on mucosal inflammation in mdr1a-/- mice that spontaneously develop colitis. Wild type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% w/w) or milk proteins (Control diet), from day 21 (weaning) until day 56. Leucocytes in mesenteric lymph nodes (MLN) and in lamina propria were determined, as was mucosal cytokine production. Neutrophil recruitment and activation in MLN and lamina propria of KO mice were increased, but were significantly reduced in both by SBI supplementation (p < 0.05). The increased neutrophil recruitment and activation observed in KO mice correlated with increased colon oxidative stress (p < 0.05) and SBI supplementation reduced this variable (p < 0.05). The Tact/Treg lymphocyte ratios in MLN and lamina propria were also increased in KO animals, but SBI prevented these changes (both p < 0.05). In the colon of KO mice, there was an increased production of mucosal proinflammatory cytokines such as IL-2 (2-fold), IL-6 (26-fold) and IL-17 (19-fold), and of chemokines MIP-1β (4.5-fold) and MCP-1 (7.2-fold). These effects were significantly prevented by SBI (p < 0.05). SBI also significantly increased TGF-β secretion in the colon mucosa, suggesting a role of this anti-inflammatory cytokine in the modulation of GALT and the reduction of the severity of the inflammatory response during the onset of colitis

Unravelling the molecular basis for regulatory T-cell plasticity and loss of function in disease

Regulatory T cells (Treg) are critical for preventing autoimmunity and curtailing responses of conventional effector T cells (Tconv). The reprogramming of T-cell fate and function to generate Treg requires switching on and off of key gene regulatory networks, which may be initiated by a subtle shift in expression levels of specific genes. This can be achieved by intermediary regulatory processes that include microRNA and long noncoding RNA-based regulation of gene expression. There are well-documented microRNA profiles in Treg and Tconv, and these can operate to either reinforce or reduce expression of a specific set of target genes, including FOXP3 itself. This type of feedforward/feedback regulatory loop is normally stable in the steady state, but can alter in response to local cues or genetic risk. This may go some way to explaining T-cell plasticity. In addition, in chronic inflammation or autoimmunity, altered Treg/Tconv function may be influenced by changes in enhancer-promoter interactions, which are highly cell type-specific. These interactions are impacted by genetic risk based on genome-wide association studies and may cause subtle alterations to the gene regulatory networks controlled by or controlling FOXP3 and its target genes. Recent insights into the 3D organisation of chromatin and the mapping of noncoding regulatory regions to the genes they control are shedding new light on the direct impact of genetic risk on T-cell function and susceptibility to inflammatory and autoimmune conditions.Timothy Sadlon Cheryl Y Brown Veronika Bandara Christopher M Hope John E Schjenken Stephen M Pederson James Breen Alistair Forrest Marc Beyer Sarah Robertson Simon C Barr

Molecular Insights Into Regulatory T-Cell Adaptation to Self, Environment, and Host Tissues: Plasticity or Loss of Function in Autoimmune Disease

There has been much interest in the ability of regulatory T cells (Treg) to switch function in vivo, either as a result of genetic risk of disease or in response to environmental and metabolic cues. The relationship between levels of FOXP3 and functional fitness plays a significant part in this plasticity. There is an emerging role for Treg in tissue repair that may be less dependent on FOXP3, and the molecular mechanisms underpinning this are not fully understood. As a result of detailed, high-resolution functional genomics, the gene regulatory networks and key functional mediators of Treg phenotype downstream of FOXP3 have been mapped, enabling a mechanistic insight into Treg function. This transcription factor-driven programming of T-cell function to generate Treg requires the switching on and off of key genes that form part of the Treg gene regulatory network and raises the possibility that this is reversible. It is plausible that subtle shifts in expression levels of specific genes, including transcription factors and non-coding RNAs, change the regulation of the Treg gene network. The subtle skewing of gene expression initiates changes in function, with the potential to promote chronic disease and/or to license appropriate inflammatory responses. In the case of autoimmunity, there is an underlying genetic risk, and the interplay of genetic and environmental cues is complex and impacts gene regulation networks frequently involving promoters and enhancers, the regulatory elements that control gene expression levels and responsiveness. These promoter–enhancer interactions can operate over long distances and are highly cell type specific. In autoimmunity, the genetic risk can result in changes in these enhancer/promoter interactions, and this mainly impacts genes which are expressed in T cells and hence impacts Treg/conventional T-cell (Tconv) function. Genetic risk may cause the subtle alterations to the responsiveness of gene regulatory networks which are controlled by or control FOXP3 and its target genes, and the application of assays of the 3D organization of chromatin, enabling the connection of non-coding regulatory regions to the genes they control, is revealing the direct impact of environmental/metabolic/genetic risk on T-cell function and is providing mechanistic insight into susceptibility to inflammatory and autoimmune conditions.Cheryl Y. Brown, Timothy Sadlon, Christopher M. Hope, Ying Y. Wong, Soon Wong, Ning Liu, Holly Withers, Katherine Brown, Veronika Bandara, Batjargal Gundsambuu, Stephen Pederson, James Breen, Sarah Anne Robertson, Alistair Forrest, Marc Beyer, and Simon Charles Barr