The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation

Multiple sclerosis (MS) is a highly prevalent demyelinating autoimmune condition; the mechanisms regulating its severity and progression are unclear. The IL-17-producing Th17 subset of T cells has been widely implicated in MS and in the mouse model, experimental autoimmune encephalomyelitis (EAE). However, the differentiation and regulation of Th17 cells during EAE remain incompletely understood. Although evidence is mounting that the antimicrobial peptide cathelicidin profoundly affects early T cell differentiation, no studies have looked at its role in longer-term T cell responses. Now, we report that cathelicidin drives severe EAE disease. It is released from neutrophils, microglia, and endothelial cells throughout disease; its interaction with T cells potentiates Th17 differentiation in lymph nodes and Th17 to exTh17 plasticity and IFN-γ production in the spinal cord. As a consequence, mice lacking cathelicidin are protected from severe EAE. In addition, we show that cathelicidin is produced by the same cell types in the active brain lesions in human MS disease. We propose that cathelicidin exposure results in highly activated, cytokine-producing T cells, which drive autoimmunity; this is a mechanism through which neutrophils amplify inflammation in the central nervous system

Exposure to inflammatory cytokines selectively limits GM-CSF production by induced T regulatory cells

Interest in manipulating the immunosuppressive powers of Foxp3-expressing T regulatory cells as an immunotherapy has been tempered by their reported ability to produce proinflammatory cytokines when manipulated in vitro, or in vivo. Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important. Here, we have studied this using induced (i) Treg cells in which de novo Foxp3 expression is driven by TCR-stimulation in vitro in the presence of TGF-β. We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN-γ, GM-CSF and TNF-α) upon secondary TCR stimulation. GM-CSF is a critical T-cell derived cytokine for the induction of EAE in mice. Despite their apparent capacity to produce GM-CSF, myelin autoantigen-responsive iTreg cells were unable to provoke EAE. Instead, they maintained strong suppressive function in vivo, preventing EAE induction by their CD4+Foxp3− counterparts. We identified that although iTreg cells maintained the ability to produce IFN-γ and TNF-α in vivo, their ability to produce GM-CSF was selectively degraded upon antigen stimulation under inflammatory conditions. Furthermore, we show that IL-6 and IL-27 individually, or IL-2 and TGF-β in combination, can mediate the selective loss of GM-CSF production by iTreg cells

Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4(+) T cells tolerized by peptide immunotherapy

Clinically effective antigen-based immunotherapy must silence antigen-experienced effector T cells (Teff) driving ongoing immune pathology. Using CD4+ autoimmune Teff cells, we demonstrate that peptide immunotherapy (PIT) is strictly dependent upon sustained T cell expression of the co-inhibitory molecule PD-1. We found high levels of 5-hydroxymethylcytosine (5hmC) at the PD-1 (Pdcd1) promoter of non-tolerant T cells. 5hmC was lost in response to PIT, with DNA hypomethylation of the promoter. We identified dynamic changes in expression of the genes encoding the Ten-Eleven-Translocation (TET) proteins that are associated with the oxidative conversion 5-methylcytosine and 5hmC, during cytosine demethylation. We describe a model whereby promoter demethylation requires the co-incident expression of permissive histone modifications at the Pdcd1 promoter together with TET availability. This combination was only seen in tolerant Teff cells following PIT, but not in Teff that transiently express PD-1. Epigenetic changes at the Pdcd1 locus therefore determine the tolerizing potential of TCR-ligation. - See more at: http://elifesciences.org/content/3/e03416#sthash.n6isQlkn.dpu

PD-1 expression is upregulated on adapted T cells in experimental autoimmune encephalomyelitis but is not required to maintain a hyporesponsive state

T cell adaptation is an important peripheral tolerogenic process which ensures that the T cell population can respond effectively to pathogens but remains tolerant to self-antigens. We probed the mechanisms of T cell adaptation using an experimental autoimmune encephalomyelitis (EAE) model in which the fate of autopathogenic T cells could be followed. We demonstrated that immunisation with a high dose of myelin basic protein (MBP) peptide and complete Freund's adjuvant failed to effectively initiate EAE, in contrast to low dose MBP peptide immunisation which readily induced disease. The proportion of autopathogenic CD4 + T cells in the central nervous system (CNS) of mice immunised with a high dose of MBP peptide was not significantly different to mice immunised with a low dose. However, autopathogenic T cells in mice immunised with high dose MBP peptide had an unresponsive phenotype in ex vivo recall assays. Importantly, whilst expression of PD-1 was increased on adapted CD4 + T cells within the CNS, loss of PD-1 function did not prevent the development of the unresponsive state. The lack of a role for PD-1 in the acquisition of the adapted state stands in striking contrast to the reported functional importance of PD-1 in T cell unresponsiveness in other disease models

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

International audienceB lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease

Evading the anti-tumour immune response - a novel role for Focal Adhesion Kinase

Here I describe a new function of Focal Adhesion Kinase (FAK) in driving anti-tumour immune evasion. The kinase activity of FAK in squamous cancer cells drives the recruitment of regulatory T-cells (Tregs) by transcriptionally regulating chemokine/cytokine and ligand-receptor networks, including the transcription of CCL5 and TGFβ, which are required for enhanced Treg recruitment. In turn, these changes inhibit antigen-primed cytotoxic CD8+ T-cell activity in the tumour microenvironment, permitting survival and growth of FAK-expressing tumours. I show that immune evasion requires FAK’s catalytic activity, and a small molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, drives depletion of Tregs and permits CD8+ T-cell-mediated tumour clearance. It is therefore likely that FAK inhibitors may trigger immune-mediated tumour regression, providing previously unrecognized therapeutic benefit

Rapid reprogramming of epigenetic and transcriptional profiles in mammalian culture systems

BackgroundThe DNA methylation profile of mammalian cell lines differs from the primary tissue from which they were derived, exhibiting increasing divergence from the in vivo methylation profile with extended time in culture. Few studies have directly examined the initial epigenetic and transcriptional consequences of adaptation of primary mammalian cells to culture, and the potential mechanisms through which this epigenetic dysregulation occurs is unknown.ResultsWe demonstrate that adaptation of mouse embryonic fibroblast, MEFS, to cell culture results in a rapid reprogramming of epigenetic and transcriptional states. We observed global 5-hydroxymethylcytosine (5hmC) erasure within three days of culture initiation. Loss of genic 5hmC was independent of global 5-methylcytosine (5mC) levels and could be partially rescued by addition of Vitamin C. Significantly, 5hmC loss was not linked to concomitant changes in transcription. Discrete promoter-specific gains of 5mC were also observed within seven days of culture initiation. Against this background of global 5hmC loss we identified a handful of developmentally important genes that maintained their 5hmC profile in culture, including the imprinted loci Gnas and H19. Similar outcomes were identified in the adaption of CD4+ T-cells to culture.ConclusionsWe report a dramatic and novel consequence of adaptation of mammalian cells to culture in which global loss of 5hmC occurs; suggesting rapid concomitant loss of methylcytosine dioxygenase activity. The observed epigenetic and transcriptional re-programming occurs much earlier than previously assumed, and has significant implications for the use of cell lines as faithful mimics of in vivo epigenetic and physiological processes.We thank Professors Adrian Bird and Nicholas Hastie for their comments on our manuscript. JT and RO are funded by IMI-MARCAR (under grant agreement number (115001) (MARCAR project)). Work in RM's lab is supported by the MRC, IMI-MARCAR and the BBSRC. This work in RM's lab was also initially funded by the Breakthrough Breast Cancer charity. Work in MB's lab was supported by Linkoping University strategic research funding and the Ake Wibergs fund (3772738). Work in SP's lab is supported by the BBSRC.</p

An unbalanced maternal diet in pregnancy associates with offspring epigenetic changes in genes controlling glucocorticoid action and fetal growth

Objective: in epidemiological studies, adverse early-life conditions associate with subsequent cardiometabolic disease. Hypothesized causes include maternal malnutrition, foetal glucocorticoid overexposure and reduced growth factors. Animal studies suggest a role for epigenetic processes in maintaining early-life effects into adulthood, but human relevance is unknown. We aimed to investigate relationships between an unbalanced maternal diet in pregnancy, neonatal and adult anthropometric variables with methylation at key genes controlling tissue glucocorticoid action and foetal growth.Design: we studied 34 individuals aged 40 from the Motherwell cohort study whose mothers ate an unbalanced diet in pregnancy, previously linked with elevated blood pressure and cortisol in adult offspring.Measurements: DNA methylation at 11?-hydroxysteroid dehydrogenase type 2 (HSD2), glucocorticoid receptor (GR) and insulin-like growth factor 2 (IGF2) was measured by pyrosequencing on buffy coat DNA.Results: methylation at specific CpGs in the HSD2 promoter and at one of the IGF2 differentially methylated regions (H19 ICR) correlated with neonatal anthropometric variables. CpG methylation within HSD2, GR and H19 ICR was positively associated with increased adiposity and blood pressure in adulthood. Methylation at GR (exon 1F) was increased in offspring of mothers with the most unbalanced diets in pregnancy.Conclusions: alterations in DNA methylation at genes important in regulating circulating cortisol levels, tissue glucocorticoid action, blood pressure and foetal growth are present in adulthood in association with both early-life parameters and cardiometabolic risk factors. The data indicate a persisting epigenetic link between early-life maternal diet and/or foetal growth and cardiovascular disease risk in human

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<p>Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3⁺ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.</p

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<p>Several inflammatory diseases including multiple sclerosis and inflammatory bowel disease have been associated with dysfunctional and/or reduced numbers of Foxp3⁺ regulatory T cells (Treg). While numerous mechanisms of action have been discovered by which Treg can exert their function, disease-specific Treg requirements remain largely unknown. We found that the integrin αv, which can pair with several β subunits including β8, is highly upregulated in Treg at sites of inflammation. Using mice that lacked αv expression or β8 expression specifically in Treg, we demonstrate that there was no deficit in Treg accumulation in the central nervous system during experimental autoimmune encephalomyelitis and no difference in the resolution of disease compared to control mice. In contrast, during a curative T cell transfer model of colitis, Treg lacking all αv integrins were found at reduced proportions and numbers in the inflamed gut. This led to a quantitative impairment in the ability of αv-deficient Treg to reverse disease when Treg numbers in the inflamed colon were below a threshold. Increase of the number of curative Treg injected was able to rescue this phenotype, indicating that αv integrins were not required for the immunosuppressive function of Treg per se. In accordance with this, αv deficiency did not impact on the capacity of Treg to suppress proliferation of naive conventional T cells in vitro as well as in vivo. These observations demonstrate that despite the general upregulation of αv integrins in Treg at sites of inflammation, they are relevant for adequate Treg accumulation only in specific disease settings. The understanding of disease-specific mechanisms of action by Treg has clear implications for Treg-targeted therapies.</p