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CD4+ T cells treated with TNFalpha blocking agents during activation.

ABSTRACT: Objective. TNFα is a potent pro-inflammatory cytokine playing a pivotal role in several autoimmune diseases. Neutralizing TNFα inhibits T cell proliferation and IFNγ production, and enhances suppressive capacity of regulatory T cells (Treg). Little is known about the mechanism of TNFα blocking agents on naïve T cell differentiation. Methods. Naïve CD4+ T cells were activated by dendritic cells (DC) in presence or absence of anti-TNFα agents. T cell polarization and activation was assessed during T cell differentiation. In addition, whole genome gene expression analysis was performed on anti-TNFα-treated T cells. Results. Neutralizing TNFα during priming of naïve CD4+ T cells by DC favors development of IL-10+ T helper (Th) cells at the expense of IFNγ induction. TNFα inhibits IL-10 via TNFRII, which becomes expressed after naïve T cell activation. While initial CD4+ T cell activation was not affected, neutralization of TNFα negatively affected later stages of T cell priming by counteracting full T cell activation and survival. Whole genome gene expression analysis revealed a regulatory gene profile of anti-TNFα-treated T cells. Indeed, neutralizing TNFα during naïve T cell priming enhanced the suppressive function of anti-TNFα-treated T cells. Conclusion. Inhibition of TNFα–TNFRII interaction affects late stage effector T cell development and shifts the balance of Th cell differentiation towards IL-10 expressing regulatory T cells, which may be one of the beneficial mechanisms in TNFα blocking therapies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE42812 | GEO | 2014/06/25

SECONDARY ACCESSION(S): PRJNA183430

REPOSITORIES: GEO

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Project description:Interferon (IFN)γ and interleukin (IL)-4 are central regulators of T helper 1 (Th1) and T helper 2 (Th2) immune responses, respectively. Both cytokines have a major impact on macrophage phenotypes: IFNγ–priming and subsequent TLR4 activation induces so called classically activated macrophages that are characterized by pronounced pro-inflammatory responses, whereas IL-4–treated macrophages, commonly called alternatively activated, are known to develop enhanced capacity for endocytosis, antigen presentation, and tissue repair and are generally considered anti-inflammatory. Considering IL-4 as priming rather than activating stimulus, we now compared the TLR4–dependent global gene activation program in IFNγ– versus IL-4–pretreated mouse macrophages, which has rarely been studied so far. Although both cytokines frequently induced opposing effects on gene transcription, the subsequent activation of bone marrow-derived macrophages by lipopolysaccharide (LPS) produced a strong, priming dependent pro-inflammatory response in both macrophage types. For example, the production of key pro-inflammatory cytokines IL-6 and IL-12 was significantly higher in IL-4– versus IFNγ–primed macrophages and several cytokine genes, including Il19, Ccl17, Ccl22, Ccl24 and Cxcl5, were preferentially induced in alternatively primed and LPS activated mouse macrophages. In a subset of genes, including IL12a, IFNγ priming was actually found to suppress LPS–induced gene expression in a Stat1–dependent manner. Our data suggest that IL-4–priming is not per se anti-inflammatory but generates a macrophage that is “tissue protective” but still capable of mounting a strong inflammatory response after TLR4–dependent activation. Keywords: Gene expression profiling Gene expression was investigated in mouse bone marrow-derived macrophages (BMM). On day 7, BMM were stimulated with either IL-4 or IFNγ overnight (18h in total). LPS treatment was performed in primed and unprimed macrophages 4 h prior to harvesting. At least three independent experiments were performed for each condition.

Project description:Purpose: In the early phases of T1D development exposure of β-cells to IFNγ, IL-1β, and TNFα is thought to induce β-cell dysfunction and the expression of chemokines and cytokines. These changes are mediated in part by post-translational histone modifications within cis-regulatory regions. The purpose of this study was to determine if prevention of these epigenetic changes can alter cytokine induced gene expression changes. Methods: Pancreatic islets were isolated from both male and female CD1 mice by collagenase digestion and were picked by hand. Hand-picked islets were cultured for four days with 1.6ng/ml IFNγ, 0.25ng/ml IL-1β and 0.16ng/ml TNFα (cytokine) or were left untreated (nocytokine). For RNA-seq islets were collected and processed for NGS using the Illumina TruSeq library prep kit. Chromatin immunoprecipitation (ChIP) was performed using 3μg of anti-H3K4me1 (Abcam) with islets from at least 10 adult ICR mice (8-10 weeks old) exposed to 1.6ng/ml IFNγ, 0.25ng/ml Il-1β and 0.16ng/ml TNFα for four days. DNA from 4 separate ChIP experiments was pooled for library construction and sequencing using the Illumina platform. Results: Comparison of gene expression levels between untreated and cytokine-treated islets resulted in the identification of 205 genes that showed an IFNγ, Il-1β, and TNFα-induced chromatin state change (from repressed or inactive to H3K4me1 enriched, i.e. the de novo regions), or increased H3K4me1 enrichment, that were associated with genes with up-regulated expression in response to IFNγ, Il-1β, and TNFα, and 111 genes with reduced H3K4me1 and down-regulated expression in response to IFNγ, Il-1β, and TNFα. Conclusions: Our study confirms that exposure of islets to pro-inflammatory cytokines induces the expression of chemokines and that these changes are the result of a switch from an inactive to an active chromatin state. Further we show that disruption of the trithorax group complex inhibits the upregulation of pro-inflammatory gene expression.

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CD4+ T cells treated with TNFalpha blocking agents during activation.

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