(61d) Epigenetic Modulation of Inflammatory T Cells in Autoimmune Disease

Introduction: A shared hallmark of gut autoimmunity is the marked decrease in the concentration of short chain fatty acids (SCFAs), which are metabolic products of gut microbiota and important immunomodulators that regulate gut health. The four and five carbon SCFAs, butyrate and pentanoate respectively, are two of the primary immune modulating agents in the gut, and potently inhibit the activity of histone deacetylases (HDAC) and thereby regulate gene transcription of key transcription factors and cytokines by modulating chromatin structure in a concentration dependent manner. HDAC inhibition by SCFAs suppress the production of pro- and enhance anti-inflammatory cytokine production by T cells in the gut. The characteristic inflammation in gut autoimmune disorders is caused, in part, by the upregulation of pro-inflammatory T cells, often accompanied by a deficiency in the number and function of immunosuppressive regulatory T cells (T_regs). An increase in the concentration of pro-inflammatory cytokines such as IL-6, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) leads to differentiation of naïve CD4+ T cells into pro-inflammatory IL-17A producing T helper (Th) 17, and IFN-γ-producing Th1 cells. We hypothesized that promoting HDAC inhibition with SCFA supplementation might restore the regulation of cytokine production by T cells, downregulate the pro-inflammatory mediators and restore the balance in T cell subsets.

Materials and Methods: To test the hypothesis, we isolated C57Bl/6J mouse T cells from the spleen and cultured them in media containing butyrate and pentanoate. T-cell media, consisting of RPMI cell media prepared with 10% Fetal Bovine Serum was used to culture the cells. In order to simulate the inflammatory milieu, the wells of T cells were activated with anti-CD3 and, anti-CD28 antibodies in media with Th17-inducing cytokines. Cells were counted at predetermined time intervals and stimulated at predetermined time intervals with phorbol myristate acetate and ionomycin to induce cytokine expression. Cytokine transport was blocked with brefeldin A and pro and anti-inflammatory cytokine expression was analyzed using flow cytometry. Tregs and Th17 cells were quantified based on FoxP3 and ROR-γt expression respectively.

Results and Discussion: Butyrate reduced IL-17A expressing CD4+ mouse T cells to 10%, compared to 17% in Th17-inducing control conditions. In Th17-inducing conditions alone, 25% of cells expressed pro-inflammatory TNF-α. Addition of Butyrate to the cell culture medium suppressed this expression to 15%. We measured the concentration-dependence of T cell expansion on pentanoate in non-inflammatory conditions. With 1 mM pentanoate, we measured 5-fold expansion over 4 days. 2 mM pentanoate induced 11-fold expansion of T-cells over 4 days, compared to 2.5-fold expansion in control conditions. 3 mM induced 6-fold expansion over 4 days, indicating that there is an optimal concentration for maximizing T cell proliferation. In non-inflammatory conditions, addition of 1 mM butyrate resulted in 40% expression of IL-10, an immunosuppressive cytokine that is associated with T_reg function, compared to ~1% in the control group. Pentanoate induced 20% IL-10 expression. However, upregulation of IL-10 was abrogated in Th17 inducing conditions, with IL-10 expression falling to 2%. To restore the production of anti-inflammatory IL-10, we tested combinations of epigenetic modulators by introducing all-trans retinoic acid (ATRA), a clinically used DNA methylating agent. Strikingly, ATRA stabilized the T_reg phenotype, increased the proportion of T_regs 3.5-fold compared to the control group in Th17 inducing conditions, and reduced IL-17A expression to 10% compared to 28% in control conditions. ATRA and butyrate in combination reduced the expression of IL-17A to 1%.

Conclusions: Our findings suggest that SCFAs potently modulate the activity of T cells and may enhance the functionality of T_regs. The metabolic modulation by pentanoate is responsible for both increased expansion and IL-10 expression, an effect which could be useful in combination with butyrate which can enhance T_reg differentiation. However, the abrogation of SCFA mediated IL-10 production in simulated inflammatory conditions suggests that restoring immune homeostasis in established disease may require a multifactor approach. The combination of ATRA and butyrate significantly promotes the differentiation of naive T cells to T_regs and inhibits IL-17 production in Th17 inducing conditions and may be a means to restore immune balance in gut-based autoimmune disorders.