Expression of the Butyrate/Niacin Receptor, GPR109a on T Cells Plays an Important Role in a Mouse Model of Graft Versus Host Disease

Abstract

INTRODUCTION: The intestinal microbiota is essential for the fermentation of fibers into the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors GPR41, GPR43 and GPR109a to activate downstream anti-inflammatory signaling pathways. In colitis or graft versus host disease (GVHD), GPR43 signaling has been reported as an important regulator of intestinal homeostasis by increasing the pool of regulatory T cells. In contrast to GPR43, which binds preferentially propionate and acetate, GPR109a is the major receptor for butyrate. We and others have demonstrated that butyrate can ameliorate gastrointestinal injury during GVHD through enterocyte protection. Therefore, we hypothesized that GPR109a plays an important role in the pathophysiology of intestinal GVHD, focusing specifically on alloreactive T cells. METHODS AND RESULTS: Using mouse models of GVHD, we examined the role of the butyrate/niacin receptor, GPR109a in allogeneic hematopoietic cell transplantation (allo-HCT). First, we studied whether a genetic knock-out (KO) of GPR109a in transplant recipient mice affected GVHD, but GPR109a-KO recipient mice did not exhibit increased mortality from GVHD compared to wild type (WT) mice. We next investigated the role of GPR109a in the donor compartment by transplanting either BM or T cells from WT or GPR109a-KO mice into major MHC mismatched BALB/c host mice. Mice transplanted with B6 BM, with T cells from a GPR109a-KO mouse into BALB/c hosts displayed a lower incidence of lethal GVHD (Fig. 1A). To determine whether the attenuation of GVHD is intrinsic to GPR109a-KO T cells, we established BM chimeras and performed a secondary transplant by transplanting B6 BM + (B6 à Ly5.1) or (GPR109a à Ly5.1) T cells into BALB/c hosts. We observed the same improvement in survival in mice that received GPR109a-KO T cells. This indicates an intrinsic role for GPR109a specifically in the donor hematopoietic compartment. Having identified a T-cell specific requirement for GPR109a we next examined expression of GPR109a on WT T cells in vitro at baseline and following stimulation with CD3/28 and found GPR109a significantly upregulated on both CD4+ and CD8+ T cells after 72 hours of stimulation (Fig 1B). At steady state in vivo, we observed the same numbers and percentages of splenic effector memory, central memory, and naïve CD4+ T cells as well as regulatory T cells in WT B6 mice and GPR109a-KO mice, suggesting normal T cell development in the knockout mice. In an in vitro mixed lymphocyte reaction (MLR), GPR109a-KO CD4+ T cells become activated, proliferate, polarize and secrete cytokine (specifically IFNg) to the same level as WT CD4+ T cells, suggesting normal functional capacity. However, after allo-HCT in mice we observed significantly fewer CD4+ and CD8+ T cells, and specifically fewer effector memory CD4+ T cells (Fig. C), in the small and large intestines of mice that received GPR109a-KO T cells at day 7 post transplant. In contrast, we found significantly more regulatory T cells in the intestines (Fig. 1D) and the spleen of GPR1091-KO T cell recipients, while numbers and percentages of polarized Th1 and Th17 T cells were similar between the two groups. We further 16S rRNA sequenced the gut microbiota of mice at day 7 after transplant and observed an increased relative abundance of bacteria from the genus Clostridium (Fig. 1D) along with an increased concentration of cecal butyrate as measured by GC-MS (Fig. 1E). In a preliminary graft versus tumor (GVT) experiment, we found that mice that received A20 tumor cells and GPR109a-KO T cells exhibited increased survival compared to mice that received A20 tumor cells and WT T cells. These preliminary findings suggest that GPR109a-KO T cells maintain their graft versus tumor response while causing less GVHD, and exclude a defective functional capacity. CONCLUSIONS: We report a novel role of the butyrate/niacin receptor GPR109a on donor T cells in allo-HCT as a genetic knock-out on T cells attenuates lethal GVHD. As these T cells are tested as functionally intact, we propose that the reduction in overall T cells of KO T cell recipients may underlie the attenuation in GVHD. Furthermore, such a reduction in allograft-induced gut injury is accompanied by maintenance of the gut commensal Clostridium and butyrate production, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. Disclosures No relevant conflicts of interest to declare.

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“… 106 The regulatory role of butyrate on T reg cells by GPR109A can also ameliorate gastrointestinal injury during graft versus host disease. 107 A previous study demonstrated that SCFA-mediated GPR109A and GPR43 activation played a role in food antigens tolerance by increasing mucosal CD103 + dendritic cells. 108 …”

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“…When transported in the systemic circulation SCFAs can interact with different immune, endocrine, neuronal, and humoral mechanisms, influencing the host gut–brain signaling pathways, thus having a central role in the maintenance of brain homeostasis and function [ 219 , 220 , 221 ]. SCFAs may accomplish their modulatory functions by acting as epigenetic modulators of gene expression, promoting histone hyperacetylation through the inhibition of histone deacetylases (HDACs), and as endogenous ligands for several G protein-coupled receptors (GPCRs), such as free fatty acid receptor 2 (FFAR2) and 3 (FFAR3) and GPR109A [ 222 , 223 , 224 ]. Expression of these receptors has been reported in several peripheral organs, including the gastrointestinal tract, and in the CNS [ 225 , 226 ].…”

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“…The GPR109A (hydroxycarboxylic acid receptor 2 (HCA2), expressed from the HCAR2 gene) GPCR is bound and activated by R-BHB (EC 50 of 0.7 mM [ 118 ]), S-BHB, or butyrate and is expressed in the lung and many types of epithelial cells, macrophages, neutrophils, and dendritic cells, but not in B or naïve T lymphocytes [ 164 ]. However, GPR109A was shown to play a role in the expansion of CD4 + and CD8 + T cells [ 165 ]. The expression pattern of GPR109A suggests that it could play a role in the protective effects of the ketogenic diet against IAV infection [ 113 ].…”