Mechanisms of Adipose Tissue Immunoregulatory T cell (Treg) Exhaustion in Obesity

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Project Abstract. Adipose tissue (AT) regulatory T cells (Tregs) are major determinants of systemic metabolism, and in lean mice, protect against obesity-associated inflammation and complications. Tregs are key in homeostatic maintenance in lean AT, but abundance profoundly decreases in obese AT leading to inflammation, insulin resistance, and other inflammatory-driven complications. Our preliminary data suggests exhaustion contributes to the loss of AT Tregs in obesity. AT vs. blood Tregs from obese humans reveal increased expression of programmed cell death protein 1 (PD-1) and other inhibitory co-receptors (OX40, CTLA4); impaired suppressive function, which is reversible; decreased liver kinase B1 (LKB1), which protects Tregs from exhaustion; and increased methylation of the CNS2 in the FOXP3 locus suggesting instability. These characteristics form our working definition of exhaustion. Moreover, RNAseq analyses of human AT PD-1 high vs. negative cells revealed downregulation of 75% of significantly changed genes, including genes involved in suppressor function, while upregulated genes included apoptosis and cell death genes. Furthermore, in cultured human Tregs, interferon gamma (IFNG) stimulated expression of inhibitory co-receptors and apoptosis markers and decreased LKB1 expression, suggesting it may mediate AT Treg exhaustion. These findings underscore the need to investigate mechanisms regulating AT Treg abundance/function. Our Specific Aims include: Aim 1. Hypothesis: Human obese AT Tregs are phenotypically exhausted, which contributes to the decline in human AT Tregs during high fat diet (HFD) ingestion. We will: A) Determine whether there are more exhausted Tregs in obese vs. lean AT; B) Utilize single cell (sc)RNAseq and Global DNA methylation to define subpopulations of human lean and obese AT Tregs that may be exhausted; and C) Determine whether pioglitazone will attenuate the HFD-induced AT Treg decline in lean humans by preventing exhaustion and impacting AT Treg transcriptional changes. Aim 2. Hypothesis: Interferon-gamma (IFNG), toxic lipids, and/or decreased Treg PPARγ activity are mediators that contribute to exhaustion and declining Tregs in obesity. To determine whether attenuation/knockout of specific pathways in Tregs (supported by evidence of in vivo metabolic changes) leads to changes in AT Treg exhaustion, AT Treg abundance, and HFD-induced systemic insulin resistance, we will use several mouse models predicted to promote Treg exhaustion: A) Treg-specific knockout of the IFNG receptor (IFNGR1); B) Treg-specific loss of serine palmitoyl transferase 2 (encoded by Sptlc2), a rate limiting enzyme required for ceramide biosynthesis; and C) Treg specific ablation of a key supportive factor for AT Tregs, PPARγ. Taken together, these investigations will shed light on a new, potentially important mechanism explaining the striking loss of AT Tregs that occurs with HFD and obesity.