Characterization of regulatory T cells in obese omental adipose tissue in humans.

Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.

IL-33 Reverses an Obesity-Induced Deficit in Visceral Adipose Tissue ST2+ T Regulatory Cells and Ameliorates Adipose Tissue Inflammation and Insulin Resistance.

Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)-localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10-expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2(+) Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2(-) Tregs. The proportion of ST2(+) Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2(+) Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance.

Insulin inhibits IL-10-mediated regulatory T cell function: implications for obesity.

Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-ß. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.

Immune regulation in obesity-associated adipose inflammation.

Adipose tissue inflammation is often a consequence of obesity and is characterized by infiltration and activation of immune cells that overproduce cytokines and chemokines. This apparent loss of immune regulation in obese adipose tissue contributes to the ongoing chronic inflammation that is thought to promote the degradation of metabolic parameters in obesity. Much recent work has sought to identify the immune cell subsets that are involved in adipose tissue inflammation, understand the mechanisms by which adipose tissue inflammation develops, and develop immunotherapeutic strategies to reverse this process. In this review, we describe the known mechanisms that underlie the loss of immune regulation in obesity-associated adipose tissue inflammation and set the stage for the development of novel therapeutic approaches.

Cutting edge: PHLPP regulates the development, function, and molecular signaling pathways of regulatory T cells.

Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.

T reg-specific insulin receptor deletion prevents diet-induced and age-associated metabolic syndrome.

Adipose tissue (AT) regulatory T cells (T regs) control inflammation and metabolism. Diet-induced obesity causes hyperinsulinemia and diminishes visceral AT (VAT) T reg number and function, but whether these two phenomena were mechanistically linked was unknown. Using a T reg-specific insulin receptor (Insr) deletion model, we found that diet-induced T reg dysfunction is driven by T reg-intrinsic insulin signaling. Compared with Foxp3cre mice, after 13 wk of high-fat diet, Foxp3creInsrfl/fl mice exhibited improved glucose tolerance and insulin sensitivity, effects associated with lower AT inflammation and increased numbers of ST2+ T regs in brown AT, but not VAT. Similarly, Foxp3creInsrfl/fl mice were protected from the metabolic effects of aging, but surprisingly had reduced VAT T regs and increased VAT inflammation compared with Foxp3cre mice. Thus, in both diet- and aging-associated hyperinsulinemia, excessive Insr signaling in T regs leads to undesirable metabolic outcomes. Ablation of Insr signaling in T regs represents a novel approach to mitigate the detrimental effects of hyperinsulinemia on immunoregulation of metabolic syndrome.

The role of FOXP3 in regulating immune responses.

Regulatory T cells (Tregs) act in trans to control immune responses. The suppressive function of Tregs relies heavily on high and stable expression of the transcription factor FOXP3, which, together with other transcription factors, activates anti-inflammatory genes and represses proinflammatory genes. FOXP3 is required to shape the unique signaling mechanisms in Tregs, creating a positive-feedback pathway to further enhance its own expression. In addition, FOXP3 is thought to switch on a complex transcriptional network that leads to the stabilization of the Treg phenotype. Emerging data reveal that FOXP3 achieves this function in concert with several other transcription factors, many of which are associated with lineages of conventional T cells. In this review, we will discuss the structural features of FOXP3 and how it functions by interacting with other transcription factors. We will also summarize the role of FOXP3 in establishing the unique signaling cascades in Tregs. Finally, we will dissect the cooperative roles of FOXP3 and other T-cell lineage-defining transcription factors and discuss how these networks not only control the ability to Tregs to suppress different types of immune responses, but also enable Treg plasticity.

Methyltransferase G9A regulates T cell differentiation during murine intestinal inflammation.

Inflammatory bowel disease (IBD) pathogenesis is associated with dysregulated CD4⁺ Th cell responses, with intestinal homeostasis depending on the balance between IL-17-producing Th17 and Foxp3⁺ Tregs. Differentiation of naive T cells into Th17 and Treg subsets is associated with specific gene expression profiles; however, the contribution of epigenetic mechanisms to controlling Th17 and Treg differentiation remains unclear. Using a murine T cell transfer model of colitis, we found that T cell-intrinsic expression of the histone lysine methyltransferase G9A was required for development of pathogenic T cells and intestinal inflammation. G9A-mediated dimethylation of histone H3 lysine 9 (H3K9me2) restricted Th17 and Treg differentiation in vitro and in vivo. H3K9me2 was found at high levels in naive Th cells and was lost following Th cell activation. Loss of G9A in naive T cells was associated with increased chromatin accessibility and heightened sensitivity to TGF-ß1. Pharmacological inhibition of G9A methyltransferase activity in WT T cells promoted Th17 and Treg differentiation. Our data indicate that G9A-dependent H3K9me2 is a homeostatic epigenetic checkpoint that regulates Th17 and Treg responses by limiting chromatin accessibility and TGF-ß1 responsiveness, suggesting G9A as a therapeutic target for treating intestinal inflammation.

The Role of the PI3K Signaling Pathway in CD4(+) T Cell Differentiation and Function.

The relative activity of regulatory versus conventional CD4(+) T cells ultimately maintains the delicate balance between immune tolerance and inflammation. At the molecular level, the activity of phosphatidylinositol 3-kinase (PI3K) and its downstream positive and negative regulators has a major role in controlling the balance between immune regulation and activation of different subsets of effector CD4(+) T cells. In contrast to effector T cells which require activation of the PI3K to differentiate and mediate their effector function, regulatory T cells rely on minimal activation of this pathway to develop and maintain their characteristic phenotype, function, and metabolic state. In this review, we discuss the role of the PI3K signaling pathway in CD4(+) T cell differentiation and function, and focus on how modulation of this pathway in T cells can alter the outcome of an immune response, ultimately tipping the balance between tolerance and inflammation.