Ex-Th17 (Nonclassical Th1) Cells Are Functionally Distinct from Classical Th1 and Th17 Cells and Are Not Constrained by Regulatory T Cells.

Th17 cells are an important therapeutic target in autoimmunity. However, it is known that Th17 cells exhibit considerable plasticity, particularly at sites of autoimmune inflammation. Th17 cells can switch to become ex-Th17 cells that no longer produce IL-17 but produce IFN-γ. These ex-Th17 cells are also called nonclassical Th1 cells because of their ability to produce IFN-γ, similar to Th1 cells; however, it is unclear whether they resemble Th1 or Th17 cells in terms of their function and regulation, and whether they have a pathogenic role in autoimmunity. We compared the phenotypic and functional features of human Th17, Th1, and ex-Th17 cell populations. Our data showed that despite their loss of IL-17 expression, ex-Th17 cells were more polyfunctional in terms of cytokine production than either Th1 or bona fide Th17 cells, and produced increased amounts of proinflammatory cytokines. The proliferative brake on Th17 cells appeared to be lifted because ex-Th17 cells proliferated more than Th17 cells after stimulation. In contrast with Th1 and Th17 cells, ex-Th17 cells were highly resistant to suppression of proliferation and cytokines by regulatory T cells. Finally, we showed that ex-Th17 cells accumulated in the joints of rheumatoid arthritis patients. Taken together, these data indicate that human ex-Th17 cells are functionally distinct from Th1 and Th17 cells, and suggest that they may play a pathogenic role at sites of autoimmunity, such as the rheumatoid arthritis joint where they accumulate. These findings have implications for therapeutic strategies that target IL-17, because these may not inhibit pathogenic ex-Th17 cells.

Polyfunctional, Pathogenic CD161+ Th17 Lineage Cells Are Resistant to Regulatory T Cell-Mediated Suppression in the Context of Autoimmunity.

In autoimmune diseases such as rheumatoid arthritis (RA), regulatory T cells (Tregs) fail to constrain autoimmune inflammation; however, the reasons for this are unclear. We investigated T cell regulation in the RA joint. Tregs from RA synovial fluid suppressed autologous responder T cells; however, when compared with Tregs from healthy control peripheral blood, they were significantly less suppressive. Despite their reduced suppressive activity, Tregs in the RA joint were highly proliferative and expressed FOXP3, CD39, and CTLA-4, which are markers of functional Tregs. This suggested that the reduced suppression is due to resistance of RA synovial fluid responder T cells to Treg inhibition. CD161(+) Th17 lineage cells were significantly enriched in the RA joint; we therefore investigated their relative susceptibility to Treg-mediated suppression. Peripheral blood CD161(+) Th cells from healthy controls were significantly more resistant to Treg-mediated suppression, when compared with CD161(-) Th cells, and this was mediated through a STAT3-dependant mechanism. Furthermore, depletion of CD161(+) Th cells from the responder T cell population in RA synovial fluid restored Treg-mediated suppression. In addition, CD161(+) Th cells exhibited pathogenic features, including polyfunctional proinflammatory cytokine production, an ability to activate synovial fibroblasts, and to survive and persist in the inflamed and hypoxic joint. Because CD161(+) Th cells are known to be enriched at sites of autoinflammation, our finding that they are highly proinflammatory and resistant to Treg-mediated suppression suggests an important pathogenic role in RA and other autoimmune diseases.

Differential Regulation of Human Treg and Th17 Cells by Fatty Acid Synthesis and Glycolysis.

In this study we examined the metabolic requirements of human T helper cells and the effect of manipulating metabolic pathways in Th17 and Treg cells. The Th17:Treg cell axis is dysregulated in a number of autoimmune or inflammatory diseases and therefore it is of key importance to identify novel strategies to modulate this axis in favor of Treg cells. We investigated the role of carbohydrate and fatty acid metabolism in the regulation of human memory T helper cell subsets, in order to understand how T cells are regulated at the site of inflammation where essential nutrients including oxygen may be limiting. We found that Th17 lineage cells primarily utilize glycolysis, as glucose-deprivation and treatment with rapamycin resulted in a reduction in these cells. On the other hand, Treg cells exhibited increased glycolysis, mitochondrial respiration, and fatty acid oxidation, whereas Th17 cells demonstrated a reliance upon fatty acid synthesis. Treg cells were somewhat reliant on glycolysis, but to a lesser extent than Th17 cells. Here we expose a fundamental difference in the metabolic requirements of human Treg and Th17 cells and a possible mechanism for manipulating the Th17:Treg cell axis.

Resolution of TLR2-induced inflammation through manipulation of metabolic pathways in Rheumatoid Arthritis.

During inflammation, immune cells activated by toll-like receptors (TLRs) have the ability to undergo a bioenergetic switch towards glycolysis in a manner similar to that observed in tumour cells. While TLRs have been implicated in the pathogenesis of rheumatoid arthritis (RA), their role in regulating cellular metabolism in synovial cells, however, is still unknown. In this study, we investigated the effect of TLR2-activation on mitochondrial function and bioenergetics in primary RA-synovial fibroblast cells (RASFC), and further determined the role of glycolytic blockade on TLR2-induced inflammation in RASFC using glycolytic inhibitor 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). We observed an increase in mitochondrial mutations, ROS and lipid peroxidation, paralleled by a decrease in the mitochondrial membrane potential in TLR2-stimulated RASFC. This was mirrored by differential regulation of key mitochondrial genes, coupled with alteration in mitochondrial morphology. TLR2-activation also regulated changes in the bioenergetic profile of RASFC, inducing PKM2 nuclear translocation, decreased mitochondrial respiration and ATP synthesis and increased glycolysis:respiration ratio, suggesting a metabolic switch. Finally, using 3PO, we demonstrated that glycolytic blockade reversed TLR2-induced pro-inflammatory mechanisms including invasion, migration, cytokine/chemokine secretion and signalling pathways. These findings support the concept of complex interplay between innate immunity, oxidative damage and oxygen metabolism in RA pathogenesis.