Metabolic and redox regulation of IL-17-producing γδ T cells

Abstract

Interleukin-17 (IL-17) is a proinflammatory cytokine that contributes to a wide range of immune responses, including host defence, tissue repair, autoimmune inflammation, and tumour progression. γδ17 T cells (CD3+TCRδ+CD27-) are a small population of unconventional T cells, which are developmentally pre- programmed to rapidly produce IL-17 in response to cytokine and T cell receptor (TCR) signals. Due to these innate response kinetics, γδ17 T cells often play a priming role in both pathogenic and protective contexts, highlighting a need to further understand how they are regulated. Immunometabolism has emerged as an exciting field in understanding the interplay between cellular metabolism and immune cell function, and identifying metabolic vulnerabilities may offer new therapeutic targets in treating disease. Data from our lab has shown that γδ17 and γδIFN T cells display distinct metabolic profiles. Whilst γδIFN T cells primarily rely on glycolysis, γδ17 T cells are characterised by high rates of oxidative metabolism. γδ17 T cells also display high rates of fatty acid uptake and storage, and are enriched in mouse models of obesity. Here, we show that γδ17 T cells display a highly proliferative phenotype compared to γδIFN T cells, associated with a significantly enhanced capacity to take up fatty acids in both Vγ4+ and Vγ6+ T cells. Unlike γδIFN T cells, γδ17 T cells displayed a high convergence between fatty acid uptake and cytokine production. Furthermore, γδ17 T cells upregulate mTORC1 signalling in response to stimulation with IL-1b and IL-23, which was required for both IL-17A and IL-17F production, but not fatty acid uptake. RNA-sequencing of unstimulated and IL- 1b+23-stimulated γδ17 T cells revealed an upregulation of gene programmes involved in lipid metabolism following activation, including fatty acid uptake, fatty acid synthesis, and PPAR signalling. Accumulation of lipids following activation was dependent on the transcription factor PPARγ, antagonism of which significantly reduced total neutral lipid content and the ability to produce IL-17A and IL-17F. Furthermore, we identify the membrane channel aquaporin 3 (AQP3), which facilitates transmembrane transport of uncharged solutes such as water, glycerol and hydrogen peroxide (H2O2), as being highly and specifically expressed by γδ17 T cells across multiple peripheral sites. Surprisingly, using CRISPR/Cas9- generated Aqp3-/- mice, we identify the H2O2-transporting capacity of AQP3 as having a role in regulating γδ17 T cell function. Ex vivo supplementation with H2O2 dose-dependently increased cellular reactive oxygen species (ROS) content in WT γδ17 T cells, whilst Aqp3-/- cells were unaffected, suggesting uptake of H2O2 by γδ17 T cells is AQP3-dependent. Treatment of cells with increasing concentrations of H2O2 during cytokine activation dose-dependently reduced fatty acid uptake, mTORC1 signalling, proliferation, and IL-17A production in an AQP3-dependent manner. Furthermore, coculture with neutrophils suppressed IL-17 production and proliferation in WT, but not Aqp3-/-, γδ17 T cells, suggesting that regulation of γδ17 T cells by neutrophils is dependent on AQP3 expression. Preliminary data from B16 tumour models suggests that γδ17 T cells retain more function in the tumours of Aqp3-/- mice in terms of Ki-67 expression and IL-17A and IL-17F production. Thus, we identify AQP3 as a specific regulator of IL-17-producing γδ T cell metabolic and functional responses via uptake of H2O2. Finally, we show that γδ17 T cells display significantly higher endogenous ROS levels than γδIFN T cells, both at rest and in response to cytokine stimulation. Whilst we have shown that γδ17 T cells are particularly susceptible to ROS- induced dysfunction, we hypothesised that metabolic programmes upregulated during activation may buffer endogenous cellular ROS to facilitate effector responses. RNAseq data revealed an upregulation of pathways involved in glutamine metabolism, which has previously been shown to regulate ROS levels in Th17 cells. Here, we show that γδ17 T cells take up more glutamine than γδIFN T cells. Inhibition of glutaminolysis in γδ17 T cells results in a significant increase in cellular ROS levels, and significantly reduces their ability to produce IL-17, but not IFN-γ, in response to IL-1b and IL-23. Scavenging of ROS using N-acetyl cysteine (NAc) rescued this defect, indicating that glutamine metabolism is required for optimal IL-17 production by γδ17 T cells through the maintenance of redox balance. Furthermore, we show that glutamine metabolism is also required for γδ17 T cell proliferation and oxidative metabolism, suggesting that glutamine is a fuel source in maintaining energy flux through the mitochondria. Overall, we have further characterised the metabolic requirements of γδ17 T cells during activation, as well as how this regulates their function, which may offer insight into how they can be regulated in pathological or protective contexts.