Fasting-induced FOXO4 blunts human CD4+ T helper cell responsiveness

Abstract

Intermittent fasting blunts inflammation in asthma 1 and rheumatoid arthritis 2 , suggesting that fasting may be exploited as an immune-modulatory intervention. However, mechanisms underpinning anti-inflammatory effects of fasting remain poorly characterized 3 , 4 , 5 . Here, we show that fasting in humans is sufficient to blunt CD4 + T helper cell responsiveness. RNA-seq and flow cytometric immunophenotyping of peripheral blood mononuclear cells (PBMCs) from volunteers subjected to overnight or 24-hour fasting, and 3-hours of refeeding implicate that fasting blunts CD4 + T helper cell activation and differentiation. Transcriptomic analysis reveal that the longer fast-duration has a more robust effect on CD4 + T cell biology. Through bioinformatic analyses, we identify the transcription factor FOXO4 and its canonical target FKBP5 as a potential fasting-responsive regulatory axis. Genetic gain- or loss-of-function of FOXO4 and FKBP5 is sufficient to modulate Th1 and Th17 cytokine production. Moreover, we find that fasting-induced or genetic overexpression of FOXO4 and FKBP5 is sufficient to downregulate mTORC1 signaling and suppress STAT1/3 activation. Our results identify FOXO4-FKBP5 as a novel fasting-induced, STAT-mediated, regulatory pathway to blunt human CD4 + T helper cell responsiveness.

Identification and Validation of Nutrient State-Dependent Serum Protein Mediators of Human CD4+ T Cell Responsiveness

“…In this study, the 24-h fast was shown to have more robust gene regulatory responses versus refeeding when compared to the overnight fast, supporting the concept that the hormetic stress of the longer fast initiates more robust immunomodulatory effects. The unbiased bioinformatic analysis of the data in the PBMCs supported the results that fasting had a robust effect on CD4 + T cells, although the study could not distinguish between cell intrinsic regulatory effects versus paracrine effects on the cells from the 24-h fast [ 32 ]. Prior studies showed that serum extracted from fasting versus refed subjects and used for the incubation of THP-1 macrophages showed differential modulation of the NLRP3 inflammasome [ 6 , 12 ], although what factors in serum orchestrated this immunomodulation has not been determined.…”

“…In the initial study on this cohort of subjects comparing RNAseq analysis and flow cytometry, it was found that the immunomodulatory effects of the 24-h fast were more robust than the overnight fast when both groups were compared to refeeding [ 32 ]. Hence, an additional integrated bioinformatics approach was employed to evaluate if we could uncover additional differences in circulating protein levels that could correlate with PBMC gene expression level changes in response to 24 h of fasting or refeeding.…”

“…The potential utility of this approach is supported by the finding that serum extracted after time-controlled fasting and refeeding in healthy volunteers and in asthma subjects differentially regulated the NLRP3 inflammasome response when used as the exclusive incubation serum in transformed THP-1 monocytic/macrophage cells [ 6 , 12 ]. A recent study identified that time-controlled fasting similarly blunted CD4 + T helper cell immune responsiveness [ 32 ]. In that study, RNA-seq and flow-cytometric analysis was performed on peripheral blood mononuclear cells from healthy volunteers in response to a baseline overnight fast, a 24-h fast and 3 h of refeeding.…”

“…In that study, RNA-seq and flow-cytometric analysis was performed on peripheral blood mononuclear cells from healthy volunteers in response to a baseline overnight fast, a 24-h fast and 3 h of refeeding. The findings included that the greater duration of the fast was linked to greater effects on blunting immune responsiveness and this fasting effect was very robust in blunting CD4 + T helper cell activation [ 32 ].…”

“…To evaluate the effects of changes in serum proteins in response to fasting and refeeding, serum from the same subjects in response to overnight fasting, 24-h fasting and refeeding [ 32 ] were analyzed using SOMAscan proteomics, and multivariate analysis was employed to identify caloric load-dependent candidate proteins. Additional candidates were identified using integrative bioinformatics by combining the SOMAscan results with the previously published peripheral blood mononuclear cell (PBMC) RNA-seq data (GEO database link: , accessed on 20 January 2021).…”