Everolimus alleviates CD4+ T cell inflammation by regulating autophagy and cellular redox homeostasis

Abstract

Aging is associated with the onset and progression of multiple diseases, which limit health span. Chronic low-grade inflammation in the absence of overt infection is considered the simmering source that triggers age-associated diseases. Failure of many cellular processes during aging is mechanistically linked to inflammation; however, the overall decline in the cellular homeostasis mechanism of autophagy has emerged as one of the top and significant inducers of inflammation during aging, frequently known as inflammaging. Thus, physiological or pharmacological interventions aimed at improving autophagy are considered geroprotective. Rapamycin analogs (rapalogs) are known for their ability to inhibit mTOR and thus regulate autophagy. This study assessed the efficacy of everolimus, a rapalog, in regulating inflammatory cytokine production in T cells from older adults. CD4⁺ T cells from older adults were treated with a physiological dose of everolimus (0.01 µM), and indices of autophagy and inflammation were assessed to gain a mechanistic understanding of the effect of everolimus on inflammation. Everolimus (Ever) upregulated autophagy and broadly alleviated inflammatory cytokines produced by multiple T cell subsets. Everolimus's ability to alleviate the cytokines produced by Th17 subsets of T cells, such as IL-17A and IL-17F, was dependent on autophagy and antioxidant signaling pathways. Repurposing the antineoplastic drug everolimus for curbing inflammaging is promising, given the drug's ability to restore multiple cellular homeostasis mechanisms.

Keywords: Autophagy; CD4+ T cells; Everolimus; Inflammaging; NRF2; ROS; Th17 cytokines.

Fig. 1

Everolimus alters T cell cytokine profiles from older adults. Cytokine production was assessed in T cells from O adults ± 0.01 µM in vitro everolimus (Ever) treatment (a). Score plot (top) and LV 1 loading plot (bottom) of the VIP PLS-DA model O vs O + Ever (1 LV, accuracy 78.3%, confidence 98.2%). Red cytokine loadings signify cytokines with a VIP score > 1. Each point represents a single sample. T cells from O adults (negative scores) feature upregulation of cytokines with negative loadings and downregulation of cytokines with positive loadings, as compared to T cells from O adults treated with Ever (positive scores on LV1) (b). Each point represents a single individual. N = 7–12 (a). Each point in the scores plot represents a sample, with blue points representing the treatment group and orange points representing the untreated group (b). The Mann–Whitney test was performed for univariate analysis, *p < 0.05 vs. O

Fig. 2

Everolimus selectively inhibits mTORC1 and promotes autophagy in T cells from older adults. Western immunoblotting quantified the protein expression of downstream mTOR targets. Representative blots (a), p-Raptor S863 (b), p-P70S6K T389 (c), p-4EBP T37 (d), and p-AKT S473 (e). Immunofluorescence and confocal microscopy assessed LC3B (f) and LAMP1 (g). Colocalization between LC3B and LAMP1 representative images (h) and quantification (i). N = 3–6 (a–e), N = 3–4 (f–i). For microscopy, at least 5–7 fields per slide with at least three cells/field were imaged at 63 × magnification with oil immersion, on a Zeiss LSM 800 confocal microscope. Images were processed as described in “Materials and methods” and brightness was adjusted to improve clarity. Student’s t test or the Mann–Whitney test or Kruskal-Wallis test, according to data set *p < 0.05 vs. O, #p < 0.05 vs. O + Ever

Fig. 3

Everolimus improves redox regulation in T cells from older adults. Heatmap showing sc-RNA sequencing data of antioxidant gene expression log2 fold change between Th17 cells from Y and O adults (a), t-SNE plot of redox genes (b), stacked violin plots of redox gene grouped by clusters from Seurat (c), and immunoblotting quantified protein levels of SOD1 (d) and SOD2 (e). Immunofluorescence and confocal microscopy quantified catalase (f) and glutathione peroxidase (g). N = 3 each Y and O (a–c), N = 4–6 (d, e), N = 3–4 (f, g). For microscopy, at least 5–7 fields per slide with at least three cells/field were imaged at 63 × magnification with oil immersion, on a Zeiss LSM 800 confocal microscope. Images were processed as described in “Materials and methods” and brightness was adjusted to improve clarity. Student’s t test or the Mann–Whitney test or Wilcoxon matched-pair signed rank test, according to data set *p < 0.05 vs. O

Fig. 4

Everolimus regulation of Th17 cytokines and redox balance is autophagy-dependent. Autophagy was silenced utilizing siRNA to Atg3 protein. Protein level of Atg3 (a) and LC3B (b). Th17 cytokine IL-17F production assessed via Luminex assay (c) and IL-17A production assessed via ELISA (d). Protein levels assessed via immunofluorescence in Atg3-silenced cells; representative images (e), SOD1 (f), SOD2 (g), catalase (h), and glutathione peroxidase (GPX) (i). N = 3–4 (a, b, e–i), N = 6–12 (c, d). For microscopy, at least 5–7 fields per slide were imaged at 63 × magnification with oil immersion, on a Zeiss LSM 800 confocal microscope. Images were processed as described in “Materials and methods” and brightness was adjusted to improve clarity. The Wilcoxon test or Kruskal–Wallis test with Dunn’s post hoc, according to data set, *p < 0.05 vs. Con siRNA+ Ever or Con siRNA.

Fig. 5

Everolimus promotes nuclear localization of Nrf2 in T cells. Immunofluorescence assessed protein levels and cellular localization of NRF2 (a); representative images (b) and quantification of colocalization of NRF2-KEAP-1 (c); representative images (d) and quantification of localization of NRF2 in the nucleus in T cells from O adults (e). NRF2 expression (f) and KEAP-1-p62 interaction in Y cells after genetic disruption of autophagy; representative images (g) and quantification (h). For microscopy, at least 5–7 fields per slide were imaged at 63 × magnification with oil immersion, on a Zeiss LSM 800 confocal microscope. Images were processed as described in “Materials and methods” and brightness was adjusted to improve clarity. The Mann–Whitney or Wilcoxon test or Kruskal–Wallis test with Dunn’s post hoc, according to data set, *p < 0.05 vs. O or Con siRNA

Fig. 6

Inhibiting Nox alleviates Th17 inflammation in autophagy-impaired cells. A Luminex assay quantified cytokine production in autophagy-deficient T cells from Y adults after treatment with mitochondria-targeted superoxide scavenger mito-Tempo (a) and NADPH oxidase inhibitor VAS2870 (b). N = 5–10 (a, b). The Kruskal–Wallis test with Dunn’s post hoc, *p < 0.05 vs. Con siRNA, #p < 0.05 vs. Atg3 siRNA