Single-cell RNA-sequencing of CD45+ splenocytes from aged WT, miR146a knockout, and miR146a-miR155 T cell conditional double knockout mice

Chronic age-dependent inflammation, or inflammaging, is a risk-factor for many disorders that emerge in aging human populations. Mechanisms underlying these aberrant immune responses are complex and remain to be elucidated. Among recently appreciated regulators of inflammaging are microRNAs; one example of which is the anti-inflammatory miR-146a, where deficient mice succumb to life-shortening chronic inflammation. In this study, we found that deletion of miR-155 in T cells significantly extends the lifespan of miR-146a-/- mice. Using single-cell RNA sequencing and flow cytometry we found that miR-155 promotes the activation of effector T cell populations, including Tfh cells, in mice aged over 15 months. This correlated with miR-155 dependent increases in germinal center B cells, autoantibody responses and serum IgG targeting tissue antigens throughout the body. Mechanistically, we found that the aerobic glycolysis genes are elevated in T cells during aging, and to even greater levels in the absence of miR-146a, and this was reduced upon deleting miR-155 in T cells. Finally, through deletion of the mitochondrial pyruvate carrier (MPC) complex in T cells, which skews metabolism towards aerobic glycolysis, we demonstrate that several of the age-dependent, activation phenotypes of miR-146a-/- T cells were recapitulated, thus revealing the sufficiency of metabolic reprogramming to influence immune cell functions during aging. Altogether, these data indicate that miRNAs play pivotal roles in regulating lifespan through T cell mediated inflammaging. WT (miR155 fl/fl), miR146a whole body knockout (146ko), and miR146-miR155 T cell conditional double knockout (tcko) (miR146ko-miR155tcko) mice were aged for 15 months. Single cell suspensions were prepared from spleens at the endpoint, pooled per each group (n=3-6 mice), followed by flow cytometric sorting of live (DAPI-) CD45+ lymphocytes (FSC-low, SSC-low). Sorted cells were processed for 10X Genomics 3' single cell sequencing.