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

慢性年龄相关性炎症（chronic age-dependent inflammation），又称炎性衰老（inflammaging），是老年人群中多发多种疾病的风险因素。此类异常免疫反应的潜在机制复杂，迄今尚未完全阐明。近年来被逐步认知的炎性衰老调节因子包括微RNA（microRNAs），其中抗炎性miR-146a便是典型代表——该基因缺陷的小鼠会因致死性慢性炎症而寿命缩短。本研究发现，在T细胞中敲除miR-155可显著延长miR-146a基因敲除（miR-146a-/-）小鼠的寿命。借助单细胞RNA测序（single-cell RNA sequencing）与流式细胞术（flow cytometry），我们证实miR-155可促进15月龄以上小鼠效应T细胞群（包括滤泡辅助性T细胞（T follicular helper cells, Tfh））的活化。该现象与miR-155依赖的生发中心B细胞（germinal center B cells）比例升高、自身抗体应答增强以及针对全身组织抗原的血清IgG水平上调密切相关。机制研究层面，我们发现衰老过程中T细胞内的有氧糖酵解（aerobic glycolysis）相关基因表达量升高，而在miR-146a缺失的情况下该上调幅度更为显著；在T细胞中敲除miR-155可逆转这一变化。最后，通过在T细胞中敲除线粒体丙酮酸载体（mitochondrial pyruvate carrier, MPC）复合物——该复合物的缺失会使细胞代谢偏向有氧糖酵解——我们重现了miR-146a-/- T细胞的多种年龄依赖性活化表型，由此证明代谢重编程足以在衰老过程中调控免疫细胞功能。综上，本研究数据表明，微RNA通过T细胞介导的炎性衰老在调控机体寿命中发挥关键作用。整体实验设计：将野生型（WT，miR155 fl/fl）、全身敲除miR-146a的小鼠（miR-146a-/-，146ko）以及T细胞条件性双敲除miR-146与miR-155的小鼠（miR146ko-miR155tcko，tcko）均饲养至15月龄。实验终点处制备脾脏单细胞悬液，每组混合3-6只小鼠的样本，随后通过流式细胞术分选出活的（DAPI阴性）CD45+淋巴细胞（FSC低、SSC低）。分选后的细胞交由10X Genomics进行3'端单细胞测序。