Single-cell RNA-sequencing of CD45+ splenocytes from aged WT, miR146a knockout, and miR146a-miR155 T cell conditional double knockout mice
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ABSTRACT: 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.
Project description:Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. Here, we identify loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure, and reduced the incidence of hematological malignancy in miR 146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR 146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.
Project description:Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. Here, we identify loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a-null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a-/- myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a-/- HSC function and subpopulation structure, and reduced the incidence of hematological malignancy in miR 146a-/- mice. miR-146a-/- HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR 146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.
Project description:Differential gene expression profile of CD4+ T cells from 10 months old Wt, miR-155-/-, miR-146a-/- and DKO mice spleens. Wt, miR-155-/-, miR-146a-/- and DKO mice were aged 10 months, CD4+ T cells were sorted from mice spleens for analyses.
Project description:Endotoxin/LPS tolerance is a tightly regulated phenomenon, which, during infection, prevents systemic hyper-inflammation. Here we report for the first time that morphine reversal of endotoxin tolerance resulting in persistent inflammation thus contributing to septicemia and septic shock. We further report that this regulation is mediated by LPS-induced down-regulation of microRNAs 146a and 155. However, only over-expression of miR-146a, but not miR-155 abrogates morphine mediated hyper-inflammation, while antagonizing miR-146a (but not miR-155) augments morphine mediated hyper-inflammation. Hence, miR-146a could be the potential therapeutic target for morphine-mediated abrogation of endotoxin tolerance.
Project description:The innate inflammatory response must be tightly regulated to ensure effective immune protection while avoiding inflammation-related pathologies. The transcription factor NF-kB is a critical mediator of the inflammatory response, and its dysregulation has been associated with immune related malignancies. We herein show that miR-155, miR-146a and NF-kB form a regulatory network that tunes the macrophage inflammatory response in mice. We show that elevated miR-155 expression potentiates NF-kB activity in miR-146a deficient mice, thus leading to an overactive acute inflammatory response and chronic inflammation. Enforced miR-155 expression overrides miR-146a-mediated repression of NF-kB activation, thus emphasizing that miR-155 plays a dominant, downstream role in promoting inflammation. We further show that miR-155 deficient macrophages exhibit a suboptimal inflammatory response when exposed to low levels of inflammatory stimuli. Importantly, we demonstrate a temporal asymmetry between miR-155 and miR-146a expression during macrophage activation, which forms a combined positive and negative feedback network on NF-kB activity. This miRNA based regulatory network enables a robust and time-limited inflammatory response essential for functional immunity.
Project description:Endotoxin/LPS tolerance is a tightly regulated phenomenon, which, during infection, prevents systemic hyper-inflammation. Here we report for the first time that morphine reversal of endotoxin tolerance resulting in persistent inflammation thus contributing to septicemia and septic shock. We further report that this regulation is mediated by LPS-induced down-regulation of microRNAs 146a and 155. However, only over-expression of miR-146a, but not miR-155 abrogates morphine mediated hyper-inflammation, while antagonizing miR-146a (but not miR-155) augments morphine mediated hyper-inflammation. Hence, miR-146a could be the potential therapeutic target for morphine-mediated abrogation of endotoxin tolerance. All treatments done in vivo. Morphine implanted subcuteniously, LPS administered as intraperitoneal injection.
Project description:Epigenetic modifications, including the role of miRs in various diseases, are key regulators of gene expression. In this study we aimed to explore whether epigenetic alterations could play a role in the TRPV4-mediated regulation of miR-146a. Here we report that there is no significant differences in the methylation status of the examined 10 CpG sites on miR146a promoter between WT and TRPV4 KO cells under LPS treatment, indicating that the regulation of miR-146a by TRPV4 may not be mediated by methylation changes at these specific sites.
Project description:small RNAseq was preformed on Wt bone marrow-derived dendritic cells (BMDC) and miR-155 and miR-146a double knockout (DKO) BMDCs that received Wt exosomes to investigate the differences in transferred miRNA
Project description:To identify pathways and processes driving the observed hematopoietic stem cell (HSC) aging-like phenotypes in miR-146a-/- vs. WT, we performed RNA-seq gene expression profiling of Lin- Sca-1+ c-Kit+ (LSK) cells isolated from miR-146a-/- or WT mouse bone marrow (BM). Differential expression analysis and EnrichmentMap network analysis identified cytokine signalling and immune pathways as potential drivers of aging-like alterations in miR-146a-/- HSC proliferation and differentiation.