ABSTRACT
Senescent cells, which accumulate with age, exhibit a pro-inflammatory senescence-associated secretory phenotype (SASP) that includes the secretion of cytokines, lipids, and extracellular vesicles (EVs). Here, we established an in vitro model of senescence induced by Raf-1 oncogene in RAW 264.7 murine macrophages (MΦ) and compared them to senescent MΦ found in mouse lung tumors or primary macrophages treated with hydrogen peroxide. The transcriptomic analysis of senescent MΦ revealed an important inflammatory signature regulated by NFkB. We observed an increased secretion of EVs in senescent MΦ, and these EVs presented an enrichment for ribosomal proteins, major vault protein, pro-inflammatory miRNAs, including miR-21a, miR-155, and miR-132, and several mRNAs. The secretion of senescent MΦ allowed senescent murine embryonic fibroblasts to restart cell proliferation. This antisenescence function of the macrophage secretome may explain their pro-tumorigenic activity and suggest that senolytic treatment to eliminate senescent MΦ could potentially prevent these deleterious effects.
ABSTRACT
Erythropoietin (EPO) is an essential hormone for erythropoiesis, protecting differentiating erythroblasts against apoptosis. EPO has been largely studied in stress or pathological conditions but its regulatory role in steady state erythropoiesis has been less documented. Herein, we report production of EPO by bone marrow-derived macrophages (BMDM) in vitro, and its further enhancement in BMDM conditioned with media from apoptotic cells. Confocal microscopy confirmed EPO production in erythroblastic island (EBI)-associated macrophages, and analysis of mice depleted of EBI macrophages by clodronate liposomes revealed drops in EPO levels in bone marrow (BM) cell lysates, and decreased percentages of EPO-responsive erythroblasts in the BM. We hypothesize that EBI macrophages are an in-situ source of EPO and sustain basal erythropoiesis in part through its secretion. To study this hypothesis, mice were injected with clodronate liposomes and were supplied with exogenous EPO (1-10 IU/mouse) to evaluate potential rescue of the deficiency in erythroid cells. Our results show that at doses of 5 and 10 IU, EPO significantly rescues BM steady state erythropoiesis in mice deficient of macrophages. We propose existence of a mechanism by which EBI macrophages secrete EPO in response to apoptotic erythroblasts, which is in turn controlled by the numbers of erythroid precursors generated.
