P21+TREM2+-Senescent Macrophages Fuel Inflammaging and Metabolic Dysfunction-Associated Steatotic Liver Disease.

Cellular senescence drives chronic sterile inflammation during aging via the senescence-associated secretory phenotype (SASP); however, the cell types responsible for this pathology remain poorly defined. Here, we identify p21?Trem2? senescent macrophages as a major source of inflammaging. Using primary mouse and human macrophages, we developed a model of DNA damage and cholesterol-induced senescence and applied multi-omic profiling to fully characterize senescent macrophages. We found that senescent macrophages exhibit a distinctive p21-TREM2 expression profile and SASP, driven in part by type-I interferon signaling via secreted mitochondrial DNA. We also found that senescent macrophage accumulation occurs in aged and MASLD mouse livers and is enriched in human cirrhotic liver tissue. Finally, senolytic treatment targeting senescent macrophages reduced liver inflammation and steatosis in both aged and MASLD mice. Together, these findings establish macrophage senescence as a central driver of chronic inflammation in aging and metabolic liver disease, highlighting a promising, tractable therapeutic target. Overall design: BMDMs used in experiments were derived from bone marrow extracted from the femurs of euthanized mice (6-12 weeks old male C57BL/6J) by mortar and pestle. Femurs were placed in the mortar and were washed with 70% ethanol to sterilize followed by two washes with complete RPMI (cRPMI; standard RPMI (Corning) supplemented with 10% fetal calf serum, penicillin-streptomycin solution (Corning), 1mM sodium pyruvate solution (Corning), 2 mM L-Glutamine solution (Corning), 10nM HEPES buffer (Corning), and 50µM 2-mercaptoethanol). After washing, 10 ml of cRPMI was added to the mortar and the femur bones were gently crushed. The resulting media was collected and filtered through a 70µm filter and placed in a conical tube. The filtered supernatant was centrifuged at 100 g (150 RCF) for 5 minutes. Cells were resuspended, counted and plated at a density of 3E6 cells/10 cm dish in 10 ml of macrophage growth media (cRPMI containing 25% M-CSF containing L929 conditioned media (made in house)). Cells were left to grow for 7 days to differentiate and were supplemented with 5 ml of macrophage growth media on day 5. On day 7, BMDMs (yielding 10-12x106 cells/10cm dish) were lifted off the plate using cold PBS containing 5mM EDTA. BMDMs were counted and replated in macrophage growth media overnight prior to experiments. On the day of experiments, macrophage growth media was replaced with cRPMI 6 hours prior to stimulation to remove M-CSF. M2 polarization was performed by stimulating macrophages with 10 ng/ml recombinant mouse IL-4 (Peprotech). For M1 polarization macrophages were stimulated with 100 ng/ml LPS (LPS EK-Ultrapure, Invivogen). To induce senescence, day 7 BMDMs were either irradiated (10 Gy) or treated with doxorubicin (Fisher) for 24 hours at 250 nM in 60% cRPMI: 40 MGM. DNA damaged cells were left in culture for 10 days and media components were replaced every 2-3 days. As a negative control, a mock irradiated or drug treated condition was cultured in parallel and required passing every 2-3 days. All senescent macrophage experiments were performed 10-13 days post DNA damage.