Lysosomal exocytosis by macrophages as a druggable mechanism for anti-inflammatory clearance of dead adipocytes in adipose tissue

The clearance of dead adipocytes in adipose tissue (AT) poses a major challenge due to their large size, which exceeds the phagocytic capacity of macrophages and prevents classical, anti-inflammatory efferocytosis. Instead, adipose tissue macrophages (ATMs) accumulate around dying adipocytes, forming crown-like structures (CLS), and engage in lysosomal exocytosis – the extracellular degradation of adipocytes. In this study, we used an ex vivo explant model of murine epididymal white AT, cultured over seven days to investigate pharmacological strategies that modulate lysosomal exocytosis. We observed a progressive increase in CLS formation, secretion of the lysosomal enzymes ß-Hexosaminidase A (HEXA) and lysosomal acid lipase (LAL), and surface abundance of LAMP1 and LAMP2, confirming ATMs as key mediators of this process. Notably, activation of lysosomal exocytosis with the mTOR inhibitor Rapamycin enhanced adipocyte clearance and significantly reduced inflammatory ATM abundance and TNF-α secretion. Bulk RNA sequencing of ATMs revealed a highly significant impact of Rapamyin on ATM proliferation. In contrast, inhibition of lysosomal exocytosis with PIKfyve inhibitor Apilimod or targeted inhibition of LAL using Lalistat-2 disrupted lysosomal function and promoted a pro-inflammatory ATM phenotype. Our findings highlight lysosomal exocytosis as a critical pathway for the resolution of dead adipocytes and the regulation of inflammation in adipose tissue. Pharmacological enhancement of this process may represent a promising therapeutic approach to attenuate inflammation in AT and its metabolic consequences, including insulin resistance and type 2 diabetes. After sacrificing the mice, the rostral eWAT was dissected under sterile conditions and cut into small pieces (explants) of ≈ 1 mm3 (10 mg per explant) in PBS. Five AT explants per well were transferred to six-well plates prepared with 1 ml cell culture medium: RPMI 1640 with 10 % fetal bovine serum and 1 % antibiotics (100 U/ml penicillin and streptomycin). Explants were over casted with cell culture inserts and cultured at 37 °C with 5 % CO2 and 21 % O2 for seven days. On day one of AT explant culture, 100 µl treatment (Rapamycin 0.5 µM or Apilimod 0.5 µM or Lalistat 10 µM or DMSO-control 0.5%) solution was added in each well. Treated AT explants were digested using collagenase. SVF was resuspended in staining buffer and the Fc-receptors blocked by anti CD16/32 (1:100) for 10 min on ice. After centrifugation (300 g, 5 min, 4 °C, slow deceleration) cells were stained with labeled F4/80 antibody (1:200) on ice for 20 min, centrifugated (300 g, 5 min, 4 °C, slow deceleration), resuspended in staining buffer. Living, F4/80+ cells were sorted (~50,000 cells per sample) using a BD FACSAria SORP and frozen at −80 °C in TRIzol. RNA isolation and purification was performed using RNeasy Micro Kit by Quiagen. RNA sequencing and analysis was performed at Leipzig University’s Core Unit ‘DNA Technologien’. RNA extraction and library preparation was performed with a sequencing depth of 10 million reads/sample. Results were then analyzed using the DESeq2 package on Galaxy2. Differentially expressed genes (DEG) were considered as significant with a p-adj. value < 0.01 and a log2(fold change) > 0.5 or < -0.5. Gene set enrichment analysis was performed with datasets from the DAVID Database. For each condition, AT from 8 animals was pooled in groups of two, so that 4 samples were analyzed for each treatment.