GSDMD-mediated metabolic crosstalk licenses a pro-regenerative niche for tissue repair

The establishment of an early pro-regenerative niche is crucial for tissue regeneration. Gasdermin D (GSDMD)-dependent pyroptosis accounts for the release of inflammatory cytokines upon various insults. However, little is known about its role in tissue regeneration followed by homeostatic maintenance. Here, we show that macrophage GSDMD deficiency delayed tissue recovery, with little impact on the local inflammatory milieu or the lytic pyroptosis process. Metabolite secretome profiling of hyperactivated macrophages unveiled the non-canonical metabolite-secreting function of GSDMD. And we further identified 11,12-epoxyeicosatrienoic acid (11,12-EET) as a bioactive pro-healing oxylipin, secreted from hyperactive macrophages in a GSDMD-dependent manner. Indeed, accumulation of 11,12-EET by direct supplementation or deletion of its hydrolytic enzyme Ephx2 accelerated muscle regeneration. We further demonstrated that the Ephx2 level accumulated within aged muscle, which led to a decreased intramuscular 11,12-EET level. And consecutive 11,12-EET treatment rejuvenated aged muscle. Mechanistically, 11,12-EET amplifies fibroblast growth factor/FGF receptor (FGF-FGFR) signaling by modulating FGF liquid-liquid phase separation, hence boosting the activation and proliferation of muscle stem cells (MuSCs). These data depict a GSDMD-guided metabolite crosstalk between macrophages and MuSCs that governs the repair process, which offers new therapeutic insights for the regeneration of injured or aged tissues. Overall design: TA muscle tissue was collected from mice with the indicated treatment or genotype. After washing with ice-cold PBS, the tissue was minced into 2-3 mm pieces and washed twice with ice-cold PBS. Digestion was performed with digestion buffer [0.5 mg/mL Collagenase I (Sigma), 0.5 mg/mL Collagenase V (Sigma), and 0.5 mg/mL Dispase (Worthington)] at 37? with 100 rpm for 10 min, and stopped with an equal volume of ice-cold PBS containing 10% FBS. Cell suspensions were passed through a 70-30 µm filter twice, counted with a cell counter (Countstar), and centrifuged at 400 g for 6 min at 12?. Cells were resuspended with RPMI 1640 (Gibco) on ice. The remaining muscle tissue was digested with 6 ml 0.25% Trypsin-EDTA at 37? with 100 rpm for 10 min, and stopped with an equal volume of ice-cold PBS containing 10% FBS, followed by 2 PBS washes and passage through 70-30 µm filters. The two cell portions were collected and red blood cells were removed using 4 ml Red Blood Cell Lysis Solution. Then the suspension was resuspended in 1× PBS (0.04% BSA) and centrifuged twice at 300 g for 3 min at 4?. The cell pellet was resuspended in 50 µL of 1× PBS (0.04% BSA). The overall cell viability was confirmed by trypan blue exclusion, which was required to be >85%, single cell suspensions were counted using a cell counter, and the final concentration was adjusted to 700-1200 cells/µL. The single-cell suspensions were loaded to 10x Chromium to capture 8,000-10,000 single cells according to the manufacturer's instructions with the 10X Genomics Chromium Single-Cell 3' kit (V3).