Fig 2: GAS6 augments homeostatic efferocytosis in murine peritoneal macrophages.A Gating strategy to identify murine large (LPM) and small (SPM) peritoneal macrophages. Cells were gated as CD19−, Ly6G− live cells. B Representative flow cytometric analysis for expression of AXL and MERTK on LPM and SPM. C Frequency of AXL or MERTK single and double positive LPM and SPM. Bar graphs show mean with SD and individual samples. D Experimental set-up to study efferocytosis in the murine peritoneum. Apoptotic cells (AC) were generated by treating murine thymocytes with dexamethasone. Afterwards, cells were labeled with pHrodo-Red and washed with EDTA/BSA and PBS. To set-up the model, initially different numbers of ACs were injected intraperitoneally. Peritoneal exudate cells (PECs) were analyzed at different time points by flow cytometry. Created with BioRender.com E Representative flow cytometric analysis of PECs for expression of F4/80 and pHrodo-Red signal. F Representative histograms for pHrodo-Red upon injection of different numbers of ACs and PECs isolated after 4 h (Gated on CD19−, F4/80+ live cells). Statistical analysis of pHrodo+ cells. Bar graphs show mean with SD and individual samples. G Representative histograms for pHrodo-Red upon injection of 2.5 × 106 ACs and PECs were isolated at indicated time points (Gated on CD19−, Ly6G−, and F4/80+ live cells). Statistical analysis of pHrodo+ cells. Bar graphs show mean with SD and individual samples. H Representative histograms for pHrodo-Red upon injection of 2.5 × 106 ACs with or without 50 nM GAS6. PECs were isolated at indicated time points (Gated on CD19−, Ly6G−, and F4/80+ live cells). Bar graphs show mean with SD and individual samples. Data were representative of two individual experiments (n = 3–5).

Fig 3: MERTK kinase activity is critical for efferocytosis in human iPSC-derived macrophages in vitro.A Flow cytometric analysis of efferocytosis in vitro using iPSC-derived macrophages. Macrophages were stimulated with Poly (I:C) or dexamethasone for 24 h prior to the assay. ACs were generated as described and labeled with 50 nM GAS6. Prior to addition of apoptotic cells, macrophages were incubated with indicated antibodies for 30 min. B Frequency of pHrodo+ macrophages. Bar graphs show mean with SD and individual samples. C Flow cytometric analysis of efferocytosis in vitro using iPSC-derived macrophages. Macrophages were stimulated with Poly (I:C) or dexamethasone for 24 h prior to the assay. ACs were generated as described and labeled with 50 nM GAS6. Prior to addition of apoptotic cells, macrophages were incubated with indicated inhibitors for 30 min. D Frequency of pHrodo+ macrophages. Bar graphs show mean with SD and individual samples. Data were representative of at least three individual experiments with technical replicates.

Fig 4: MERTK kinase activity is essential for clearance of apoptotic cells in vivo.A (top) targeted codon in Mertk for the generation of MertkK614M/K614M is indicated in blue. A KpnI restriction enzyme site was engineered to aid PCR-based genotyping. (bottom) Amino acid sequence of the targeted region, indicating the introduced mutation at Lysine (K) 614 for Methionine (M) 614. B Diagnostic PCR results, after DNA amplification and digestion with KpnI. The WT allele yields one 548 bp product and the mutant allele yields a 184 and a 364 bp band. C Representative histograms showing expression of MERTK in bone marrow-derived macrophages from mice with the indicated genotypes. Bar graphs show geometrical mean fluorescence of MERTK. Shown is mean with SEM and individual samples. D Representative flow cytometric analysis of efferocytosis with bone marrow-derived macrophages from mice with the indicated genotypes. Macrophages and ACs were co-incubated for 1 h in the presence of serum at a ratio of 1:6 and analyzed afterwards for pHrodo signal. Bar graphs show mean with SEM and individual samples. E Experimental set-up to study contribution of AXL and MERTK for efferocytosis in the murine peritoneum. ACs (2.5 × 106) were injected intraperitoneally into mice of indicated genotype. After 4 h PECs were analyzed via flow cytometry. Created with BioRender.com. F Representative histograms for pHrodo-Red upon injection of ACs into mice of indicated genotype (Gated on CD19−, Ly6G−, and F4/80+ live cells). Frequency of pHrodo+ cells. Bar graphs show mean with SD and individual samples. Data were representative of two individual experiments (n = 3–5).

Fig 5: IL-17A Trans-Signaling through Cardiac Fibroblasts Downregulates MerTK expression on Monocytes and MDMs(A and B) Representative EM images showing (A) apoptotic cells or apoptotic cellular debris internalized by Ly6Chi MDMs (arrowheads) and (B) engulfed cellular debris were largely absent in Ly6Clo MDMs. Scale bars: 2 µm.(C) Macrophage phagocytic index was calculated using the following formula: (number of engulfed apoptotic cells/total number of macrophages) × (number of macrophages with engulfed apoptotic cells/total number of macrophages) × 100.(D–G) Hearts from day 21 EAM mice.(D) Frequencies of F4/80hiCD64+ macrophages out of viable CD45+Ly6G–CD11b+ cells were assessed by flow cytometry.(E and F) MerTK MFI of F4/80hiCD64+ macrophages (E) and F4/80–CD64+ monocytes (F) in the hearts.(G) ELISA showing soluble Mer (sMer) in WT and IL-17Ra-/- EAM mice sera.(H and I) Cardiac fibroblasts were harvested from WT naive mice, whereas monocytes were sorted from EAM IL-17Ra-/- mice.(H) MerTK MFI of Ly6Chi or Ly6Clo monocytes and MDMs in vitro after 160 h post-co-culture with cardiac fibroblasts stimulated with or without IL-17A.(I) sMer detected in supernatants of the monocyte-fibroblast co-culture by ELISA.(J) Flow cytometric analysis of the frequencies of FITC+F4/80hiCD64+ macrophages in the myocardium of WT, IL-17Ra-/-, and non-treated controls.(K) Percentages of FITC+F4/80hiCD64+ macrophages out of viable CD45+Ly6G–CD11b+ cells.(L) MerTK MFI in patients with either myocarditis or ischemic cardiomyopathy.(D–G) Data are representative of five independent experiments. n = 8 – 9. (H and I) Data are representative of three independent experiments with technical triplicates. n = 3. (J and K) Data are representative of two independent experiments. n = 3. (C–G, I, K, and L) Groups were compared using Student’s t test. *p < 0.05; **p < 0.01. (H) Groups were compared using one-way ANOVA followed by Dunnett test. **p < 0.01; ****p < 0.0001. All data were presented as mean ± SD.See also Figure S7 and Tables S2 and S3.