Fig 2: Liver pathology in aged Axl−/−Mertk−/− mice.(A) 9–12-mo-old Axl−/−Mertk−/− mice accumulate approximately ninefold more cCasp3+ apoptotic cells than WT mice. (B) 9–12-mo-old Axl−/−Mertk−/− exhibit an approximately fourfold increase in MARCO staining in liver than WT. (C) 9–12-mo-old Axl−/−Mertk−/−Cx3Cr1+/GFP mice have approximately fivefold more infiltrating GFP+ immune cells in liver than Cx3Cr1+/GFP. (D) Levels of the indicated mRNAs, isolated from 8 to 12 mo livers, and quantified by qRT-PCR relative to WT. (E) Serum ALT and AST activity from 7-mo-old WT and Axl−/−Mertk−/− mice. (A, B, C) Representative images from 3 to 4 mice per genotype. (D, E) Representative data from two independent experiments (n = 3–4 mice per genotype). Bars (A, B, C): 100 μm; *P < 0.05. t test.

Fig 3: STAT1 activation mediates Gas6-induced increases in LXRα and LXRβ as well as ABCA1, ABCG1, ApoE, AIM, Arg2, and VEGF expression.BMDM from C57BL/6 mice (a) or from Mer−/− and WT mice (b) were stimulated with 400 ng/ml Gas6 for the indicated times. BMDM from WT or STAT1−/− mice were stimulated with 400 ng/ml Gas6 for the indicated times (c–g). (h) RAW 264.7 cells were co-transfected with an LXRE-driven luciferase reporter vector and a Renilla control plasmid. After 24 h, cells were treated with 400 ng/ml Gas6 in the absence or presence of fludarabine at the indicated concentrations. LXR activity was then measured by the dual luciferase assay. (i,j) BMDM from C57BL/6 mice were pretreated with vehicle or the indicated concentrations of the JAK2 inhibitor tyrphostin AG490 (Tyr) for 1 h before being treated with 400 ng/ml Gas6. (a,b,e,f) The relative abundances of total STAT1, phosphorylated STAT1 (Y701), LXRα, and LXRβ proteins were determined by Western blotting analysis. The relative densitometric intensity was determined for each band and normalized to the indicated proteins. (c,d,g,i,j) The amounts of the LXRα, LXRβ, ABCA1, ABCG1, ApoE, AIM, Arg2, and VEGF mRNAs were analyzed by real-time PCR and normalized to that of Hprt mRNA. Data in all bar graphs are means ± SEM of three independent experiments. *P < 0.05 compared with control; +P < 0.05 as indicated.

Fig 4: Evaluating the outcome on efferocytosis after treatment with MER-specific antibodies, Mertk gene ablation or Pros1 overexpression. (A) Immunohistochemical staining for cleaved Caspase-3 in knee joints of collagen-induced arthritis (CIA) mice injected with IgG or anti-MER. Shown are representative pictures of knee joint sections with similar microscopic scores of inflammation, in two different magnifications. (B) Pictures of knee joint sections shown in (A) were quantified and the cleaved Caspase-3 positive area was corrected by the total inflamed area (n = 10–12 knee joints). (C) Quantification of immunohistochemical staining for cleaved Caspase-3 in knee joints of wild-type (WT) or Mertk−/− mice with KRN serum transfer arthritis (STA) mice (n = 10–18 knee joints). (D) Quantification of immunohistochemical staining for cleaved Caspase-3 in knee joints of CIA mice intravenously injected with Ad Luc or Ad Pros1 (n = 18 knee joints). (E) Quantification of immunohistochemical staining for cleaved Caspase-3 in knee joints of KRN STA mice intra-articularly injected with Ad Luc or Ad Pros1 (n = 12 knee joints). P, patella; F, femur; S, synovium. For (B–E), data are presented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001 with unpaired t-test. See also Figure S5 in Supplementary Material.

Fig 5: Effect of MER-specific antibody treatment on clinical severity and pathology of collagen-induced arthritis (CIA). (A) Mice with CIA were intravenously injected with IgG or anti-MER (n = 11–12 mice). Two mice were euthanized 1 h after injection. Lung, liver, and spleen were isolated and samples were immunoprecipitated (IP) for MER and immunoblotted for MER or phosphorylation (pY) (n = 1). (B) Macroscopic score of knee joints as determined by swelling and blood vessel formation (n = 20–22 knee joints). (C) Shown are representative pictures of histological knee joint sections stained with safranin O and fast green of each group. (D) Histological knee joint sections were scored for the arthritis parameters depicted. Three semi-serial sections per joint were scored in a random and blinded manner with an arbitrary score from 0 to 3 (n = 20–22 knee joints). P, patella; F, femur; S, synovium; JC, joint cavity. For (B–D), data are presented as mean + SEM. *p < 0.05, **p < 0.01 with unpaired t-test. See also Figure S4 in Supplementary Material.