Fig 2: GAS6 protects PMHs against cell death induced by palmitic acid via MERTK and bemcentinib blocks LPS-induced inflammation in KCs. (A) Activating antibodies against AXL (a-AXL) or MERTK (a-MERTK) were used in primary fibroblast from WT, AXL, and MERTK KO mice. AXL and MERTK activators (10 nM) were exposed for 1 hour and p-AKT analyzed in cell extracts by Western blot. (B) Cell death after 18 hours in PMHs exposed to palmitic acid (0.75/1.0/1.25 mM) pretreated with recombinant GAS6 or activating antibodies against AXL or MERTK. Results are expressed as mean ± SD. *P = .05 vs palmitic acid-treated cells (n = 3). (C) p-AKT and p-STAT3 levels in PMHs after exposure to GAS6, AXL, or MERTK activators in the presence or absence of palmitic acid (0.75 mM). (D) Changes in p-AXL and p-MERTK levels after KC exposure to GAS6, AXL, or MERTK activators. (E, F) mRNA expression levels of IL-1ß and IL-6 in KCs exposed to LPS (50 ng/mL, 2 hours), activating antibodies, or bemcentinib (0.25 µM). *P = .05 vs control cells (n = 6–8).

Fig 3: AXL inhibition reduces liver fibrosis and inflammation in MCD-fed mice. (A) Representative images of liver sections after H&E and Sirius Red staining; bar (200 µm). Sirius Red quantifications using ImageJ software in 6 random sections from each animal are shown below the respective pictures. Student’s t test; *P = .05 vs control mice, #P = .05 vs MCD-fed mice; n = 5–6 independent samples. (B) Collagen determination by hydroxyproline quantification in liver samples (n = 4–5) and (C) mRNA expression level of MCP-1, TNF, a-SMA, MPO, F4/80, and ß-actin in liver samples from treated mice. Results are expressed as mean plus standard deviation (n = 4–5). *P = .05 vs control mice; #P = .05 vs MCD-fed mice; Student’s t test. (D, E) Body and liver weight were measured after sacrifice in mice fed for 6 weeks with chow and MCD diet that received vehicle or bemcentinib (BGB324) oral gavages for the last 2 weeks. *P = .05 vs control; n = 4–5. The results shown are representative for 2 independent experiments.

Fig 4: Rules of engagement for TAM receptor ligand interaction and signaling.Gas6 activates all three TAM receptors independently, but Axl is uniquely dependent on Gas6: Pros1 activates Tyro3 and Mer but not Axl. Optimal activation of any receptor by any ligand requires the simultaneous presence of PtdSer, which binds to the Gla domain of the ligands, and calcium ions (Ca2+). Gla-less Gas6 is dead as a ligand when assayed against Axl in isolation. Its Axl-bound orientation is schematized differently from Tyro3- and Mer-bound Gla-less Gas6, since the latter two result in partial kinase activation, although there are no structural data for these complexes. The right-most signaling configuration, in which full-length Gas6 is also inactive as an Axl ligand in the absence of PtdSer and Ca2+, is speculation based on the results of this paper and data in (Zagórska et al., 2014). See text for details.DOI: http://dx.doi.org/10.7554/eLife.03385.016

Fig 5: Axl-/- mice and Cd11c-Cre+Axlfl/fl mice mount impaired T cell responses to IAV infection.(A) Representative plots (left) and percentage (middle) of CD8+IFN-?+ T cells in the lung of WT and Axl-/- mice after 9 days of infection with 10 PFU of PR8. 4–5 mice per genotype, representative of 4 independent experiments. Right, quantification of IFN-?-producing H-2Db-restricted CD8+ T cells specific for IAV PA amino acids 224–233 in the lung 9 days post infection with 10 PFU of PR8. 7–8 mice per genotype, 2 independent experiments. (B) Representative plots (left) and percentage (right) of CD4+IFN-?+ T cells in the lung of WT and Axl-/- mice infected as in (A). (C) Number of CD8+IFN-?+ T cells (left), CD8+PA+IFN-?+ T cells (middle), and CD4+IFN-?+ (right) in the lung 9 days post-infection with 10 PFU of PR8 in Cre-Axlfl/fl, Cd11c-Cre+Axlfl/fl, and Csf1r-Cre+Axlfl/fl mice, as indicated. 5–10 mice per genotype, representative of 2–3 independent experiments. ns, non-significant; *p<0.05; **p<0.01; ***p<0.001DOI: http://dx.doi.org/10.7554/eLife.12414.014