Fig 2: Complement component C4 and C5 expression in WT and Mdr2-KO mouse livers. (A) Liver sections were stained with Clec4f (green) for KCs, C4 (red), and DAPI for nuclei (blue). Dotted lines demarcate Clecf+ cells as well as immune cell infiltrates in portal triads from adjacent liver parenchyma. (B) Liver sections were stained with Clec4f (green) for KCs, C5 (red), and DAPI (blue) for nuclei. (C) Complement component C3, C1q, CD68 expression in WT and Mdr2-KO livers. Liver sections were stained with C3 (red), C1q (white), and CD68 (green) for macrophages/KCs and DAPI for nuclei (blue). Dotted lines demarcate immune cell infiltration sites from adjacent liver parenchyma. White squares represent high magnification images shown separately. Representative images of WT livers (n=6); Mdr2-KO livers (n=6) are shown. Scale bars 100 µm in images of low and high magnification.

Fig 3: Translational studies in healthy and diseased human livers. (A) Normal liver sample Nr. 5 ( Supplementary Table 1 ), NAFLD sample Nr. 9 ( Supplementary Table 1 ) and viral hepatitis sample Nr. 7 ( Supplementary Table 1 ) tissues were stained with ORO for lipid (red) and HE for nuclei (blue). (B) Human livers - as described in Figure 6A - were stained with ApoE (red), C1q (white), and DAPI (blue) showing similar staining patterns vs mouse livers. Dotted lines demarcate immune cell infiltration sites from adjacent liver parenchyma. (C) Human livers – as described in Figure 6A - were stained with C5 (red), CD68 (green) and DAPI (blue) indicating nuclei. Representative images of human samples ( Supplementary Table 1 ) are shown. Dotted lines demarcate immune cell infiltration sites from adjacent liver parenchyma. Scale bars 100 µm.

Fig 4: Axl signaling promotes microglial survival in the absence of CSF1R signaling.(A) Dosing regimen of PLX3397 to various genotypes. (B) Confocal images of microglia in the NFL/GCL in all genotypes from central to mid-periphery of the dorsal leaf. C1q (mono). Scale bars 100 µm. (C) Ratio of density of CD45+CX3CR1-gfp+ or CD45+CD11b+ (microglia/singlets) in PLX treated retinas over genotype-matched controls by flow cytometry. (n = 17 CX3CR1-GFP/+, n = 5 Bax KO, n = 11 CX3CR1 KO, n = 5 CR3 KO CX3CR1-GFP/+, n = 9 Mertk KO, n = 8 Axl KO, n = 7 Mertk Axl dKO; ± SEM) = 2 litters collected for each genotype. Line demarcates data from CX3CR1-GFP/ + and Bax KO previously published in Anderson et al., 2019a. Welch’s ANOVA W(6,18.55) = 16.53, p < 0.0001 and Dunnett’s T3 multiple comparisons test. Not all comparisons shown but can be found in Supplementary file 7.

Fig 5: Neuroinflammation in the ventral thalamus of aged GrnR493X/R493X mice. a Representative hippocampal/thalamic tilescans co-stained for Iba1/Pgrn show severe microgliosis in the brain of 18 month old GrnR493X/R493X mice (scale bar, 500 µm). b Representative Iba1/Pgrn co-stained immunofluorescence images of hippocampal CA3 and thalamic VPM/VPL regions in 18 month old Grn+/+ and GrnR493X/R493X brain sections (scale bar, 20 µm). c Quantification of microglial density (i-ii) and branching morphology (iii-iv) in the CA3 and thalamic VPM/VPL regions. d, Microglial Pgrn fluorescence intensity in the CA3 (i) and VPM/VPL (ii). e–f, Representative images and quantification of C1qa staining in the VPM/VPL. n = 10 mice were used per sex/genotype (except male GrnR493X/R493X n = 8 and male Grn+/+ Iba1-Pgrn staining of VPM/VPL n = 9); values are shown as mean ± SEM; ns not significant, *p < 0.05, ***p < 0.0001 was determined by Student’s t-test