Fig 2: Accumulation of TIM4neg Macrophages in a Mouse Model of NASH(A) Body weight of C57BL/6 male mice fed a HFD for the indicated time.(B–D) Liver triglyceride (B) and mRNA markers of steatosis (C) and fibrosis (D) were quantified.(E) H&E (top panels) and picrosirius red (bottom panels) from liver tissue.(F) Flow-cytometry plots are shown for CD45+, live, singlets (top panels). Representative plots of F4/80hi cells (red gate) were analyzed for expression of TIM4 (bottom panels).(G) Quantification of flow data per gram of liver tissue.(H) Percentage of F4/80hi cells that are TIM4neg over course of HFD feeding.In (A), each dot represents the mean ± SEM and in (B)–(D). In (G), each dot represents a single mouse, and the mean is shown (n = 7–8 mice/group). The data represent the results of one independent experiment with n = 7–8 mice per group. *p < 0.05 for HFD versus STD. Scale bars, 250 µm

Fig 3: TLR Activation Induces Spic in the Tissue Macrophages(A) SpicGFP/GFP mice were treated with i.p. LPS (75 µg in PBS) or control (PBS), and lungs were harvested 48 h after treatment. Top: FCS with indicated markers on singlets, highlighting SPIC expression. Bottom: distribution of indicated markers on SPIC+ and SPIC- cells (gating shown in top panels, arrow).(B) FCS with indicated markers in CD45+ live (7AAD-) singlets from large intestine of SpicGFP/GFP and wild-type (WT) mice, showing Tim4 and CD4 expression largely restricted to SPIC+ cells.(C) Published gene expression profiles (microarray based) of murine ileum and colon were downloaded from a public database (GEO: GSE32513). Shown are expression values (linear scale) of Spic.(D) qRT-PCR-based expression of Spic (normalized to Hprt) in colon obtained from conventionally raised (Cont) and germ-free (GF) mice.(E) Lung macrophages (CD45+CD64+CD11C+) were isolated by FACS, cultured with M-CSF for 12 h, and treated with LPS (1 µg/ml). RNA was extracted 10 h after LPS treatment, and the expression (normalized to Hprt)ofSpic was measured by qRT-PCR.(F) Peritoneal cells (PECs) were cultured with M-CSF for 12 h, followed by LPS (1 µg/ml) treatment. RNA was extracted 20 h after treatment, and the expression (relative to Hprt)ofSpic was measured by qRT-PCR.(G) CD45+F4/80+GFP+ and CD45+F4/80+GFP- liver macrophages were purified by FACS, cultured with M-CSF for 12 h, and treated with LPS (1 µg/ml). RNA was extracted 14 h after LPS treatment, and the expression (normalized to 18S rRNA)ofSpic was measured by qRT-PCR.(H) RPMs from SpicGFP/GFP spleen were purified by FACS, cultured with M-CSF for 12 h, and treated with LPS (1 µg/ml). RNA was extracted 16 h after LPS treatment, and the expression (normalized to Hprt)ofSpic was measured by qRT-PCR.FCS, numbers represent percentage of cells within indicated gate. (A and B) Represent =3 experiments with =3 mice per group. qRT-PCR, data representative of = 3 independent experiments; and graphs show a single experiment with n = 2 per group. Results expressed as mean ± SEM. p = 0.05 (*), p = 0.01 (**), p = 0.001 (***), and p = 0.0001 (****). See also Figures S1, S6, and S7

Fig 4: scRNA-seq analysis reveals adrenal gland leukocyte diversity and monocyte contribution to the macrophage pool(A) scRNA-seq analysis of adrenal gland CD45+ cells from 7-week-old male and female wild-type mice.(B) Proportion of each cluster identified in scRNA-seq analysis.(C) Heatmap showing normalized expression levels of cluster-specific genes.(D) Violin plots showing Cx3cr1, Timd4, and Lyve1 expression by cells from clusters 1–6.(E) Flow-cytometry plot showing Ly6C and CCR2GFP expression among adrenal gland CD45+CD11b+CD64+ cells in male and female CCR2GFP/+ mice. Data are representative of three independent experiments.(F) Flow-cytometry plot showing Ly6C and TdTomato expression among adrenal gland CD45+CD11b+CD64+ cells from male and female CCR2creERT2 x R26TdTomato mice 48 h after TAM gavage. Data are representative of two independent experiments.(G) Quantification of TdTomato+ macrophages in 16- to 20-week-old male and female heterozygous (CCR2+/-, ? n = 6 and ? n = 6) or double knockin (CCR2-/-, ? n = 4 and ? n = 3) CCR2creERT2 x R26TdTomato mice 48 h after TAM gavage. Data are pooled from two independent experiments.(H) Histograms representing R26TdTomato expression in AGMs from 10-week-old female CCR2creERT2/+ x R26TdTomato mice 2, 7, and 14 days after TAM gavage. Data are representative of one (days 7 and 14) or two (day 2) experiments.(I) Proportions of TdTomato+ macrophages from 10-week-old female CCR2creERT2/+ x R26TdTomato mice 2 (n = 8), 7 (n = 4), and 14 (n = 3) days after TAM gavage. Data from one (days 7 and 14) or two (day 2) experiments.(J) Proportions of TdTomato+ macrophages from 8 (? n = 2, ? n = 6) or 16 (? n = 5, ? n = 5)-week-old male and female Ms4a3cre/+ x R26TdTomato mice. Data are from one experiment.(K) Quantification of AGMs in male and female CCR2+/- (? n = 18, ? n = 21) and CCR2-/- (? n = 10, ? n = 10) mice. Data are pooled from four independent experiments.(L) Proportions of Ki67+ AGMs in male and female CCR2+/- (? n = 11, ? n = 11) and CCR2-/- (? n = 6, ? n = 5) mice. Data are pooled from two independent experiments.Statistical analysis was performed using two-way ANOVA with Bonferroni’s post-test.See also Figure S2.

Fig 5: Gating strategy for Kupffer cell and LSEC identification in LNPC samples isolated from C57BL/6 miceRepresentative dot plots of LNPC preparation obtained with the quick ex vivo digestion protocol (A) or with the in situ digestion protocol (B). Single, live cells can be divided in LSEC (CD45-, CD31+) and in hepatic leukocytes (which are CD45+). Once discarded the lineage positive cells (CD3+, CD19+, CD49+, Ly6G+), the F4/80+CD11bint macrophage population is pre-gated to further distinguish capsular macrophages (TIM4-, I-A/I-E+) from Kupffer cells (TIM4+, I-A/I-Eint). Kupffer cells (KCs) can be divided in KC1 (CD206-, ESAM-) and in KC2 (CD206+, ESAM+) subpopulations.