Fig 1.
Flow cytometry characterization of resKCs and recMφs from mouse livers with or without collagenase pre-treatment.
(A) Gating strategy of liver recMφs and resKCs with four-color analysis. Livers from B6 mice were minced and treated with collagenase. Non-parenchymal cells were extracted and subjected to flow cytometry analysis. The extracted cells were gated with FSC and SSC, and CD45-positive immune cells were selected. Ly6G-negative cells, excluding neutrophils, were used for the experiments. Based on FACS analysis of F4/80 and CD11b expression, F4/80low CD11bhigh cells were recMφs (blue dots)., and F4/80high CD11blow cells were defined as resKCs (green dots). (n = 13). (B) The expression of Ly6C, MerTK, as well as intracellular and surface CLEC4F in recMφs (upper panels with blue histogram) and resKCs (lower panels with green histogram) was demonstrated (n = 5). (C) The FACS analysis of liver MNCs obtained without collagenase treatment (left panel) and after clodronate liposome administration (200 μl per mouse) are displayed (right panel) (n = 6). Data are presented as the mean ± SE.
Fig 2.
Effect of low-dose irradiation on the proportion and function of resKCs and recMφs in WT mice.
Irradiation-induced changes in the proportion and functions of resKCs and recMφs. Liver MNCs harvested from normal mice and mice 2 days after irradiation (2 Gy) were analyzed. (A) FACS analysis of F4/80 and CD11b in liver MNCs isolated from control and irradiated mice, with recMφs and resKCs displayed as blue and green dots, respectively. (B) Bar graph of recMφ (blue columns) and resKC (green columns) abundance in control and irradiated mice (n = 13 and 6). (C) Phagocytic activity of recMφs (upper panels) and resKCs (lower panels) against intravenously injected FITC-E. coli in mice with or without irradiation. The percentage of FITC-E. coli-positive cells was calculated relative to non-injected control mice (gray area in each panel). (D) Bar graph of E. coli phagocytosis by recMφs (blue columns) and resKCs (green columns) in control and irradiated mice (n = 5 in each group). (E) Intracellular TNF-α production in recMφs (upper panels) and resKCs (lower panels) stimulated with LPS in vitro. Liver MNCs from control or irradiated mice were stimulated with LPS in vitro for 1.5 h, and intracellular TNF-α was labeled (n = 5 in each group). (F) Bar graph of intracellular TNF-α expression in recMφs (blue columns) and resKCs (green columns) in control and irradiated mice (n = 5 in each group). Data are presented as the mean ± SE (*p <0.05, unpaired Student’s t-test).
Fig 3.
The increased in vitro phagocytic activity of resKCs and recMφs against FITC-E. coli and the decreased TNF-α production by LPS-stimulated liver MNCs following pre-treatment with a synthetic LXR ligand (T0901317).
(A) The phagocytic activity of recMφs (upper panels, blue line) and resKCs (lower panels, green line) against E. coli. Negative controls were incubated with E. coli for 1 h on ice (red line). (B) Bar graph of the in vitro phagocytic activities of recMφs (blue columns) and resKCs (green columns) in control and T0901317-treated MNCs (n = 12 in each group). (C) The in vitro TNF-α production by MNCs with or without T0901317 pre-treatment. Adherent cells were obtained after incubation with LPS for 45 minutes, mRNA was isolated, and the relative expression TNF mRNA was compared between the two groups (left panel, n = 5 in each group). After incubation with LPS for 6 h, culture supernatant was collected, and TNF-α concentration was measured and compared (left panel, n = 14 in each group). (D) IL-12 production by liver MNCs stimulated with LPS for 12 h with or without T0901317 pre-treatment (n = 9 in each group). Data are presented as the mean ± SE (*p <0.05, unpaired Student’s t-test).
Fig 4.
The proportions of resKCs and recMφs, in vivo phagocytic activity of resKCs against E. coli, and plasma inflammatory cytokine levels in WT and LXR-deficient mice.
(A) The proportions of resKCs and recMφs in the livers of WT, LXRα KO, and LXRα/β KO mice were determined via flow cytometry (n = 4 in each group). (B) Bar graph of recMφ (blue columns) and resKC (green columns) proportions in WT, LXRα KO, and LXRα/β KO mice (n = 4 in each group) Data are presented as the mean ± SE (*p <0.05, one-way ANOVA followed by Tukey’s multiple comparisons). (C) In vivo phagocytic activity of WT mice (n = 6), LXRα KO mice (n = 3), and LXRα/β KO mice (n = 3). Mice were injected with FITC-E. coli, and the phagocytic activity of resKCs (green line) was analyzed 15 min after injection. ResKCs from a mouse without E. coli injection were used as negative controls (gray line). (D) Bar graph of the phagocytotic activity of resKCs (green columns) in WT, LXRα KO, and LXRα/β KO mice (n = 6, 3, 3 in each). Data are presented as the mean ± SE (*p <0.05, one-way ANOVA followed by Tukey’s multiple comparisons) (E, F) WT mice and LXRα/β KO mice were injected with live E. coli, and plasma TNF-α and IL-12 levels were measured at the indicated time points after E. coli injection (n = 3 in each group). Data are presented as the mean ± SE. (*p <0.05, unpaired Student’s t-test).
Fig 5.
The decreased in vitro phagolytic activity of resKCs from LXRα KO mice against pHrodo E. coli.
(A) The phagolytic activity of recMφs (upper panels, blue line) and resKCs (lower panels, green line). WT and LXRα KO mouse recMφs and resKCs incubated with pHrodo E. coli on ice were used as negative controls (gray line). (B) Bar graph of the phagolytic activity of recMφs (blue columns, n = 4) and resKCs (green columns, n = 4). Data are presented as the mean ± SE. (*p <0.05, unpaired Student’s t-test).
Fig 6.
The decreased in vivo phagocytic activity of resKCs for acetylated LDL in LXRα KO and LXRα/β KO mice.
(A) WT mice (n = 6), LXRα KO mice (n = 3), and LXRα/β KO mice (n = 3) were injected with DIO-labeled acetylated LDL, and the endocytosis of acetylated LDL by recMφs (upper panels, blue line) and resKCs (lower panels, green line) was analyzed via flow cytometry. ResKCs and recMφs from WT mice injected with control acetylated LDL (without fluorescence) were used as negative controls (gray line). (B) Bar graph of DIO-labeled acetylated LDL endocytosis by recMφs (blue columns) and resKCs (green columns). Data are presented as the mean ± SE (*p <0.05, one-way ANOVA followed by Tukey’s multiple comparisons test).
Fig 7.
CD36 expression in resKCs and recMφs from WT, LXRα, and LXRα/β KO mice.
(A) The expression of CD36 on recMφs and resKCs from WT, LXRα, and LXRα/β KO mice (n = 4 in each group) was analyzed and compared. (B) Bar graph of CD36+ cells among recMφs (blue columns) and resKCs (green columns). Data are presented as the mean ± SE. (*p <0.05, one-way ANOVA followed by Tukey’s multiple comparisons test).