Fig 2: Blockade of TIM-4 improves hepatic acute reaction response injury and reduces the secretion of inflammatory cytokines. (A) KCs were isolated from mice in the sham, control mAb and TIM-4 mAb groups on day 7 post-surgery. Cells were stained with specific secondary antibodies labeled with tetramethylrhodamine (red) and DAPI (blue) and then examined using laser confocal microscopy (magnification ×800). (B) KCs were isolated from each group and stained with FITC-TIM-4 mAb for flow cytometry. (C) Blood samples were obtained (0.5 ml/mouse) from murine abdominal aortas in each group on day 7 post-surgery. Levels of AST, ALT and TBIL were determined in a clinical biochemical laboratory. (D-F) Levels of inflammatory cytokines, including TNF-a, IFN-?, CCL2, CXCL2 and TGF-ß were detected using ELISA and western blotting. (G) Representative micrographs of the pathological damage observed in each group post-transplantation following staining with hematoxylin and eosin (magnification ×400). (H) Donor mice received CL treatment to destroy KCs prior to surgery. orthotopic liver transplantation or sham surgery was subsequently performed and mice were treated either with or without TIM-4 mAb. All mice underwent analysis to determine the RAI score on day 7 following surgery. (I) The expression of key phosphoproteins involved in the NF-?B and p38 MAPK signaling pathways, including p65 and p38, respectively, were analyzed using western blotting. Data are presented as the mean ± standard deviation. aP<0.05 vs. sham group; bP<0.05 vs. control mAb group. TIM-4, T cell immunoglobulin-domain mucin-domain-4; mAb, monoclonal antibodies; KCs, kupffer cells; FITC, fluorescein isothiocyanate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TBIL, total bilirubin in serum; TNF-a, tumor necrosis factor-a; IFN-?, interferon-?; CCL2, chemokine ligand ; CXCL2, C-X-C motif chemokine ligand 2; TGF-ß, transforming growth factor-ß; CL, clondronate liposomes; NF-?B, nuclear factor-?B; MAPK, mitogen-activated protein kinase.

Fig 3: Decreased miR-4512 expression in monocytes and macrophages contributes to a pro-inflammatory state. (A–C) Relative expression of miR-4512 (A), CXCL2 (B), and TLR4 (C) in PBMC, monocytes, and neutrophils isolated from SLE patients and control subjects, as determined by RT-qPCR. Data are presented as the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. (D–E) Concentrations of Th1 (including IL-1ß, IL-12, TNF-a, and IFN-?), Th2 (including IL-4, IL-6, and IL-10), and Th17 (IL-17) cytokines in the supernatants obtained from monocytes and macrophages transfected with NC/miR-4512-agomir/antagomir (D) or in the serum from SLE patients and control subjects (E), as determined by ELISA.

Fig 4: Treatment with CXCL2 antibody inhibits inflammatory cytokine production in the MRL/lpr lupus model. (A, C, E) Representative images of CXCL2 (A), CD45 (C), and MPO (E) staining of kidney sections of mice from different groups. Inserts, details of a typical staining of the glomerulus. (B, D, F) Quantitative analysis of CXCL2 (B), CD45 (D), and MPO (F) deposition in the glomeruli in kidney sections. The glomeruli were classified into four categories according to the degree of straining. The glomerulus ratios in each category in different groups are summarized (n = 4 animals/group, 3 sections/animal). Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05. (G) Concentrations of Th1 (including IL-1ß, IL-2, IL-12, TNF-a, and IFN-?), Th2 (including IL-4, IL-6, and IL-10) and Th17 (IL-17) cytokines in the plasma and renal tissue homogenates of mice from different groups, determined by ELISA (n = 4 animals/group). Data are shown as the mean ± SEM. ****P < 0.0001; ***P < 0.001; *P < 0.01; *P < 0.05.

Fig 5: miR-4512 directly targets TLR4 and CXCL2. (A, B) Luciferase activity of WT/MUT-CXCL2 and WT/MUT-TLR4 plasmids in HEK293T following transfection with an miR-4512 agomir. Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001; NS = not significant. (C–F) RT-qPCR analysis of CXCL2 and TLR4 transcript levels in human primary monocytes and macrophages 48 h after transfection with NC/miR-4512 agomiR/antagomiR. Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001. (G, H) CXCL2 concentration in the supernatants obtained from human primary monocytes (G) and macrophages (H) 48 h after transfection with NC/miR-4512-agomir/antagomir. Data are presented as the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05; NS, not significant. (I) Western blot analysis of TLR4 expression in monocytes and macrophages 72 h after transfection with NC/miR-4512-agomir/antagomir. Results are representative of three independent experiments, with actin used as an internal control. (J, K) CXCL2 concentration in the plasma of SLE and control subjects from Tibetan (J) and Han (K) populations. Data are presented as the mean ± SEM. ****P < 0.0001; ***P < 0.001. (L) Western blot analysis of TLR4 expression in PBMCs isolated from SLE patients (A1–A4) and healthy controls (B1–B4). Results are representative of three independent experiments.