Macrophage Sphingosine 1-Phosphate Receptor 2 Blockade Attenuates Liver Inflammation and Fibrogenesis Triggered by NLRP3 Inflammasome - PubMed (original) (raw)

Macrophage Sphingosine 1-Phosphate Receptor 2 Blockade Attenuates Liver Inflammation and Fibrogenesis Triggered by NLRP3 Inflammasome

Lei Hou et al. Front Immunol. 2020.

Abstract

NLR family pyrin domain containing 3 (NLRP3) inflammasome accompanies chronic liver injury and is a critical mediator of inflammation-driven liver fibrosis. Sphingosine 1-phosphate (S1P)/S1P Receptor (S1PR) signaling participates in liver fibrogenesis by affecting bone marrow (BM)-derived monocytes/macrophage (BMM) activation. However, the relationship between S1P/S1PR signaling and NLRP3 inflammasome in BMMs remains unclear. Here, we found significantly elevated gene expression of NLRP3 inflammasome components (NLRP3, pro-interleukin-1β, and pro-interleukin-18) and the activation of NLRP3 inflammasome significantly elevated during murine chronic liver injury induced by a bile duct ligation operation, a methionine-choline-deficient and high-fat diet, or carbon tetrachloride intraperitoneal injection. Moreover, the increased expression of sphingosine kinase 1 (SphK1), the rate-limiting synthetic enzyme of S1P, was positively correlated with NLRP3 inflammasome components in both patients and mouse model livers. Flow cytometry analysis and immunofluorescence staining showed BMMs contributed to the significant proportion of NLRP3+ cells in murine inflammatory livers, but not Kupffer cells, dendritic cells, endothelial cells, T cells, and hepatocytes. Focusing on macrophages, S1P promoted NLRP3 inflammasome priming and activation in a dose-dependent manner. Blockade of S1PR2 by JTE-013 (antagonist of S1PR2) or S1PR2-siRNA inhibited S1P-induced NLRP3 inflammasome priming and inflammatory cytokine (interleukin-1β and interleukin-18) secretion, whereas blockade of S1PR1 or S1PR3 had no such effect. in vivo, a β1,3-d-glucan-encapsulated siRNA particle (GeRP) delivery system is capable of silencing genes in macrophages specifically. Treatment with S1PR2 siRNA-GeRPs markedly reduced NLRP3 inflammasome priming and activation and attenuated liver inflammation and fibrosis. Together, the conclusions indicated that targeting macrophage S1PR2 retarded liver inflammation and fibrogenesis via downregulating NLRP3 inflammasome, which may represent an effective therapeutic strategy for chronic liver injury.

Keywords: NLR family pyrin domain-containing 3; S1P receptor 2; inflammation; liver injury; macrophage; sphingosine 1-phosphate.

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Figures

Figure 1

The dynamic changes of NLRP3 inflammasome priming and activation during chronic liver injury. The adult mice received an operation of bile duct ligation (BDL), methionine-choline-deficient and high-fat (MCDHF) diet, or carbon tetrachloride (CCl4) treatment to induce liver injury. (A) Expressions of NLRP3, pro-IL-1β, and pro-IL-18 in mouse livers were measured by RT-qPCR. (B) NLRP3, pro-IL-1β, pro-IL-18, and caspase-1 (cleaved caspase-1 p20 and un-cleaved caspase-1) protein expression from sham-operated and BDL-treated mouse livers were analyzed and quantified by Western blot. (C) ELISA analysis of IL-1β and IL-18 levels in serum. Data are presented as the mean ± SEM. n = 6 per group. Comparisons between two independent groups were performed using a student's _t_-test. One-way ANOVA was used for panels (A–C). *p < 0.05 vs. control group.

Figure 2

The cell-specific expression pattern of NLRP3 in the fibrotic liver of BDL-treated mice. (A) Mice were anesthetized and sacrificed at 2 weeks after sham or BDL operation. NLRP3 positive cells of the mouse liver non-parenchymal cells were analyzed for T cells, endothelial cells, dendritic cells, and macrophages-specific markers (CD3e, CD31, CD11c, and F4/80, respectively). Mice were lethally irradiated and received whole bone marrow cell transplants from EGFP transgenic mice, followed by sham or BDL operation. At 2 weeks after sham or BDL operation, the proportion of NLRP3+ cells in BMMs (F4/80+ EGFP+) was analyzed by FACS. (B) Representative images of immunofluorescence analysis by confocal microscopy to track the expression of NLRP3 (red) in macrophages (F4/80+, pink) of bone marrow origin (EGFP+, green) in mouse liver tissue. Data are presented as the mean ± SEM. n = 6 per group. Student's _t_-test was used in panel (A). *p < 0.05 vs. sham group.

Figure 3

S1P increased NLRP3 inflammasome priming and activation in dose-dependent manner. (A) NLRP3, pro-IL-1β, and pro-IL-18 mRNA expression were examined by RT-qPCR in non-serum-starved BMMs (NSS), and serum-starved BMMs treated with the indicated concentrations of S1P for 2 h. (B) NLRP3, pro-IL-1β, IL-1β, pro-IL-18, IL-18, and caspase-1 (cleaved caspase-1 p20 and un-cleaved caspase-1) protein expression were examined and quantified by Western blot in serum-starved BMMs treated with S1P for 12 h. (C) ELISA measurements of IL-1β and IL-18 in culture supernatants from serum-starved BMMs treated with S1P for 12 h. Data are presented as the mean ± SEM. n = 6 per group. One-way ANOVA was used for all comparisons. *p < 0.05 vs. control.

Figure 4

S1P induced IL-1β and IL-18 secretion in NLRP3 inflammasome-dependent manner. (A) Representative images of immunofluorescence analysis by confocal microscopy in serum-starved BMMs treated with 1 μmol/L S1P for 6 h. White arrows indicate oligomerized ASC. (B) The relative expression of NLR and non-NLR PRR family members were examined by qRT-PCR in serum-starved BMMs treated with S1P for 2 h. (C) ELISA measurements of IL-1β and IL-18 in culture supernatants from serum-starved BMMs pretreated with 100 nmol/L MCC950 for 1 h, followed by treatment with 1 μmol/L S1P for 12 h, or 100 ng/ml LPS for 4 h, then 5 mmol/L ATP for 30 min. Data are presented as the mean ± SEM. n = 6 per group. Student's _t_-test was used in panel (B) and one-way ANOVA was used in panel (C). *p < 0.05 vs. control. #p < 0.05 vs. S1P treatment alone. &p < 0.05 vs. LPS plus ATP treatment.

Figure 5

Blockade of S1PR2 by JTE-013 reduced NLRP3 inflammasome priming and secretion of mature IL-1β and IL-18 in BMMs. (A) The mRNA expression of NLRP3, pro-IL-1β, and pro-IL-18 in serum-starved BMMs treated with the indicated concentrations of S1P or H2S1P for 2 h was examined by qRT-PCR. (B) serum-starved BMMs were pretreated with S1PR1 antagonist W146 (5 μmol/L), S1PR2 antagonist JTE-013 (10 μmol/L), and S1PR3 antagonist CAY-10444 (10 μmol/L) for 1 h, followed by 1 μmol/L S1P treatment for 2 h. NLRP3, pro-IL-1β, and pro-IL-18 mRNA were examined by qRT-PCR. (C) IL-1β and IL-18 in culture supernatants were examined by ELISA. Data are presented as the mean ± SEM. n = 6 per group. Student's _t_-test was used in panel (A) and one-way ANOVA was used in panels (B,C). *p < 0.05 vs. control, #p < 0.05 vs. S1P treatment alone, ns, not significant.

Figure 6

S1PR2-siRNA reduced NLRP3, pro-IL-1β, and pro-IL-18 expression in BMMs. (A) The efficiency of S1PR1, S1PR2, and S1PR3 knockdown by their siRNAs in serum-starved BMMs. (B) Effects of S1PR1, S1PR2, and S1PR3 siRNA on NLRP3, pro-IL-1β, and pro-IL-18 mRNA expression in response to 1 μmol/L S1P for 2 h. (C) Mice received an operation of BDL to induce liver injury, then were anesthetized and sacrificed at 1 or 3 days or at 1 or 2 weeks after BDL operation. The relationship between S1PR2 and NLRP3, pro-IL-1β, and pro-IL-18 in mouse liver was analyzed by regression analysis. Data are presented as the mean ± SEM. Student's _t_-test was used in panel (A) and one-way ANOVA was used in panel (B). n = 6 per group. *p < 0.05 vs. control; #p < 0.05 vs. S1P treatment alone.

Figure 7

S1PR2 siRNA-GeRPs was able to induce macrophage-specific knockdown of S1PR2 and downregulate NLRP3 inflammasome in BDL-injured livers. (A) Strategy for loading Cy3-labeled glucan shells with siRNA-EP complexes and representative images of GeRPs (red). (B–D) Representative images and quantitative analysis of immunofluorescence analysis by confocal microscopy to track the selective uptake of GeRPs (red) in macrophages (F4/80+, green) in mouse livers at 2 weeks after BDL operation. (E) mRNA expression of S1PR2 in macrophages and hepatocytes from mice treated with scrambled or S1PR2-siRNA GeRPs. (F) mRNA expression of NLRP3, pro-IL-1β, and pro-IL-18 in liver tissues from mice treated with scrambled or S1PR2-siRNA GeRPs. (G) ELISA analysis of IL-1β and IL-18 levels in serum. Data are presented as the mean ± SEM. n = 6 per group. Student's _t_-test was used in panel (E) and two-way ANOVA was used in panels (F,G). *p < 0.05 vs. sham group; #p < 0.05 vs. BDL-treated alone.

Figure 8

Effects of macrophage-selective siRNA knockdown of S1PR2 in BDL-injured livers. (A) Representative H&E and Sirius Red staining and quantitative analysis in mouse livers at 2 weeks after BDL operation. Inset: H&E and Sirius Red staining in sham-treated livers. (B) Relative mRNA levels of hepatic α-SMA, Col α1(I), and Col α1(III) in mouse livers at 2 weeks after BDL operation. Data are presented as the mean ± SEM. n = 6 per group. Student's _t_-test was used in panel (A) and two-way ANOVA was used in panel (B). *p < 0.05 vs. sham group; #p < 0.05 vs. BDL-treated alone.

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Macrophage Sphingosine 1-Phosphate Receptor 2 Blockade Attenuates Liver Inflammation and Fibrogenesis Triggered by NLRP3 Inflammasome - PubMed (original) (raw)