Gal-3 Deficiency Suppresses Novosphyngobium aromaticivorans Inflammasome Activation and IL-17 Driven Autoimmune Cholangitis in Mice

Abstract

Gal-3 has the role in multiple inflammatory pathways. Multiple-hit etiology of primary biliary cholangitis (PBC) and evolving immune response at various stages of the disease includes involvement of Gal-3 in PBC pathogenesis. In this study we aimed to clarify the role of Gal-3 in Novosphingobium aromaticivorans (N. aromaticivorans) induced biliary disease. Autoimmune cholangitis was induced in mice by two intra-peritoneal injections of N. aromaticivorans within 2 weeks. The role of Gal-3 was evaluated by using Lgals3^-/- mice and mice treated with Gal-3 inhibitor. The histological and serological parameters of disease, phenotype of dendritic, NK, NKT, and T cells and inflammasome expression were evaluated. Marked attenuation of the disease in Lgals3^-/- and Gal-3 inhibitor, DAVANAT^®, treated mice is manifested by the absence of bile duct damage, granulomas and fibrosis. Liver infiltrates of N. aromaticivorans infected wild type mice had higher incidence of pro-inflammatory macrophages, dendritic cells, NK, NKT, and T cells. Lgals3 deletion and treatment with Gal-3 inhibitor reduced inflammatory mononuclear cell infiltrate, expression of NLRP3 inflammasome in the liver infiltrates and interleukin-1β (IL-1β) production in the livers of N. aromaticivorans infected mice. In vitro stimulation of wild type peritoneal macrophages with N. aromaticivorans caused increased NLRP3 expression, caspase-1 activity and IL-1β production compared with Lgals3^-/- cells. Our data highlight the importance of Gal-3 in promotion of inflammation in N. aromaticivorans induced PBC by enhancing the activation of NLRP3 inflammasome and production of IL-1β and indicate Gal-3 as possible therapeutical target in autoimmune cholangitis. Galectin-3 appears involved in inflammatory response to gut commensal leading to PBC.

Keywords: C57BL/6 mice; NLRP3; Novosphingobium aromaticivorans; galectin-3; galectin-3 inhibitor; primary biliary cholangitis.

Figure 1

Deletion of Gal-3 significantly attenuates PBC in C57BL/6 mice. C57BL/6 WT and Lgals3^−/− mice received two intra-peritoneal injections of N. aromaticivorans (10⁷ CFU) and 8 weeks after liver tissues were fixed, sectioned and stained with hematoxylin and eosin (A) Histological score for parameters of liver inflammation, granuloma formation, and fibrosis presented per mouse. (B) Representative liver sections of immunized WT and Lgals3^−/− infected mice. (C) Total score I (liver infiltration and bile duct damage) and score II (liver fibrosis and granuloma formation). (D) Serum levels of anti-PDC-E2 antibodies in non-infected mice (week 0) and 4, 6, and 8 weeks after N. aromaticivorans infection. (E) Significant increase of levels of AST and ALT in sera of N. aromaticivorans infected WT mice. (F) Representative images of Picrosirius staining of liver sections obtained from WT and Lgals3^−/− mice 8 weeks after N. aromaticivorans infection. Data are from representative experiment, presented as mean + SE, 8 mice/group, *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 2

Gal-3 deletion decreases Th17 and Tc17 lymphocytes in livers after PBC induction. Eight weeks after N. aromaticivorans infection mononuclear cells were isolated from livers and used for flow cytometric analysis after intracellular staining for inflammatory cytokines. (A) Percentages of CD4+ and CD8+ cells containing IL-17 and IFN-γ in the liver in WT and Lgals3^−/− mice. (B) Representative dot plots of CD4+ and CD8+ cells positive for IFN-γ and IL-17. Data are presented as mean+SE, 8 mice/group, *p < 0.05, **p < 0.005.

Figure 3

Gal-3 deficiency attenuates inflammatory and cytotoxic capacity of NK cells. Eight weeks after N. aromaticivorans infection mononuclear cells were isolated from livers and used for flow cytometric analysis of NK and NKT cells (A) Percentages of NK and NKT cells per liver. (B) Representative dot plots displaying the frequencies of NK and NKT cells in infected and untreated wild type and Lgals3^−/− mice. (C) Percentages of NKG2D and perforin positive cells in NK and NKT gated cells in liver in wild type and Gal-3 KO mice. (D) Expression of markers of activation and cytotoxicity on CD49b+CD3+ and CD49b+CD3- gated cells. (E) Percentages of IL-17 and IFN-γ positive cells in NK and NKT gated cells in the livers in WT and Lgals3^−/− mice. (F) Expression of IL-17 and IFN-γ on CD49b+CD3+ and CD49b+CD3- gated cells. Data are presented as mean+SE, 8 mice/group. NK cells were isolated from the livers of untreated WT and Lgals3^−/− mice (six mice in each group) cultured in vitro with N. aromaticivorans (cell/bacteria ratio 1:10) for 24 h and flow cytometric analysis was done. (G) Percentages of activated and IL-17 containing NK cells isolated from livers of untreated wild type and Lgals3^−/− mice and in vitro cultured with NA for 24 h., **p < 0.005; *p < 0.05.

Figure 4

Gal-3 deficiency attenuates inflammatory DCs in liver. Four and eight weeks after N. aromaticivorans infection mononuclear cells were isolated from the livers and used for flow cytometric analysis of dendritic cells (A) Percentages of CD11c+, CD11c+CD11b+, and CD11c+CD1d+ dendritic cells per liver in WT and Lgals3^−/− mice. (B) Percentages of activated (CD86+) CD11c+, CD11c+CD11b+, and CD11c+CD1d+ dendritic cells per liver in WT and Lgals3^−/− mice. (C) Percentages of inflammatory (IL-12+, TNF-α, and IL-1β) CD11c+, CD11c+CD11b+, and CD11c+CD1d+ cells per liver in WT and Lgals3^−/− mice. Dendritic cells isolated from spleens of untreated WT and Lgals3^−/− mice, six mice per group, cultured in vitro with N. aromaticivorans (cell/bacteria ratio 1:10) for 24 h were analyzed for expression of markers of activation and cytokines using flow cytometry. (D) Percentages of CD86+, IL-12+, IL-4+, and NLRP3 expressing N. aromaticivorans stimulated WT and Lgals3^−/− dendritic cells. Data are presented as mean+SE, 8 mice in each group, ***p < 0.001,**p < 0.005; *p < 0.05.

Figure 5

Gal-3 deficiency significantly attenuates activation of dendritic cells early after N. aromaticivorans infection, in spleen and liver. Three days after N. aromaticivorans infection mononuclear cells were isolated from the livers and spleens of WT and Lgals3^−/− mice and flow cytometric analysis was done. Percentages of CD11c+ dendritic cells, and CD11c+ dendritic cells expressing markers of activation and inflammatory cytokines per spleen (A) and liver (B). Percentages of NLRP3+ dendritic and myeloid cells in spleen (C) and liver (D) in WT and Lgals3^−/− mice with representative dot plots (E), 3 days after N. aromaticivorans infection. Data are presented as mean+SE, 8 mice/group, ***p < 0.001,**p < 0.005; *p < 0.05.

Figure 6

Novosphyngobium aromaticivorans stimulated Gal-3 deficient peritoneal macrophages have increased NLRP3 inflammasome expression and IL-β production. Immunofluorescence staining for F4/80 (red) and NLRP3 inflammasome (A), or IL-1 (B) (green) with DNA staining with DAPI (blue) in peritoneal macrophages from wild type (upper two panels) and Lgals3^−/− mice (lower two panels) cultured for 7 days with Novosphyngobium aromaticivorans (first and third panel) or in medium only (second and forth panel). (C) Significantly higher production of IL-1β by WT vs. Gal-3 KO peritoneal macrophages upon simulation with Novosphyngobium aromaticivorans (cell: bacteria ratio 1:10), whereas the production was significantly reduced in the presence of the caspase-1 inhibitor Z-YVAD (10 mmol/L). Wild type macrophages stimulated with bacteria produce more IL-6 when compared with WT macrophages but the production was not affected by caspase-1 inhibitor Z-YVAD. (D) Significantly increased caspase-1 activity in cell lysates of wild type peritoneal macrophages stimulated with Novosphyngobium aromaticivorans in comparison with Lgals3^−/− peritoneal macrophages. (E) Significantly lower percentage of F4/80+ NLRP3+ and F4/80+IL-1β+ macrophages in Gal-3 KO vs. WT mice when stimulated with Novosphyngobium aromaticivorans. Data are presented as mean+SD, 5 mice/group, ***p < 0.001,**p < 0.005; *p < 0.05.

Figure 7

DAVANAT treatment attenuates PBC in C57BL/6 mice. C57BL/6 WT and Lgals3^−/− mice received two intraperitoneal injections of N. aromaticivorans (10⁷ CFU). One group of WT mice was treated with Gal-3 inhibitor from the first day of experiment (three times per week, in the course of 4 weeks). Four, eight and 24 weeks after N. aromaticivorans infection liver tissues were fixed, sectioned and stained with hematoxylin and eosin (A) Histological score for parameters of liver inflammation, granuloma formation, and fibrosis 8 weeks after infection presented per mouse. (B) Representative liver sections of WT, Lgals3^−/−, and Gal-3 inhibitor treated WT mice, four (5 mice per group) and 24 (5 mice per group) weeks after N. aromaticivorans infection, arrows indicate: bile duct mononuclear cell infiltrate (yellow); parenchymal infiltration (black); organized parenchymal infiltration (green), parenchymal necrosis (violet), perivascular infiltrates (orange), bile duct obliteration (red); no infiltration (blue). (D) Serum levels of anti-PDC-E2 antibodies (C) in WT, Lgals3^−/−, and Gal-3 inhibitor treated WT mice 4 weeks after infection with NA (5 mice per group). (E) Serum levels of AST and ALT in WT, Lgals3^−/−, and Gal-3 inhibitor treated WT mice 4 (5 mice per group) and 8 weeks (5 mice per group) after infection with N. aromaticivorans. Data presented as mean + SE, *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 8

DAVANAT treatment decreases expression of NLRP3 inflammasome and IL-1β in liver infiltrates of N. aromaticivorans infected WT mice. Seven days after N. aromaticivorans infection of WT, Lgals3^−/− and Gal-3 inhibitor treated mice (7 or 8 mice per group) immunohistochemical staining of liver sections with analysis of the percentage of NLRP3 (A) and IL-1β (B) positive cells in liver infiltrates was done. (C) Percentages of F4/80 macrophages positive for NLRP3, IL-1β, and IL-18 in liver infiltrates 7 days after infection determined by flow cytometry. (D) IL-1β production determined by ELISA in liver tissue homogenate 7 days after infection. Data are presented as mean+SD. (E) NLRP3 and ASC inflammasome components expression in livers determined using real-time qRT-PCR, 15 weeks after infection, presented as mean+SE, *p < 0.05, **p < 0.005, ***p < 0.001.

Figure 9

DAVANAT treatment decreases Gal-3 expression in liver infiltrates of N. aromaticivorans infected WT mice. (A) Representative sections of Gal-3 immunohistochemical staining in liver sections obtained from WT, Lgals3^−/− and Gal-3 inhibitor treated mice 7 days after N. aromaticivorans infection. (B) Gal-3 levels in cell culture supernatants of splenocytes obtained from WT mice, stimulated in vitro with LPS (1 μg/ML) for 24 h, or treated with Gal-3 inhibitor (100 μM) for 2 h before LPS stimulation, or left untreated, presented as mean+SD, **p < 0.005, ***p < 0.001.

Figure 10

DAVANAT treatment decreases liver fibrosis N. aromaticivorans induced cholangitis. (A) Representative images of Picrosirius staining and quantitaive analysis of fibrosis in liver sections obtained from WT, Lgals3^−/− and Gal-3 inhibitor treated mice 3 months after infection, indicating negliglable fibrosis in Lgals3^−/− and Gal-3 inhibitor treated WT mice and marked fibrosis in WT mice. (B) Pro-collagen and α-SMC expression in livers determined using real-time qRT-PCR, 15 weeks after infection. Data are presented as mean+SE, *p < 0.05, ***p < 0.001.