Galectin-3 Deficiency Facilitates TNF-α-Dependent Hepatocyte Death and Liver Inflammation in MCMV Infection

Abstract

Galectin-3 (Gal-3) has a role in multiple inflammatory pathways. Various, opposite roles of Gal-3 in liver diseases have been described but there are no data about the role of Gal-3 in development of hepatitis induced with cytomegalovirus infection. In this study we aimed to clarify the role of Gal-3 in murine cytomegalovirus (MCMV)-induced hepatitis by using Gal-3-deficient (Gal-3 KO) mice. Here we provide the evidence that Gal-3 has the protective role in MCMV-induced hepatitis. Enhanced hepatitis manifested by more inflammatory and necrotic foci and serum level of ALT, enhanced apoptosis and necroptosis of hepatocytes and enhanced viral replication were detected in MCMV-infected Gal-3 deficient mice. NK cells does not contribute to more severe liver damage in MCMV-infected Gal-3 KO mice. Enhanced expression of TNF-α in the hepatocytes of Gal-3 KO mice after MCMV infection, abrogated hepatocyte death, and attenuated inflammation in the livers of Gal-3 KO mice after TNF-α blockade suggest that TNF-α plays the role in enhanced disease in Gal-3 deficient animals. Treatment with recombinant Gal-3 reduces inflammation and especially necrosis of hepatocytes in the livers of MCMV-infected Gal-3 KO mice. Our data highlight the protective role of Gal-3 in MCMV-induced hepatitis by attenuation of TNF-α-mediated death of hepatocytes.

Keywords: TNF-α; galectin-3; hepatitis; hepatocyte death; murine cytomegalovirus infection.

FIGURE 1

Acute MCMV infection increases Gal-3 expression in hepatocytes. (A) Representative sections of Gal-3 expression in the liver of WT and KO mice 36 and 72 h after MCMV infection with 1 × 10⁵ PFU/animal and the control uninfected liver from WT and KO mice. (B) Liver tissue was sectioned at three non-subsequent depths. Gal-3 positive hepatocytes were counted in ten different fields and presented as mean number + SD of positive cells per section, six mice per group, ^∗∗∗p < 0.001.

FIGURE 2

Galectin-3 deficiency enhances MCMV-induced hepatitis. WT and Gal-3 KO mice were i.p. infected with 1 × 10⁵ PFU/animal. Livers were analyzed 36 and 72 h after virus infection. (A) H&E staining of paraffin embedded liver sections. Black arrows highlight necrotic areas, and white arrows indicate inflammatory foci. (B) Scores of cumulative liver pathology for apoptosis/necroptosis and inflammation; the height of each bar represents the mean of the total histological score (6 animals per group). (C) ALT levels were determined in the serum 36 and 72 h after virus infection. (D) TUNEL staining of liver sections 72 h after MCMV infection. (E) Quantitative analysis of cell death rate: TUNEL-positive nuclei (brown) were counted in five random fields, and the data were summarized as the mean number of positive cells. (F) Apoptotic cells were visualized by anti-caspase-3 staining (shown in red). Two representative images are shown/group. (G) Apoptosis of hepatocytes isolated from MCMV infected WT and KO mice was analyzed by flow cytometry using Annexin V (FITC) and PI double staining. (H) Concentration of HMGB1 in the liver tissue homogenate 36 h p.i. determinated by ELISA. (I) Percentage of hepatocytes expressing membrane surface calreticulin isolated from the livers 36 h p.i. determined by flow cytometry. (J) Viral titres in liver is determined by standard plaque assay at 3 days p.i. (K) Virus-infected cells are revealed by anti-IE1 staining (shown in brown). Two representative images are shown/group. The data are presented as means +SE, or means+SD, 7 mice per group, ^∗∗∗p < 0.001; ^∗p < 0.05; two tailed, unpaired Student’s t-test.

FIGURE 3

Liver inflammatory NK cells are attenuated in Gal-3 KO mice. Flow cytometry of mononuclear cells isolated from livers of WT and KO 36 and 72 h after i.p. infection with 1 × 10⁵ PFU/animal MCMV was done. Percentages (A) and absolute numbers (B) of NK cells calculated per liver. (C) Total numbers of IFN-γ+, IL-17+, and IL-10+ NK cells 36 and 72 h after infection calculated per liver. (D) Images of H&E stained liver sections obtained from C57BL/6 and KO mice 72 h after intravenous infection with MCMVΔm157 (2 × 10⁵ PFU/animal). (E) Number of inflammatory cell infiltrates (indicated by black arrows in D) determined by manual counting. Data are presented as the mean + SE, 15 mice per group, ^∗∗∗p < 0.001; ^∗∗p < 0.005; ^∗p < 0.05; two tailed, unpaired Student’s t-test.

FIGURE 4

Galectin-3 deficiency enhances the expression of TNF-α in hepatocytes of infected animals. (A) Sections of TNF-α liver immunohistochemistry 36 and 72 h after MCMV infection. (B) Quantitative analysis of TNF-α positive hepatocytes: positive hepatocytes were counted in five random fields, and the data were summarized as the mean number of positive cells. (C) Concentration of TNF-α in the liver tissue homogenate determinated 72 h p.i. by ELISA. Percentage of TNF-α+ (D) and NF-κB+ (E) hepatocytes and (F) total number of CD11c+ TNF-α+ cells determined by flow cytometry 72 h after infection. Data are presented as the mean + SE, six mice per group, ^∗p < 0.05, ^∗∗∗p < 0.001, two tailed, unpaired Student’s t-test.

FIGURE 5

TNF-α blockade attenuates liver inflammation and necrosis in MCMV-infected Gal-3 KO mice. WT and KO mice were i.p. infected with 1 × 10⁵ PFU/animal. Chimeric monoclonal antibody, Infliximab (5 mg/kg) was administrated intraperitoneally 1 h before MCMV infection. Mice were analyzed 48 h p.i. (A) H&E staining of paraffin embedded liver sections. Black arrows highlight necrotic areas, and white arrows indicate inflammatory foci. (B) Number of inflammatory cell infiltrates per section (indicated by white arrows in A) was determined by manual counting. (C) Number of necrotic foci per section (indicated by black arrows in A) was determined by manual counting. (D) ALT levels determined in the serum. Data are presented as mean + SE of n = 6 mice. ^∗∗∗p < 0.001; ^∗p < 0.05 from two tailed, unpaired Student’s t-test.

FIGURE 6

Gal-3 is protective in MCMV-induced hepatocyte damage. (A) H&E staining of paraffin embedded liver sections in WT and KO mice treated with recombinant Galectin-3 (50 μg/mL) intraperitoneally 2 h before MCMV infection, analyzed 72 h after infection. (B) Scores of cumulative liver pathology for apoptosis/necroptosis and inflammation. (C,D) Number of inflammatory cell infiltrates and necrotic areas per section determined by manual counting. Data are presented as mean + SE of n = 8 mice. ^∗∗∗p < 0.001; ^∗p < 0.05 from two tailed, unpaired Student’s t-test.

FIGURE 7

Role of Gal-3 in MCMV-induced hepatitis. In the presence of Gal-3 (left panel) liver infiltrating NK cells contain IFN-γ and take a role in viral clearance. MCMV in hepatocytes induce apoptosis and necroptosis, membrane expression of calreticulin (CALR), release of pro-inflammatory molecule HMGB-1, and TNF-α production. TNF-α binds TNFR and additionally stimulates apoptosis, but intracellular Gal-3 protects hepatocytes from cell death. Result is controlled liver damage and lower viral titers. In Gal-3 KO mice (right panel) NK cells that contain more IL-10 are poorer in controlling the virus, leading to increased virus-induced apoptosis and necroptosis. Increased death of Gal-3 KO hepatocytes is accompanied with higher expression of CALR and HMGB1 release that stimulates TNF-α in CD11c+ cells. More MCMV in Gal-3 KO hepatocytes induce higher NF-κB mediated production of TNF-α that augments apoptosis of hepatocytes, and in the absence of protective effect of Gal-3 results in more severe liver damage in Gal-3 KO mice.