Gal-3–deficient mice on the C57BL/6 background (Gal-3 KO) and wild-type (WT) C57BL/6 mice (6–8 weeks of age) were used in the experiments. Breeding pairs of WT and Gal-3 KO mice were obtained from the University of California, Davis, United States (Davis, CA, United States; by courtesy of D.K. Hsu and F.T. Liu). All animal procedures were approved by the Ethics Committee of Faculty of Medical Sciences, University of Kragujevac and conducted in accordance with the National Institutes of Health guidelines for humane treatment of laboratory animals. Unless otherwise stated each experimental group in each experiment contained six animals.

The bacterial artificial chromosome (BAC)-derived MCMV strain MW97.01 has previously been shown to be biologically equivalent to MCMV strain Smith (VR-1399) and is hereafter referred as WT MCMV (Wagner et al., 1999). Mice were injected intraperitoneally (i.p.) with 1 × 10⁵ PFU of MCMV strain MW97.01 in a volume of 200 μL of diluent (PBS). Mice were also infected with MW97.01, the mutant virus lacking m157 gene (Δm157 MCMV) intravenously (i.v.) with 2 × 10⁵ PFU in a volume of 100 μL of diluent (PBS).

Serum levels of asparate aminotransferase (AST) and alanine aminotransaminase (ALT) were measured 36 and 72 h after MCMV infection by standard photometric method using the automated biochemistry analyzer Olympus AU 400 (Olympus Diagnostica GMBH, Hamburg, Germany) and Olympus AU reagents, according to the manufacturer’s instructions, expressed in U/L.

The isolated livers were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and consecutive 4 μm tissue sections were cut at various depths and mounted on slides. Sections were stained with Hematoxylin and Eosin (H&E) and every fourth (six slides) was evaluated for inflammation and necrosis in the liver. Section examined under low-power light microscopy (BX51; Olympus) equipped with digital camera. Scores of cumulative liver pathology for inflammation and necrosis were presented using the following scoring system: 0, normal (no pathology); 1, mild (1–3 abnormal areas); 2, moderate (3–5 abnormal areas); 3, severe (>5 abnormal areas). To determine the number of inflammatory infiltrates, whole liver tissue was sectioned at three non-subsequent depths and ten different fields were counted/section. Histological samples were blinded prior to evaluation.

Formalin-fixed, paraffin-embedded mouse liver tissue sections were incubated with rabbit anti-TNF-α (ab66579, Abcam), rabbit anti-caspase-3, active/cleaved (NB100-56113, Novus Biologicals), anti-caspase 3 and rabbit anti-Gal-3 (ab53082, Abcam). Sections were visualized by rabbit-specific conjugate (Expose Mouse and RabbitSpecific HRP/DAB Detection IHC Kit; Abcam) and photomicrographed with a digital camera mounted on light microscope (BX51; Olympus). Virus-infected cells were visualized by anti-IE1 staining (MCMV protein expressed with early kinetics).

TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nickend labeling) staining was performed to assess death hepatocytes in livers sections. Formalin-fixed, paraffin-embedded tissue sections were stained with in situ Cell Death Detection Kit, POD (Roche) following the instructions of manufacturer. DAB (3,3′-diaminobenzidine) as peroxidase substrate, was used to yield the characteristic brown color for nuclei. Slides were counterstained with hematoxylin solution and photomicrographed with a digital camera mounted on light microscope. The TUNEL-positive nuclei (brown) were quantified under ×400 magnification in five randomly fields and the data were summarized as the mean number of positive cells.

The isolation of liver-infiltrating inflammatory mononuclear cells was conducted as previously described (Volarevic et al., 2012). The isolated liver-infiltrating mononuclear cells were stained with fluorochrome-conjugated antibodies, including CD3, CD49b, CD8, NKG2D, CD69, perforin, granzyme B, NF-κB, IFN-γ, IL-10, IL-17, and TNF-α. Isotype Abs with matching conjugates were used as negative controls. For intracellular staining, cells were activated with PMA/ionomycin and processed as previously described (Milovanovic et al., 2012). Cells were analyzed with the FACSCalibur Flow Cytometer (BD Biosciences), and analysis was conducted with FlowJo (Tree Star).

In order to inhibit production of TNF-α, mice were injected with chimeric monoclonal antibody, Infliximab (Remicade, JANSSEN BIOLOGICS B.V.), 5 mg/kg in 200 μL of saline intraperitoneally 1 h before MCMV infection. Mice were sacrificed 48 h after infection.

WT and galectin-3 KO mice were treated with recombinant Galectin-3, 5 μg per mouse (Peprotech, Rocky Hill, NJ, United States) intraperitoneally, 2 h before MCMV infection. Mice were sacrificed 36 h after MCMV infection.

Hepatocytes were isolated as previously described (Li et al., 2010). Briefly, extirpated livers were transferred HBSS, cutinto 1 mm³ size pieces and washed in complete DMEM. Dissected tissue was centrifuged at 800 × G for 4 min, pellet resuspended in digestion medium (0.6% NaCl, 0.05% KCl, 1.2% HEPES, 0.07% CaCl2, 3 g/mL collagenase type I) and incubated for 20 min at 37°C. After incubation cells centrifuged at 800 × G for 4 min, pellet was washed twice in a complete DMEM, passed through the 100 μm filter and cells centrifuged at 600 × G for 4 min. Pellet that contains hepatocytes was resuspended in DMEM medium with FBS. Isolated hepatocytes were washed in cold PBS and resuspended in 1X binding buffer (10X binding buffer: 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at concentration 1 × 10⁶/mL. Annexin FITC and propidium iodide (PI) were added to the 100 μL of cell suspension and incubated for 15 min at room temperature (25°C) in the dark. After incubation 400 μL of 1X binding buffer was added to each tube and stained cells were analyzed within 1h using FACSCalibur (BD, San Jose, United States) and FlowJo software (Tri Star). For detection of cell surface expression of calreticulin, isolated hepatocytes were stained with anti-calreticulin antibody (Abcam) and analyzed by FACSCalibur (BD, San Jose, United States) and FlowJo software (Tri Star).

Levels of TNF-α and HMGB1 in the liver homogenate were measured using ELISA kits (R&D Systems, Minneapolis, MN, United States for TNF-α and Elabscience for HMGB1) according to the manufacturer’s instructions.

All statistics were carried out using SPSS 18.0 for Windows software. Results were analyzed using the Student’s t-test or Mann–Whitney test and ANOVA or Kruskal–Wallis. Data in this study were expressed as the mean + SE or +SD. Values of P < 0.05 were considered significant.

Previously, we have shown very weak expression of Gal-3 in the liver parenchyma and biliary epithelial cells in healthy C57BL/6 mice (Arsenijevic et al., 2016). Also we found strongly enhanced expression of Gal-3 in patients with virus induced hepatitis (Volarevic et al., 2015). To explore the effect of MCMV infection on Gal-3 expression in mouse livers, immunostaining of Gal-3 in the livers of WT and Gal-3 KO mice was done 36 and 72 h after MCMV infection. Time-dependent increase of Gal-3 expression in hepatocytes after MCMV infection was noticed in the livers of WT mice (Figure 1A). Significantly higher number of Gal-3 expressing hepatocytes per field was noticed 72 h after infection when compared with liver sections obtained 36 h after MCMV infection (Figure 1B). As a control, Gal-3 was not detected in the livers of untreated and KO infected mice (Figure 1A).

In light of increased Gal-3 expression in hepatocytes of MCVM-infected mice, we wanted to explore the role of Gal-3 in overall severity of MCMV-induced hepatitis. For this, histological and serological parameters of liver damage were analyzed in WT and Gal-3 KO mice, 36 and 72 h after MCMV infection. Histological parameters related to MCMV-induced hepatitis, liver inflammation and necrosis were more pronounced in Gal-3 KO mice, 36 and 72 h after infection (Figure 2A,B). No difference in the architecture of liver tissue was noticed between WT and Gal-3 KO uninfected mice (Figure 2A). Bigger necrotic areas and inflammatory foci were observed in the livers of Gal-3 KO mice in comparison with WT mice, 36 h after infection (Figure 2A,B). Similar differences between Gal-3 KO and WT mice in the size of necrotic areas were observed 72 h after infection also (Figure 2A,B). At this time point, there was no difference in the size of inflammatory foci between the two groups (Figure 2A), but Gal-3 KO mice had higher number of smaller inflammatory foci compared with WT mice (Figure 2A). Although HCMV infection in immunocompetent hosts is subclinical, it is often accompanied with elevated serum levels of transaminases. Thus, we examined the level of alanine transaminase (ALT) in the sera of infected mice. In line with histological findings, we observed significantly higher level of ALT in the sera of Gal-3 KO mice 36 h after MCMV infection, compared to WT mice (Figure 2C).

In order to further analyze the liver damage in WT and KO mice after MCMV infection we used TUNEL assay. As shown in Figure 2D,E, 72 h after MCMV infection, the livers of Gal-3 KO mice contains significantly higher number of TUNEL positive (brown nuclei) hepatocytes than the livers of WT mice. Moreover, higher number of apoptotic (caspase 3 positive) cells per one inflammatory infiltrate was detected in Gal-3 KO compared to the WT livers (Figure 2F, red dots). To confirm the increased apoptosis of hepatocytes in Gal-3 KO mice, we isolated hepatocytes from WT and Gal-3 KO mice 72 h after MCMV infection and measured the percentage of apoptotic cells by flow cytometry. In line with histological observations, we detected significantly higher percentage of apoptotic (Annexin V positive) hepatocytes isolated from infected Gal-3 KO mice, compared with those from WT mice (Figure 2G). Given the marked necrotic fields in the liver sections from Gal-3 KO mice (Figure 2A,B) and the ability of MCMV to induce necroptosis (Upton et al., 2017), we examined markers of necroptotic death, HMGB1 in liver tissue homogenates and membrane expression of calreticulin on hepatocytes, 36 h after infection. We have found significantly higher concentration of HMGB1 in the liver homogenates (Figure 2H) and higher percentage of hepatocytes expressing calreticulin on membrane surface (Figure 2I) in Gal-3 KO mice in comparison with infected WT mice.

In the livers of Gal-3 KO mice, there is a significant increase in viral titers compared to the livers of wild-type mice (Figure 2J). There was no significant difference in viral titers in lung and spleen between WT and Gal-3 KO mice, 72 h after infection (Supplementary Figure S1A). At 8 days post-infection (Supplementary Figure S1B), viral plaques in spleen were not detected with the exception of a very low titer in one Gal-3 KO animal. In lungs, liver and salivary gland, viral plaques were readily detected but no significant differences were observed between the two groups. Lastly, to demonstrate the presence of virus-infected cells, sections of liver were stained with an antibody against IE1, a MCMV protein expressed with early kinetics. As shown in Figure 2K, in the liver tissue of Gal-3 KO mice, higher number of infected cells was observed compared to the WT animals.

Together, previous data indicate protective role of Gal-3 in MCMV-induced hepatitis, possibly relating to its increased expression in hepatocytes and known role of Gal-3 in attenuation of cell death (Takenaka et al., 2004).

Taking into account the facts that NK cells play a crucial role in the early immune response against MCMV in C57BL/6 mice (Scalzo and Yokoyama, 2008) and that NK cells contribute to liver damage in viral infections (Zheng et al., 2015), we explored the possibility that bigger liver damage in infected Gal-3 KO mice is a consequence of stronger NK cell activity. We analyzed the presence and the phenotype of NK cells in liver mononuclear infiltrates in WT and Gal-3 KO mice, 36 and 72 h after MCMV infection. There was no statistically significant increase in the percentage and total number of NK cells in the livers of infected Gal-3 KO mice in comparison with uninfected Gal-3 KO mice (Figure 3A,B). On the other hand, MCMV infection induced a significant increase in both total number and percentage of NK cells in the livers of WT mice (Figure 3A,B). Total number of IFN-γ expressing NK cells 36 h after MCMV infection was significantly higher in the livers of WT mice in comparison with Gal-3KO mice, while 72 h after infection this difference lost significance (Figure 3C). No significant difference in the percentage and total number of NK cells expressing IL-17 was noticed between the groups. Lastly, total number of IL-10 positive NK cells was significantly higher in the liver of Gal-3 KO mice in comparison with WT mice, 36 h after infection (Figure 3C).

In order to exclude the role of NK cells in the higher liver damage in Gal-3 KO mice, we analyzed liver damage in WT and KO mice after infection with Δm157 MCMV, the mutant virus lacking m157 gene, which does not stimulate NK cells. As observed for WT MCMV, the inflammatory cell infiltrates were readily observed in the liver tissue of both wild-type and Gal-3 KO infected mice (Figure 3D, black arrows). No difference in the size of infiltrates was observed between the two groups but, interestingly, the number of infiltrates was significantly higher in Gal-3 KO mice compared to the wild-type mice (Figure 3E). Thus, enhanced liver damage in MCMV-infected Gal-3 KO mice does not appear to relate to NK cell activity.

Based on our finding of enhanced necoptosis in Gal-3 KO mice and the fact that TNF-α signaling triggers necroptosis (Vandenabeele et al., 2010) and is required for MCMV-induced liver damage (Orange et al., 1997), TNF-α detection in the livers of MCMV-infected mice was done. The expression of TNF-α in hepatocytes was detected by immunostaing the livers of MCMV-infected mice both, WT and Gal-3 KO (Figure 4A). The number of TNF-α positive hepatocytes was significantly higher in the livers of Gal-3 KO mice compared to the group of infected WT mice, 36 and 72 h after infection (Figure 4B). Also, the concentration of TNF-α in the liver tissue homogenate was significantly higher in the group of Gal-3 KO mice compared to the group of WT mice, 72 h after MCMV infection (Figure 4C). Further, significantly higher percentage of TNF-α+ hepatocytes (Figure 4D), and total number of TNF-α+ CD11c+ cells (Figure 4E), analyzed by flow cytometry 72 h after MCMV infection, were found in the group of Gal-3 KO mice in comparison with WT mice. In accordance with the role of NF-κB in the promotion of TNF-α expression and its role in TNF-α mediated actions, significantly higher percentage of NF-κB+ hepatocytes was found in the group of Gal-3 KO mice in comparison to WT mice, 72 h after infection (Figure 4F). These results suggest that higher TNF-α expression in hepatocytes and immune cells could be the cause of enhanced MCMV-induced hepatocyte death observed in Gal-3 KO mice.

In order to further explore the possibility that TNF-α plays a role in enhanced MCMV-induced liver damage in Gal-3 deficient animals, pharmacological inhibition of TNF-α with infliximab was done before infection. In the control group of WT mice, TNF-α blockade before MCMV infection did not alter liver inflammation or necrosis, while a pre-treatment of MCMV-infected Gal-3 KO mice with infliximab significantly decreased liver inflammation and necrosis (Figure 5A–C). Liver of untreated MCMV-infected Gal-3 KO mice contained bigger inflammatory and necrotic foci in comparison to the liver of infliximab-pretreated mice (Figure 5A). Also, the TNF-α blockade before MCMV infection significantly decreased serum levels of ALT in Gal-3 KO mice (Figure 5D). Ameliorated MCMV-induced hepatitis in Gal-3 KO mice treated with infliximab supports the role of TNF-α in enhanced disease in Gal-3 deficient animals.

To confirm the protective role of Gal-3 in MCMV-induced hepatitis disease severity in WT and Gal-3 KO mice treated with recombinant Gal-3 was evaluated. Administration of recombinant Galectin-3 did not significantly alter inflammation and necrosis in WT mice (Figure 6A,B). Although we have not observed an altered number of inflammatory foci per liver in WT mice, there was an increase in their size (Figure 6A, white arrows). Gal-3 KO mice treated with recombinant Galectin-3 had lower histological score of necrosis and inflammation and lower number of inflammatory and necrotic foci per section, 72 h after MCMV infection (Figure 6A–D). Alleviated disease in Gal-3 KO mice treated with recombinant Galectin-3 confirms the protective role of Gal-3 in MCMV induced hepatitis.