The imbalance of liver resident macrophages polarization promotes chronic autoimmune hepatitis development in mice

Plamids and in vivo gene transfection

Plasmids pCYP2D6, psTLR2 and psTLR4 are expression vectors (pcDNA3.1; Invitrogen, Carlsbad, CA, USA) carrying the cDNA encoding human cytochrome P450 2D6 (CYP2D6), the extracellular domain of murine TLR2 and TLR4, respectively as described previously (Yan et al., 2013; Li et al., 2017). For in-vivo gene transfection, plasmids were prepared and analyzed as described previously (Geng et al., 2006). Mice received the injection of plasmid DNA via the tail vein (i.v. injection) using the hydrodynamics-based gene delivery technique, which delivers the transgene mainly into the liver (Geng et al., 2006).

Animal models and treatment

Female 7–8 weeks old C57BL/6 mice were purchased from the Center of Medical Experimental Animals of Hubei Province (Wuhan, China) for investigation. Animals were housed in a 12:12-h light-dark cycle with free access to food and animal care was supervised by veterinarians and trained animal care staff in animal care facilities. The Changzhi Medical College’s Institutional Animal Care and Use Committee approved all animal studies (No. DW2022073). All animal experiments were performed under S2-conditions in the central animal facility and were carried out in compliance with accepted standards of humane animal care as described in the Guide for the Care and Use of Laboratory Animals (National Research Council of the National Academics, 1996). The euthanasia procedures were in accordance with the AVMA Euthanasia Guidelines. Euthanasia was performed using carbon dioxide suffocation and cessation of breathing and any surviving mice at the conclusion of the experiment were euthanized. To induce hepatic inflammation, the mice were intravenously (i.v.) injected with adenovirus or intraperitoneally (i.p.) injected with clodronate liposomes. Data were collected as previously described (Chi et al., 2020). To express human CYP2D6, the mice received i.v. injection of pCYP2D6 plasmid. To induce autoimmune response and AIH, pCYP2D6 plasmid along with adenovirus was injected into the mice. To block the ligands for TLR2 and TLR4 in the liver, mice received the i.v. injection of plasmids psTLR2/psTLR4. To deplete Kupffer cells, 100 μl of clodronate liposomes (Liposoma BV, Amsterdam, Netherlands) per mouse were administered intraperitoneally every 4 days from d20 after AIH mice model establishment. The protocols for the injection of different agents are shown in Supplemental Figures, and indicated in the corresponding figure legends.

Isolation and culture of mouse Kupffer cells

A modification method of the type IV collagenase digestion in vitro was used to dissociate liver tissue (Kitani et al., 2011; Mo et al., 2020). For isolation of Kupffer cells, the livers of anesthetized mice were perfused as described above. After resuspension of the liver homogenate with 10 ml RPMI 1640 and centrifuged at 300×g for 5 min at 4 °C, the top aqueous phase was discarded, and the cell sediments was reserved. And then, cell sediments were resuspended with 10 ml RPMI 1640 and centrifuged at 50×g for 3 min at 4 °C. The top aqueous phase (cleared cell suspension) was transferred into a new 10 ml centrifuge tube and centrifuged at 300×g for 5 min at 4 °C, the top aqueous phase was discarded, and the cell sediments were reserved. The cell sediments mainly contained non-parenchymal cells of the liver that were KCs, sinusoidal endothelial cells and satellite cells. To purify the obtained cell population further, the method of selective adherence to plastic was used (Li et al., 2014). The cells were then seeded into 6-well plate at a density of 1–3 × 10⁷/well in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 100 U/ml Penicillin/Streptomycin (Sigma, St. Louis, MI, USA) and incubated for 2 h in a 5% CO2 atmosphere at 37 °C. Non-adherent cells were then removed from the dish by gently washing with PBS, the adherent cells were KCs.

Histology

Liver tissues from median and left lobes were collected, and embedded in paraffin according to standard histological procedures. Tissue sections were prepared and subjected to H&E staining for observation under a light microscope. To evaluate AIH inflammation, an inflammation score was performed as previously described (Bulau et al., 2011). For lobular inflammation, no inflammation was counted as 0, mild lobular inflammation (<10% of liver parenchyma) as 1, moderate lobular inflammation (10–50% of liver parenchyma) as 2, and a score of 3 was given for severe lobular inflammation (>50% of liver parenchyma). For portal inflammation, no portal inflammation was counted as 0, mild portal inflammation (<1/3 of portal tracts) as 1, moderate portal inflammation (approximately 1/2 of portal tracts) as 2, and a score of 3 was given for severe portal inflammation (>2/3 of portal tracts). The scores for portal and lobular inflammation were added, representing the AIH inflammation score.

Analysis of liver fibrosis

The liver tissues were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5-μm thickness. Picrosirius staining was performed for detection of fibrosis. For quantitative assessment of fibrosis, a fibrosis score was performed as described previously (Deng et al., 2013). Briefly, a fibrosis score was performed by using a 0–4 scale: 0, no fibrosis; 1, minimal portal fibrosis; 2, portal fibrosis with septa formation; 3, localized bridging fibrosis; and 4, extensive bridging fibrosis.

Real-time RT-PCR

The quantification of the expression of genes was performed using real-time RT-PCR. The sequences of the primers used for detecting gene expression were as follows:

For sample analysis, the threshold was set based on the exponential phase of products, and C_T value for samples was determined. The resulting data were analyzed with the comparative C_T method for relative gene expression quantification against house keeping gene GAPDH (Yan et al., 2013).

Immunohistochemistry

Liver tissue sections were prepared and subjected to immunohistochemical analysis as described earlier (Kitani et al., 2011). Anti-rabbit F4/80, Anti-rabbit CD86 and anti-rabbit CD163 (servicebio biotechnology, Wuhan, China) were used as primary Abs for detecting Kupffer cells, M1 phenotype macrophages and M2 phenotype macrophages, respectively. HRP conjugated secondary Ab was used for detecting macrophages, M1 macrophages and M2 macrophages in the liver tissues.

Statistical analysis

All experiments were performed in triplicate and repeated three times, including three biological replicates and three technical replicates. The results were expressed as mean values ± SD and interpreted by using one-way analysis of variance (ANOVA). The differences were considered statistically significant when P < 0.05.

Liver resident macrophages are involved in AIH chronic inflammatory processes

In order to explore the effect of liver resident macrophages on the liver inflammation in AIH, the clodronate liposomes were used to eliminate liver resident macrophages (Figs. S1A and S1B) based on our previous AIH mouse models (Chi et al., 2018). The lymphocyte infiltration was significantly increased by decreasing liver resident macrophages (Figs. 1A and 1B), and AIH inflammatory response, evaluated by the expression of Il1b and Tnfa genes in the liver, was also effectively increased by macrophage clearance (Fig. 1C). Further studies are needed to explore whether liver resident macrophages could effectively delay the development of liver fibrosis in AIH mice. The liver fibrosis induced by sustained AIH inflammation became more severe after liver resident macrophages depletion, evaluated by Picrosirius staining (Figs. 1D and 1E). Same results were observed when the expression of Col1a1 and Col1a2 genes in the liver was also detected (Fig. 1F). These results indicated that liver resident macrophages could effectively delay the development of AIH.

The intensity of immune response is regulated by liver resident macrophages

It is urgently needed to confirm whether liver resident macrophages could involve in the regulation of autoimmune response in AIH. Liver resident macrophages depletion could increase the intrahepatic expression of IL-6 and IL-12 that promote IFN-γ production, which favors Th1 response (Fig. 2A). Furthermore, IL-4 and IL-13 expression was also elevated to favor Th2 responses in AIH mice with liver resident macrophages depletion (Fig. 2B). These results suggested that liver resident macrophages could limit the intensity of autoimmune response, resulting in progressive and chronic AIH development.

The effect TLR2 and TLR4 ligands on liver resident macrophages polarization in AIH development

The effect of TLR2 or TLR4 ligands on liver macrophage polarization was further identified in vivo to better understand the underlying mechanisms of chronic inflammatory processes in AIH. We firstly detected the macrophage polarization marker in liver tissue by immunohistochemistry. The expression of CD163 (M2 marker) was decreased slowly in liver tissue of AIH mice. However, the expression of CD86 (M1 marker) was increased slowly in liver tissue during AIH development (Fig. 3A). Furthermore, the expression of M1 macrophage marker (iNOS and TNF-a) was markedly upregulated during AIH development. However, the expression of M2 macrophage marker (Arg-1 and Ym-1) was significantly downregulated in liver tissue during AIH development (Fig. 3B). To further determine the effect TLR2/4 ligands on liver resident macrophages polarization, we expressed sTLR2 and sTLR4 to block TLR2 and TLR4 ligands (Yan et al., 2013; Li et al., 2017). In AIH mice, blocking TLR2 ligands resulted in M2 macrophage marker was significantly downregulated while blocking TLR4 ligands resulted in M1 macrophage marker was dramatically downregulated (Fig. 3C). These results indicated that TLR2 and TLR4 ligands might regulate liver macrophage polarization to influence AIH development.

Changes in the cytokine secretion profile of liver resident macrophages following TLR2 and TLR4 ligand exposure and activation in vitro

In order to confirm the involvement of TLR2 and TLR4 ligands in liver macrophage polarization and cytokine secretion by liver resident macrophages in vitro, Kupffer cells were isolated from liver tissues of AIH mice. The activation of TLR4 with LPS led to a significant upregulation of M1 macrophage markers, whereas expression of M2 macrophage markers was no significant difference compared with the control group (Fig. 4A). In contrast, TLR2 stimulation with Pam3 significantly upregulated M2 macrophage markers, whereas expression of M1 macrophage markers showed no significant change compared with control group (Fig. 4A). The stimulation of Kupffer cells with LPS, but not with Pam3, strongly induced the production of proinflammatory cytokines IL-1β and IL-6 (Fig. 4B). In contrast, the stimulation of Kupffer cells with Pam3, but not with LPS, strongly induced the production of anti-inflammatory cytokines IL-10 and TGF-β (Fig. 4B). TLR2 expression increased first and then decreased in AIH development. By contrast, TLR4 remained highly expressed in AIH development (Fig. 4C). These results indicated that TLR4 ligand could promote M1 Kupffer cells producing proinflammatory cytokines, whereas TLR2 ligand could promote M2 Kupffer cells producing anti-inflammatory cytokines.