CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease

Abstract

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

Fig. 1. Patients with alcohol-related liver disease have less CRIg expression on liver macrophages.

a, d, e RNA sequencing data of liver tissues from subjects without liver disease (Control; n = 10), patients with early alcoholic steatohepatitis (Early ASH; n = 12), non-severe alcoholic hepatitis (Non-severe AH; n = 11) and severe alcoholic hepatitis (Severe AH; n = 18). b and c Single-cell RNA sequencing data of liver tissues from subjects without liver disease (uninjured; n = 5) and alcohol-related cirrhotic patients (cirrhotic; n = 2). a mRNA level of CRIg. b UMAP annotating different macrophage cell types as KC (Kupffer cell), TMo (tissue monocyte) and SAMac (scar-associated macrophage). c Dot plot showing expression of CRIg and other stated marker genes in human liver macrophage subpopulations. d mRNA level of TIMD4 (marker for Kupffer cells). e mRNA level of TREM2 (marker for scar-associated macrophages). f Representative liver sections of CD68 and CRIg immunofluorescence staining. g Quantification of the stained liver sections. Scale bar = 100 μm. Results are expressed as median with interquartile range (a, d, e), or mean ± s.e.m. (g). P value is determined by two-sided Kruskal–Wallis test with Dunn’s post-hoc test (a, d, e), or two-sided Student t-test (g). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. Deficiency of CRIg causes progression of ethanol-induced liver disease in mice.

Wild-type (WT), CRIg^−/−, and CRIg^−/−/Tlr2^−/− mice were fed the Lieber DeCarli ethanol diet for 8 weeks. a Hepatic level of mRNA encoding CRIg in WT mice. b Serum levels of ALT. c Hepatic triglyceride content. d Representative oil red O-stained liver sections. e and f Hepatic levels of Il1b and Col1a1 mRNAs. g Representative sirius red-stained liver sections. h Quantification of the sirius red-stained liver sections. i and j Hepatic DNA levels of Firmicutes and Proteobacteria, normalized to total amount of bacteria using universal 16S primers. Scale bar = 100 μm. Results are expressed as mean ± s.e.m. (a–c, e, f, h–j). P values among groups of mice fed with control diet or ethanol diet are determined by two-sided Student t-test (a) or one-way ANOVA with Tukey´s post-hoc test (b, c, e, f, h–j). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. CRIg^−/− mice are more susceptible to E. faecalis- and ethanol-induced liver disease.

WT, CRIg^−/− and CRIg^−/−/Tlr2^−/− mice were placed on the chronic–binge ethanol diet and gavaged with a cytolytic E. faecalis strain (5 × 10⁸ colony forming units (CFUs)) every third day. a Serum levels of ALT. b Hepatic triglyceride content. c Representative oil red O-stained liver sections. d Hepatic levels of mRNAs encoding inflammatory cytokines and chemokines IL1B, CXCL1, and CXCL2. e Serum levels of ethanol in ethanol-fed mice. f Hepatic levels of Adh1 and Cyp2e1 mRNAs. Scale bar = 100 μm. Results are expressed as mean ± s.e.m. (a, b, d–f). P values among groups of mice fed with control diet or ethanol diet are determined by one-way ANOVA with Tukey´s post-hoc test (a, b, d–f). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. CRIg is required for capturing of E. faecalis translocated from gut to the liver.

a and b CRIg^−/− mice and their WT littermates were placed on a chronic–binge ethanol diet and GFP-E. faecalis (2 × 10⁷ CFUs) was intravenously injected 1 h after ethanol-binge. Mice were sacrificed 1 h after injection and Kupffer cells were isolated. a Quantification of the stained cells. b Representative microscopy pictures. c and d CRIg^−/− mice and their WT littermates were fed the chronic-binge ethanol diet. One hour after the ethanol binge, mice were gavaged with E. faecalis (5 × 10⁹ CFUs). Bacterial load (c) and DNA levels (d) were determined 20 min after bacterial gavage. Scale bar = 10 μm. Results are expressed as mean ± s.e.m. (a, c, d). P values determined by two-sided Mann–Whitney test (a, c, d). *P < 0.05, **P < 0.01.

Fig. 5. Extracellular region of CRIg protects mice from ethanol-induced liver disease.

WT mice were fed the chronic–binge ethanol diet. a One hour after the ethanol binge, mice were given intravenous injections of control-Ig or CRIg-Ig (12 mg/kg body weight [BW]) and gavaged with E. faecalis (5 × 10⁹ CFUs). Bacterial load was determined 20 min after bacterial gavage. b–k One day before the ethanol binge, mice were given intravenous injections of control-Ig or CRIg-Ig (12 mg/kg body weight [BW]). b Serum levels of ALT. c Hepatic triglyceride content. d Representative oil red O-stained liver sections. e–g Hepatic levels of mRNAs encoding inflammatory cytokines and chemokines IL1B, CXCL1, and CXCL2. h Serum levels of inflammatory cytokines and chemokines TNF and CXCL1. i Hepatic level of mRNA encoding CRIg. j Serum levels of ethanol. k Hepatic levels of Adh1 and Cyp2e1 mRNAs. Scale bar = 100 μm. Results are expressed as mean ± s.e.m (a–c, e–k). P values determined by two-sided Student’s t-test (a–c, e–k). *P < 0.05, **P < 0.01.

Fig. 6. Alcohol reduces the clearance of pathobionts by Kupffer cells and contributes to liver disease.

Left panel: During homeostasis, translocation of pathobionts from the gut to the liver is a rare event. Translocated E. faecalis are cleared and phagocytosed by CRIg on Kupffer cells. Right panel: Chronic alcohol consumption results in intestinal expansion of E. faecalis. These pathobionts escape the intestinal barrier and translocate to the liver. Alcohol changes hepatic macrophage composition and phenotype, and downregulates CRIg on Kupffer cells, thus contributing to reduced CRIg expression in the liver and reducing clearance of translocated E. faecalis. Hepatic and circulating E. faecalis are recognized by TLR2, a pattern recognition receptor that binds Gram-positive bacteria, resulting in hepatic inflammation and progression of alcoholic liver disease.