Scavenger receptor a is a major homeostatic regulator that restrains drug-induced liver injury

Abstract

Background and aim: Drug-induced liver injury occurs frequently and can be life threatening. Although drug-induced liver injury is mainly caused by the direct drug cytotoxicity, increasing evidence suggests that the interplay between hepatocytes and immune cells can define this pathogenic process. Here, we interrogate the role of the pattern recognition scavenger receptor A (SRA) for regulating hepatic inflammation and drug-induced liver injury.

Approach and results: Using acetaminophen (APAP) or halothane-induced liver injury models, we showed that SRA loss renders mice highly susceptible to drug hepatotoxicity, indicated by the increased mortality and liver pathology. Mechanistic studies revealed that APAP-induced liver injury exaggerated in the absence of SRA was associated with the decreased anti-inflammatory and prosurvival cytokine IL-10 concomitant with excessive hepatic inflammation. The similar correlation between SRA and IL-10 expression was also seen in human following APAP uptake. Bone marrow reconstitution and liposomal clodronate depletion studies established that the hepatoprotective activity of SRA mostly resized in the immune sentinel KCs. Furthermore, SRA-facilitated IL-10 production by KCs in response to injured hepatocytes mitigated activation of the Jun N-terminal kinase-mediated signaling pathway in hepatocytes. In addition, supplemental use of IL-10 with N -acetylcysteine, only approved treatment of APAP overdose, conferred mice improved protection from APAP-induced liver injury.

Conclusion: We identify a novel hepatocyte-extrinsic pathway governed by the immune receptor SRA that maintains liver homeostasis upon drug insult. Giving that drug (ie, APAP) overdose is the leading cause of acute liver failure, targeting this hepatoprotective SRA-IL-10 axis may provide new opportunities to optimize the current management of drug-induced liver injury.

FIGURE 1

Loss of scavenger receptor A (SRA) exacerbates drug-induced liver injury in mice. (A) wild type (WT) or SRA^−/− mice (n = 10) were challenged with acetaminophen (APAP) (600 mg/kg), followed by monitoring of mouse survival. (B) Mice (n = 7) were injected with APAP (400 mg/kg) and liver histopathological changes were analyzed by hematoxylin and eosin staining 18 hours after APAP challenge (Bar = 500 μm). (C) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was performed to assess hepatocyte death (Bar = 100 μm). (D) Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured at 18 hours after APAP challenge. (E) Levels of phosphorylated c-Jun N-terminal kinase (c-JNK) and cleaved poly (ADP-ribose) polymerase (PARP) in the liver tissues were examined by immunoblotting 6 hours after APAP administration. Data are representative of at least 6 independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001

FIGURE 2

Lack of scavenger receptor A (SRA) amplifies hepatic inflammation associated with drug-induced liver injury. Wild type (WT) or SRA^−/− mice were treated with acetaminophen (APAP) (400 mg/kg). (A) Frequencies of neutrophils in the liver were examined by flow cytometry. Expression of TNF-α, interferon-γ (IFN-γ), interleukin (IL)-17A, or IL-10 in the liver tissues (B) and levels of IFN-γ or IL-10 in the serum (C) were determined by ELISA after APAP challenge. (D) RNA was extracted from hepatic nonparenchymal cells (NPCs) derived from APAP-treated WT or SRA^−/− mice. RNA-sequencing was performed to examine the global gene changes followed by REACTOME pathway analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 3

Scavenger receptor A (SRA) protects mice from halothane-induced liver injury. Wild type (WT) or SRA^−/− mice (n=5) received halothane (30 mmol/kg in 2 mL olive oil) or olive oil as control. Serum alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B) liver infiltration by neutrophils (C) were determined 20 hours later. Data are representative of at least 3 independent experiments. *p < 0.05; **p < 0.01.

FIGURE 4

IL-10 is essential for hepatoprotective activity of scavenger receptor A (SRA) in acetaminophen (APAP)-induced liver injury (AILI). Wild type (WT), SRA^−/−, IL-10^−/−, or SRA^−/−IL-10^−/− mice (n=5) were challenged with APAP, followed by analyses of liver histopathological changes using hematoxylin and eosin staining (A). Serum alanine aminotransferase (ALT) (B) and liver-infiltrating neutrophils (C) were determined. WT or SRA^−/− mice (n=5) received IL-10 (200 ng, i.p.) or PBS (vehicle) 0.5 hours before or 4 hours after APAP challenge. Serum alanine aminotransferase (ALT) (D) and neutrophil infiltration (E) were assessed. WT or SRA^−/− mice (n=5) received oxidized low-density lipoprotein (oxLDL) (100 μg, i.v.) or PBS (vehicle) 5 hours before APAP challenge. Serum ALT (F) and neutrophil infiltration (G) were assessed. Data are representative of at least 3 independent experiments. Abbreviation: NS, not significant. *p< 0.05; **p< 0.01; ***p< 0.001; ****p<0.0001.

FIGURE 5

Scavenger receptor A (SRA)-conferred hepatoprotective activity in acetaminophen (APAP)-induced liver injury (AILI) is dependent on hematopoietic cells and KCs. Bone marrow (BM) cells isolated from donor WT or SRA^−/− mice were transferred into the recipient SRA^−/− mice (n=5). Twelve weeks after BM reconstitution, mice were subjected to APAP challenge. Liver pathology (A), serum ALT (B), and liver-infiltrating neutrophils (C) were analyzed. Wild type (WT). or SRA^−/− mice were depleted of KCs by injection of liposomal clodronate (LC) prior to APAP challenge, followed by analyses of serum ALT (D) and neutrophils in the liver (E). Data are representative of at least 3 independent experiments. Abbreviation: NS, not significant. *p<0.05; **p<0.01; ***p < 0.001; ****p<0.0001.

FIGURE 6

Hepatoprotective activity of scavenger receptor A (SRA) involves IL-10 production by KCs. (A) Wild type (WT) mice were injected with acetaminophen (APAP), followed by analysis of IL-10 expression in KCs using intracellular staining and flow cytometry. (B) WT or SRA^−/− mice were depleted of KCs by injection of liposomal clodronate (LC) 48 hours before APAP challenge. Transcription of il10 in the liver tissue was assayed by realtime PCR. (C) WT and SRA^−/− bone marrow-derived macrophages (BMMϕ) were stimulated with APAP (5 mmol/l)-injured hepatocytes for 24 hours. Levels of IL-10 in the culture media was examined by ELISA. (D) WT or SRA^−/− BMMϕ were co-cultured with APAP-injured FL83B cells at 1:2 ratio for 24 or 48 hours. The production of IL-10 and IL-12p40 in the culture media was determined by ELISA. (E) WT or SRA^−/− BMMϕ were stimulated with APAP-injured FL83B cells for 72 hours to prepare BMMϕ conditional media (CM). FL83B cells were treated with APAP for 2 hours followed by extensive washing. FL83B cells were then cultured in the presence or absence of BMMϕ CM for additional 18 hours. Jun N-terminal kinase (JNK) phosphorylation and poly (ADP-ribose) polymerase (PARP) cleavage were examined by immunoblotting. (F) APAP-stressed FL83B cells were cultured in the presence of WT BMMϕ CM prepared as described above with or without addition of IL-10-neutralizing antibodies. Immunoblotting was performed to examine JNK phosphorylation or PARP cleavage. (G) Mouse primary hepatocytes were treated with APAP in the presence or absence of IL-10 (20 ng/mL) for overnight, JNK activation was examined by immunoblotting. (H) Correlation between SRA expression and IL-10 levels in human blood samples after low-dose APAP exposure from NCBI data set (n=60, GSE30418). Data are representative of at least 3 independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001. WT indicate Wild type.

FIGURE 7

Supplementing IL-10 in combination with N-acetylcysteine (NAC) reduces acetaminophen (APAP)-induced liver injury (AILI). C57BL/6 mice (n=5) received APAP to induce liver injury. Two hours after APAP injection, mice were treated with a single dose of NAC (300 mg/kg). IL-10 treatment (400 ng) was started from 2 hours post-APAP injection for 3 doses at 4-hour intervals. Liver pathology (A), alanine aminotransferase (ALT) or aspartate aminotransferase (AST) (B), and neutrophil in the liver (C) were analyzed. (D) Six hours after APAP injection, C57BL/6 mice (n=5) were treated with NAC or combination regimen. (E) A proposed model for scavenger receptor A (SRA) activity in drug-induced liver injury. SRA regulates KC response to drug-stressed hepatocytes by facilitating the production of anti-inflammatory and prosurvival cytokine IL-10, which can directly mitigate the injury of hepatocytes in a trans-acting manner and also antagonize proinflammatory response or cytotoxic effects mediated by neutrophils or cytokines such as TNF-α, interferon-γ (IFN-γ), or IL-17. A defect in the hepatic SRA-IL-10 pathway results in exacerbation of pathogenic inflammation and hepatotoxicity. Data are representative of at least 3 independent experiments. Abbreviation: NS, not significant. *p< 0.05; **p< 0.01; ***p < 0.001; ****p < 0.0001.