Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization

Ai Xiaoming; Jia Wenbo; Wang Jinyi; Wu Bin; Hu Chunyang; Chen Qi; Kong Lianbao

doi:10.3389/fphys.2020.582347

Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage Polarization

Front Physiol. 2020 Oct 29:11:582347. doi: 10.3389/fphys.2020.582347. eCollection 2020.

Authors

Ai Xiaoming^{1

2}, Jia Wenbo¹, Wang Jinyi¹, Wu Bin¹, Hu Chunyang¹, Chen Qi¹, Kong Lianbao¹

Affiliations

¹ Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
² Department of General Surgery, BenQ Medical Center, The Affiliated BenQ Hospital of Nanjing Medical University, Nanjing, China.

Abstract

Background: Regnase-1 (MCPIP) has been identified as an anti-inflammatory agent, but little is known about its influence on liver ischemia/reperfusion (I/R) injury. Macrophages can evolve biphasic responses and differentiate into remarkable polarizations, contributing greatly to the uncontrolled inflammatory cascades during liver I/R injury. Therefore, the aim of this study was to explore whether regnase-1 participated in liver I/R via manipulating macrophage polarization.

Materials and methods: C57BL/6 mice were randomly divided into five groups: Sham, I/R, Clodronate, Clo + BMDM, and Clo + LV MCPIP BMDM. A liver I/R model was established, and histopathological and immunostaining examinations were performed for the liver specimens; double immunofluorescence staining was used to localize MCPIP in the liver. Primary hepatocytes were isolated to simulate a hypoxia and reoxygenation (H/R) model in vitro. Bone marrow-derived macrophages (BMDM) were extracted and subjected to lentiviral transduction to knockdown MCPIP expression. BMDM with or without MCPIP deletion were exposed to H/R supernatants, and the polarized states were measured by flow cytometry. RT-PCR analysis and Western blot were also conducted.

Results: Compared to those in the Sham group, liver functions and Suzuki's scores were deteriorated in the I/R group, which were reversed in the Clodronate group. The increased expression of regnase-1 in the I/R group diminished with pretreatment of clodronate liposomes. Subsequent double immunofluorescence staining established the localization of regnase-1 in macrophages in the liver. The insulted lesions in the Clodronate group became progressively aggravated with adoptive transfer of BMDM in the Clo + BMDM group, and they were further exacerbated with the transfusion of BMDM with MCPIP knockdown in the Clo + LV MCPIP BMDM group. Gene expressions of M1 and M2 markers were detected by RT-PCR, suggesting that MCPIP knockdown tended to favor the M1 transformation. Subsequently, ex vivo flow cytometrical detection showed that, upon stimulation by H/R supernatants, LV-MCPIP BMDM posed a higher ratio of M1/M2 than BMDM. Finally, we found that MCPIP participated in macrophage M1/M2 polarization through the NF-κB, C/EBPβ, and PPARγ signaling pathways during liver I/R.

Conclusion: Our study confirms that regnase-1 plays a critical role in liver I/R via regulation of macrophage polarization and, thus, might offer a potential therapeutic target.

Keywords: ischemia/reperfusion injury; liver; macrophage; polarization; regnase-1 (MCPIP).