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Macrophage Regnase-1 Deletion Deteriorates Liver Ischemia/Reperfusion Injury Through Regulation of Macrophage
Ai Xiaoming1,2, Jia Wenbo1, Wang Jinyi1
1Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China.
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.
Insights
Regnase-1 (MCPIP) regulates macrophage polarization during liver ischemia/reperfusion (I/R) injury. Targeting MCPIP may offer a novel therapeutic strategy for liver I/R.
Area of Science:
- Immunology
- Hepatology
- Inflammation Research
Background:
- Regnase-1 (MCPIP) is an anti-inflammatory agent with an unclear role in liver ischemia/reperfusion (I/R) injury.
- Macrophages exhibit dynamic polarization, significantly impacting inflammatory responses in liver I/R.
- This study investigates regnase-1's involvement in liver I/R by examining its effect on macrophage polarization.
Purpose of the Study:
- To determine if regnase-1 influences liver I/R injury.
- To elucidate the mechanism by which regnase-1 modulates macrophage polarization in the context of liver I/R.
Main Methods:
- Established a liver I/R model in C57BL/6 mice, including sham, I/R, and clodronate-treated groups.
- Utilized primary hepatocytes for *in vitro* hypoxia/reoxygenation (H/R) modeling.
- Investigated MCPIP knockdown in bone marrow-derived macrophages (BMDM) and their subsequent polarization response to H/R supernatants.
- Employed histopathology, immunostaining, double immunofluorescence, RT-PCR, Western blot, and flow cytometry for analysis.
Main Results:
- Liver function and injury scores deteriorated post-I/R but improved with clodronate treatment.
- Regnase-1 expression increased in I/R livers and localized to macrophages.
- MCPIP knockdown in BMDM exacerbated liver injury and promoted M1 polarization.
- MCPIP deficiency in BMDM led to a higher M1/M2 ratio upon H/R stimulation.
- MCPIP influences macrophage polarization via NF-κB, C/EBPβ, and PPARγ signaling pathways.
Conclusions:
- Regnase-1 plays a crucial role in liver I/R injury by regulating macrophage polarization.
- MCPIP's modulation of macrophage polarization presents a potential therapeutic target for liver I/R.
- Understanding MCPIP's role in macrophage polarization offers insights into mitigating liver I/R damage.
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