Nuclear Receptor Small Heterodimer Partner (SHP) Regulates Hepatic Macrophage Differentiation Through Posttranscriptional Regulation of PPARg

Abstract

BackgroundHepatic macrophages, consisting of resident Kupffer cells and infiltrating monocyte‐derived macrophages (MDMs), are central players of innate immunity involved in hepatic inflammation. The significance of macrophage differentiation towards M1‐like pro‐inflammatory or M2‐like anti‐inflammatory phenotypes in liver inflammatory diseases is widely appreciated; however, the mechanisms controlling this process are not well understood. SHP is a negative regulator of inflammatory signaling. We recently found that Shpis downregulated in M1 and upregulated in M2‐like macrophages, linking Shp to the regulation of macrophage differentiation. In this study, we uncovered a novel regulatory network consisting of Shp, miR‐34a, and peroxisome proliferator–activated receptor gamma (Pparg) in hepatic macrophage differentiation.MethodsWe generated transgenic mice by knocking out Shp from the cells of myeloid origin using Lyz2‐Cre (Shp‐MKO represents Shpflox/flox; Lyz2‐Cre positive). The littermates Lyz2‐Cre negative mice were used as wild‐type controls (WT represents Shpflox/flox; Lyz2‐Cre negative). The in vitro M1 and M2 macrophage differentiation was conducted using primary hepatic macrophages isolated from WT and Shp‐MKO mice and RAW 264.7 mouse macrophage cell line overexpressing Shp(RAW‐Shp). Lipopolysaccharide (LPS) was injected into WT and Shp‐MKO mice to induce acute liver inflammation. Hepatic non‐parenchymal cells were isolated by liver perfusion and subjected to Flow Cytometry to determine immune cell heterogeneity. Hepatic infiltrating CD11b+ MDMs were isolated using microbeads followed by determination of inflammatory gene expression by qPCR.ResultsThe in vitro macrophage differentiation study showed that Shp deletion promoted M1‐like but interfered M2‐like macrophage polarization. In contrast, Shp‐overexpression inhibited M1‐like polarization of RAW‐Shp cells. Flow cytometry analysis revealed a significant increase in the infiltration of CD11b+ MDMs into the liver of Shp‐MKO in comparison to WT after LPS injection. Additionally, the percentage of pro‐inflammatory CD11b+Ly6c+MDMs population doubled in Shp‐MKO liver after LPS treatment. Accordingly, LPS injection significantly induced hepatic Tnfa and Ccl2expressions in Shp‐MKO, which supports the overall increase in hepatic infiltration of pro‐inflammatory macrophages observed in Shp‐MKO. On the molecular level, SHP overexpression significantly induced Pparg, the master regulator of M2‐like macrophage polarization. To explore the molecular basis of Shp regulation of Pparg, we performed small RNA sequencing of Shp‐knockout liver. A total of 53 different microRNAs (miRNAs) were upregulated in Shp‐knockout liver. Using miRNA target prediction tool, we found that 5 of these upregulated miRNAs (miR‐34a, mir‐27a, miR‐130b, miR‐673, miR‐540) can target Pparg‐3´‐UTR. We confirmed that miR‐34a‐5p, the negative regulator of Pparg, was increased in Shp deficient macrophages but decreased in Shpoverexpressing macrophages.ConclusionOur study revealed a previously unknown role of SHP in macrophage differentiation. We found that Shp promotes the anti‐inflammatory macrophage differentiation by posttranscriptional regulation of Pparg through inhibition of miR‐34a. Targeting SHP may be useful for developing effective treatment for liver inflammatory diseases.