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

Hepatic 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.We 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.The 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.Our 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.