Androgen Receptor And PPARγ Signaling Pathways Regulates Proinflammatory Activation Of Human Macrophage

Androgen receptors(androgen receptor,AR)is a nuclear receptor.AR play an important role to control the development and function of both male and female reproductive systems.Increasing studies have showed that AR plays distinct roles in individual immune cells,and may modulate the development and function of the immune system.In addition,some studies have shown that AR might enhance the inflammatory response within tissues damage.Clinical evidence shows that hepatitis,liver cirrhosis,liver failure and hepatocellular carcinoma(HCC)caused by liver inflammation in patients with chronic hepatitis b(CHB)are mainly male.Foreign studies have confirmed that hepatitis B virus(HBV)interacts with androgens and AR.Our previous research found that there was an increase in androgen pulses during the activation of chronic hepatitis B,and the CAG repeat fragment polymorphism of AR-related exons is significantly associated with Acute-on-chronic liver failure(ACLF)in patients with chronic hepatitis B,which suggests AR-mediated signaling pathway is likely to promote liver damage in chronic hepatitis B.Macrophages are derived from monocytes and play a major regulatory role in the inflammatory response.Previous reports have showed macrophages play an important role in regulating inflammatory response and tissue regeneration during wound healing and traumatic hemorrhagic shock.Macrophages(including Kupffer cells and mononuclear derived macrophages)play a crucial regulatory role in the process of liver injury,which can not only promote the inflammatory response to aggravate liverinjury,but also inhibit the inflammatory response and promote liver tissue repair.Studies of mouse skin injury models have shown AR expression of macrophages can promote the inflammatory response at the site of injury and delay the repair process of skin injury,which suggested that AR may be involved in the regulation of liver injury through macrophages.Macrophages express HLA Class II molecules and participate in immune responses as antigen-presenting cells.Studies have showed that HLA-Ⅱ genes have gender-specific parental origin effects during transmission to their offspring,indicating the presence of gender-specific genomic imprinting.Androgen signaling can promote the expression of HLA Class II molecules,and the major histocompatibility complex(MHC)genotype can also affect male serum testosterone levels.We previously analyzed the HLA-Ⅱ gene region and found that there is an androgen response element(ARE)in the HLA-Ⅱ gene promoter region,suggesting that there is a basis for differential androgen transcription regulation in the HLA-Ⅱ gene region.Peroxisome proliferator activated receptor gamma(PPAR-γ)is a member of the ligand-dependent activated nuclear receptor superfamily.Studies have shown that PPAR-γ is a key transcription factor that controls the activation of macrophages.On one hand,it can inhibit the intracellular signal transduction pathway of NF-κB to inhibit the inflammatory response.On the other hand,it can promote the differentiation of macrophages into tissue-repair macrophages through the signaling axis of IL-4 / IL-13 /STAT6/PPAR-γ.Notably,AR has been found to inhibit PPAR expression in human primary T cells and human prostate cancer cell lines,thereby blocking its mediated intracellular signaling pathway.Based on the above results,macrophages may inhibit the expression of PPAR-γ through AR-mediated signaling,thereby promoting the inflammatory response.This study focuses on the molecular mechanism of human monocyte derived macrophage activation and HLA Class II molecular expression regulated by AR.To isolate monocytes from human peripheral blood of healthy people,and the separation purity was measured.Monocytes differentiate into macrophages stimulated by Macrophage colony-stimulating factor(M-CSF).The expression patterns of HLA Class II molecules,AR,PPAR-γ,and estrogen receptors in monocytes and macrophages were analyzed.Use IFN-γ to stimulate macrophages to promote inflammatory differentiation,and use ASC-J9,GW9662 to block the expression ofAR and PPAR-γ in macrophages,or use DHT to stimulate macrophages.Analysis of HLA Class II molecules,AR,PPAR-γ expression and the distribution of AR and PPAR-γ in macrophages.The expression of AR,PPAR-γ and inflammatory factors at the transcriptional level of macrophages was measured.Our results are as follows:1.Isolate and purify human monocytes.The purity of the isolated monocytes is basically greater than 90%,and the highest can reach 93%.Analysis of the expression of HLA Class II molecules,AR,and estrogen receptors in monocytes revealed that their HLA Class II molecules were low-expressed and did not express AR and estrogen receptors ERα and ERβ.M-CSF induced monocytes to differentiate into macrophages.It was found that during the differentiation of monocytes into macrophages,PPAR-γ was not significantly expressed,while HLA Class II molecules continued to be up-regulated,and AR expression was gradually up-regulated.Analyze variable spliceosome transcription showed that AR mRNA in macrophages was mainly full-length mRNA.In addition,the estrogen receptors ERα and ERβ were also significantly up-regulated,and the pattern of ERα up-regulation was similar to that of AR,which up-regulated the expression of whole cells.ERβ up-regulation was only observed in some cells,and expression of ERβ + and ERβ-two groups in monocyte derived macrophage were clearly visible.2.After human monocytes are induced to differentiate into macrophages,IFN-γstimulates macrophages to induce inflammatory differentiation and ASC-J9 blocks AR.It was found that the monocyte derived macrophage without IFN-γ stimulation,after blocking AR with ASC-J9,the expression of PPAR-γ was up-regulated,and there was a dose-response relationship between the up-regulation of the expression and the blocker.In monocyte derived macrophage without IFN-γ stimulation,AR is distributed in the cytoplasm and nucleus.After IFN-γ stimulation induced pro-inflammatory differentiation of macrophages,AR entered the nucleus in large quantities,and PPAR-γ mRNA was significantly down-regulated compared with the unstimulated group.IFN-γ stimulated macrophages were given ASC-J9 at the same time to block AR,and AR entering the nucleus was inhibited.PPAR-γ mRNA was significantly up-regulated compared to the unstimulated group and the IFN-γ alone stimulation group,and the expression of PPAR-γ protein was also higher than the unstimulated group and the IFN-γ alone stimulation group.3.IFN-γ stimulates monocyte derived macrophage to induce pro-inflammatory differentiation,block the expression of AR and PPAR-γ with ASC-J9 and GW9662 respectively.The analysis revealed that macrophages without IFN-γ stimulation had low-expression of HLA-DP,while both HLA-DR and HLA-DQ were highly expressed.After stimulation of IFN-γ to induce pro-inflammatory differentiation of macrophages,HLA-DR,HLA-DP,and HLA-DQ expressions were all significantly up-regulated.However,when IFN-γ stimulated macrophages and blocked AR at the same time,the up-regulation of three types of HLA Class II molecules was inhibited,indicating that AR-mediated signaling pathways are involved in HLA-Ⅱ molecular up-regulation in macrophages by IFN-γ stimulation.IFN-γ stimulated macrophage and blocked both AR and PPAR-γ at the same time,and the up-regulation of three HLA-Ⅱ class molecules was not restored,indicating that AR upregulates the expression of HLA Class II molecules in proinflammatory inflammatory macrophages stimulated by IFN-γ is not achieved by inhibiting PPAR-γ-mediated signaling pathways.4.After IFN-γ stimulation induces pro-inflammatory differentiation of macrophages,the macrophage pro-inflammatory cytokines TNF-α,IL-6,IL-12p35,CXCL10,CXCL11 mRNA levels are up-regulated.While IFN-γ stimulated macrophages to block their AR at the same time,the levels of TNF-α,IL-6,CXCL10,and CXCL11 mRNA in macrophages were down-regulated compared with the IFN-γalone stimulation group,indicating that AR-mediated signaling pathways are involved in up-regulation of pro-inflammatory cytokines on transcription levels in IFN-γ-stimulated macrophages.IFN-γ stimulated macrophages simultaneously blocked expression of AR and PPAR-γ,TNF-α,IL-6,IL-12p35,CXCL10,CXCL11 mRNA levels were significantly up-regulated compared with IFN-γ stimulation and alone blocked AR group,indicating that AR promotes the up-regulation of proinflammatory cytokines by inhibiting PPAR-γ-mediated signaling pathways in IFN-γ-stimulated macrophages.IFN-γ and DHT together stimulate macrophages,it was found that the levels of other proinflammatory cytokine m RNAs except IL-6 did not increase significantly compared with the IFN-γ alone stimulation group,indicating that AR-mediated up-regulation of proinflammatory cytokine expression in IFN-γ-stimulated macrophages is independent of androgen signaling.