The Role And Mechanism Of A2aR In Regulating Macrophage M1 Polarization In The Pathogenesis Of Psoriasis

Background:Psoriasis is a common chronic,recurrent,inflammatory,systemic disease,that is maintained by the interaction of innate immune cells,acquired immune cells and non-immune cells.In recent years,studies have confirmed that the IL-23/Th17 immune axis plays a central role in the immune response of psoriasis,and the biological preparations developed for key cytokines such as IL-23 and IL17A in this link are clinically efficient,but these cytokines are downstream of the immune pathway and cannot control the onset of psoriasis from the source.Therefore,once the biological agent is stopped,psoriasis will recur or even worsen.Macrophages,as one of the important players in the natural immune system,also play a key role in inflammatory skin diseases.Macrophages exhibit significant plasticity and can polarize into classically activated pro-inflammatory M1 and alternately activated anti-inflammatory M2.An imbalance of M1 and M2 leads to inflammation of the skin.Recent studies have revealed that macrophages in psoriasis patients prefer M1 polarization to M2 polarization,but it is unclear how M1 polarization mediates the psoriasis immune response and its regulatory mechanism.Therefore,it is important to clarify the importance of M1 polarization in the pathogenesis of psoriasis,and to find the key molecules of M1 polarization,and to provide new therapeutic methods for controlling the source of the disease from the root and solving the problems of psoriasis treatment.Chapter I The role and mechanism of M1 polarization in the pathogenesis of psoriasisObjectives:To clarify the pathogenesis of M1 polarization in psoriasis and to identify the key molecules of M1 polarization.Methods:1.The skin lesions and paralesion tissues of psoriasis patients are collected,and the infiltration of macrophages and M1 in the skin lesions and adjacent lesions of psoriasis patients is detected by Real Time Polymerase Chain Reaction(RT-PCR),flow cytometry and immunohistochemistry.2.Imiquimod(IMQ)is used to construct the mouse model of psoriasis for collecting the skin lesion tissue of psoriasis mice and the skin tissue of control mice,and the infiltration of macrophages and M1 in the skin of psoriatic-like dermatitis mice and control mice is detected by RT-PCR,flow cytometry and immunohistochemistry.3.The use of macrophage scavenger clodronate liposomal Clodronate Liposomes is to intervene in the IMQ-induced psoriatic-like dermatitis mice model process.Psoriasis Area and Severity Index(PASI)score,HE staining,immunohistochemistry and flow cytometry and other methods are used to evaluate psoriasis inflammatory indexes,proliferation indexes,macrophage and M1 changes.4.Bone marrow-derived macrophages(BMDM)are extracted from mice to induce M1 polarization in vitro.In this study,BMDM that induced M1 polarization is named BMDM1,however,BMDM that do not induce M1 polarization is named BMDMO.Transcriptomic sequencing analyzed the differential genes between the BMDMO group and the BMDM1 group to explore the key molecules that regulate M1 polarization.RT-PCR,immunohistochemical staining and flow cytometry are used to verify it.Results:macrophages1.The infiltration of and M1 in the lesion tissues of psoriasis patients is significantly higher than that in the paralesion tissues,and the infiltration of macrophages is positively correlated with the PASI score of psoriasis patients.Psoriasis patients have significantly higher mRNA levels of macrophages and M1-associated inflammatory factors in lesion tissues than paralesions.2.The infiltration of macrophages and M1 and the mRNA levels of related inflammatory factors in the skin lesions of psoriatic-like dermatitis mice are significantly higher than those in the skin tissues of control mice.3.The macrophage scavenger clodronate liposomes improve inflammation and epidermal proliferation in psoriatic-like dermatitis mice,reducing macrophage and M1 infiltration in mouse skin lesions and spleen.4.Transcriptomic sequencing find that the expression levels of 3817 genes in the BMDM1 group are upregulated,compared with the BMDMO group,and the RNA level of Adenosine A2a receptor(A2aR)in BMDM1 is significantly higher than that of BMDMO.In vitro experimental,RTPCR verification find that the mRNA expression level of A2aR in BMDM1 is significantly higher than that of BMDMO.Conclusion:1.Macrophage and M1 infiltration are increased in psoriasis skin lesions,And the removal of macrophages can improve psoriasis-like inflammation.2.A2aR may be a key molecule in regulating M1 polarization.Chapter Ⅱ The role and mechanism of A2aR regulation of M1 polarization in the pathogenesis of psoriasisObjectives:To clarify the role and mechanism of A2aR regulation of M1 polarization in the pathogenesis of psoriasis,and to provide new therapeutic methods for controlling the source of the disease from the root and solving the problems of psoriasis treatment.Methods:1.Immunohistochemistry and flow cytometry are used to detect A2aR expression on macrophages and M1 in skin lesions and paralesion tissues in psoriasis patients.2.The psoriatic-like dermatitis mice models induced by IMQ and Recombinant Mouse IL-23 Protein(rmIL-23)are used to explore the effect of the A2aR agonist CGS 21680 HC1 on the psoriasis mouse models,and the A2aR knockout mice A2a-/-are induced psoriatic-like dermatitis mice to evaluate inflammation indexes,proliferation indexes,macrophage and M1 changes in psoriatic-like dermatitis mice by PASI score,HE staining,immunohistochemistry and flow cytometry.3.BMDM is extracted in vitro and induced to polarize to BMDM1,the effect of A2aR agonist CGS 21680 HCl on BMDM1 polarization is detected by flow cytometry.Transcriptomics sequencing analyzed the signaling pathway of A2aR regulating BMDM1 polarization and its downstream molecules.RT-PCR,ELISA and Western Blot are used to verify the signaling pathway and expression of downstream molecules of signaling pathway in BMDM1 treated with the A2aR agonist CGS 21680 HCl and A2aR antagonist CPI-444.Flow cytometry is used to detect the proportion of BMDM 1 polarization after knockdowning the downstream molecules by si-RNA.4.Single cell sequencing technology is used to analyze the distribution of chemokines to chemotactic T cells in macrophage population in the lesion tissues of psoriasis patients.Flow cytometry is used to analyze the effect of A2aR regulation of M1 polarization on Naive CD4+T cell differentiation and inflammatory response of keratinocyte(KC).Results:1.The amount of A2aR expressed on macrophages in skin lesions in psoriasis patients is higher than that in paralesion tissues,and is significantly positively correlated with PASI score.The amount of A2aR expressed by M1 in psoriasis lesions is higher than in paralesion tissue.2.In vivo experiments,we find that the A2aR agonist CGS 21680 HCl can improve IMQ and rmIL-23-induced psoriasis-like inflammation in mice,reduce the amount of macrophage and M1 in skin lesions and spleens.However,the A2a-/-gene mice show a more severe psoriasis inflammatory response induced by IMQ,and have the more infiltration of macrophages and M1 in skin lesions.3.We extract mouse primary macrophage BMDM in vitro,and successfully induce M1 polarization.A2aR agonist CGS 21680 HC1 can inhibit BMDM1 polarization,transcriptomic sequencing analysis reveals that the A2aR agonist CGS 21680 HCl regulates M1 polarization through the nuclear factor-activated B cell κ-kappa B(NF-κB)pathway.Western Blot experiments show that the A2aR agonist CGS 21680 HCl inhibits the phosphorylation of protein p65 in NF-κB pathway protein p65 in BMDM1,and the A2aR antagonist CPI-444 can promote the phosphorylation of protein p65 in NF-κB pathway in BMDM1.A2aR agonist CGS 21680 HCl can inhibit M1 polarization of BMDM,however,A2aR antagonist CPI-444 can promote it.It is proved that A2aR regulates M1 polarization through the NF-κB pathway.4.In the signaling pathway study,we use the A2aR agonist CGS 21680 HCl to inhibit BMDM1 polarization,transcriptomics sequencing analysis show that the changes in KRT16 are the most obvious among the top 20 genes with the most obvious downregulation,and in vitro experiments,we find that the mRNA level and protein level of KRT16 are upregulated in BMDM1,then are down-regulated in BMDM1 treated with A2aR agonist.And knock down the NF-KB-p65 gene of BMDM1 by si-RNA and inhibit the NF-κB pathway by QNZ,the expression of KRT16 mRNA and protein level in BMDM1 are down-regulated.Further knock down the the KRT16 gene of in BMDM1,the proportion of BMDM1 polarization is reduced.It is proved that A2aR inhibited the expression of KRT16 to inhibit BMDM1 polarization through the NF-κB signaling pathway.5.Regulation of chemokines:Transcriptomics sequencing analysis find that the changes of CXCL10/11 are more obvious in the first 20 genes with the most obvious downregulation of BMDM1 treated with the A2aR agonist CGS 21680 HCl,and single cell sequencing analysis find that the expression of CXCL10/11 on dendritic cells(DC)/macrophages are significantly higher than that of other types of cells in the skin lesions of psoriasis patients.In vitro experiments show that the mRNA level and protein level expression of CXCL10/11 are down-regulated in BMDM1 treated with the A2aR agonist CGS 21680 HCl.However,the mRNA level and protein level expression of CXCL10/11 are upregulated in BMDM1 treated with the A2aR antagonist CPI-444.And the mRNA level and protein level expression of CXCL10/11 are down-regulated after knocking down the NF-KB-p65 gene of BMDM1 by si-RNA.It is demonstrated that A2aR inhibited CXCL10/11 expression in BMDM1 through the NF-κB signaling pathway.6.Regulation of T cell homing and differentiation:CXCL10/11 are expressed highly in M1 in psoriasis lesions and mainly chemotaxis T cells to peripheral tissues.CD4+T cell infiltration is increased in the skin lesions of mice with psoriasis-like dermatitis.The A2aR agonist CGS 21680 HCl could reduce the infiltration of CD4+T cells.In vitro experiments,we find that BMDM1 supernatant promotes Naive CD4+T cell differentiating to Th1/17,and the A2aR agonist CGS 21680 HCl can inhibit BMDM1 supernatant-induced Th1/17 differentiation.In vitro coculture experiments,we find that BMDM1 promotes Naive CD4+T cell differentiating to Th1/17,and the A2aR agonist CGS 21680 HCl can inhibit BMDM1-induced Th1/17 differentiation.7.Regulation of inflammatory factor expression in KC:In vitro experiments,we find that the supernatant of BMDM1 induces the inflammatory response of KC,and the A2aR agonist CGS 21680 HCl can inhibit the expression of inflammatory factors of KC induced by BMDM1.Conclusion:1.A2aR inhibits the expression of KRT16 through the NF-κB signaling pathway,thereby inhibiting M1 polarization.2.A2aR regulates the CD4+T cell homing,Th1/17 differentiation and KC inflammatory factor expression through M1 polarization to participate in the whole process of psoriasis occurrence and development.Figures 33,Tables 24,References 120...