The Preliminary Research On The Role And Mechanism Of Histone 2A Deubuiquitinase MYSM1 In Macrophage

BACKGROUND Macrophages were located in all tissues and shown great functional diversity.They had roles in development,homeostasis,tissue repair and immunity.The innate immune response was the first line of defensing against infection.Macrophages were located throughout the body tissues,acting as professional phagocytic cells which expressed a multitude of receptors on their surfaces.During and following phagocytosis,PRRs recognized signals associated with invading pathogens and foreign substances.They ingested and processed foreign materials and formed antigen-MHCII complex to present foreign antigen in response to inflammatory signals.Meanwhile,activated macrophages produced reactive oxygen and nitrogen intermediates,including NO and superoxide which contributed to kill invading organisms.Macrophages expressed a variety of PPRs,which were vital for host to detect the pathogen.The Toll-like receptors located on the cells surface or in the cytoplasm of cells were the most important PPRs,sensing the microbial products.When stimulated,these receptors activated signal cascades to initiate the inflammatory response.TLR signal activated transcriptional mechanisms that leaded to release cytokines,chemokines and interferon.Following infection,the first-responder macrophages usually exhibited an inflammatory phenotype and secreted pro-inflammatory mediators such as TNF and IL-6,which participated in the activation of various anti-infection mechanisms.They were highly heterogeneous and palstic so that they could rapidly change their function in response to microenvironmental signals.Classically activated macrophages mediated the protection of the host from a variety of bacteria and viruses and had roles in antitumour immunity.Alternatively activated macrophages had anti-inflammatory function and regulated wound healing.Epigenetic modifications were hereditable transcriptional regulation of gene expression without altering genome DNA sequence and played essential roles in a variety of physilogical and pathological processes,including DNA methylation,histone covalent modification and non-coding RNA.Epigenetic modifications were increasingly shown to be involved in the flexible regulation of TLR dependent innate immune responses.Epigenetic modifications such as histone modifications became increasingly involved in the transcriptional regulation of multiple TLR-induced genes.Histone lysine ubiquitination was an reversible form of histone modifications.In particular,MYSM1 mediated H2 A deubiquitination associated with transcriptional activation.Although published studies found that Mysm1 deficiency resulted in obvious developmental defects especially in immune system,indicating it was a key epigenetic modificator.But the function of MYSM1 especially in immune responses and inflammation remained unclear.OBJECTIVES To clarify the potential roles of MYSM1 in the development and differentiation of myeloid lineage.To study the function and mechanism of MYSM1 in macrophage in innate immunity.METHODS To better characterize the function of MYSM1,we generated Mysm1-deficient mice by crossing Mysm1 floxed mice with Lyz2-Cre transgenic mice,leading to the absence of MYSM1 in myeloid cells specifically.We detected the deficiency in myeloid cell sorted from bone marrow after conditionally deleting MYSM1 by q PCR.Furthermore,we analyzed the development and differentiation of myeloid cell lineages in bone marrow and spleen by FACS.Granulocyte-macrophage colony stimulating factor(GM-CSF)were able to induce bone marrow cells differentiation towards macrophage in vitro.Phagocytosis-promoting activity of BMDM incubated with zymosan A bioparticles was evaluated by a phagocytosis assay followed by microscopic observation.The antigen presenting function of BMDM were detected through FACS.BMDM were stimulated with different TLRs agonists,then analyzed by quantitative real-time PCR for the transcripts of proinflammatory cytokines and chemokines,enzyme linked immunosorbent assay for their secreting level.We next investigated that whether Mysm1 deficiency affected neutrophil recruitment in vivo.WT and c KO mice were injected via intraperitoneal route with carrier buffer or LPS for 4h,then cells harvested from peritoneal cavity were analyzed by FACS.We further investigated the effect of Mysm1 on inflammatory disease comparing the severity of 3% DSS-induced IBD in Mysm1-deficinecy with control mice.To investigate how MYSM1 might control the proximal TLR signaling cascades in macrophage,we next selected several well-known regulators of TLR signals and analyzed their expression in the levels of transcripts in Mysm1-silenced macrophages.BMDMs either uninfected or infected with with LPS or poly(I:C)for indicated time points were analyzed by immunoblot analysis for MYSM1 in the cytoplasm and nucleus,PPAR? and the MAPK and NF-kappa B signal molecular in lysates of WT and Mysm1-silenced macrophages.RESULTS FACS revealed no significant defects in the percentages of myeloid lineage cells compared with control in bone marrow and spleen.Efficient deletion of Mysm1 was verified at the level of both m RNA and protein in macrophages.FACS showed that in vitro differentiated bone-marrow derived macrophage(BMDMs)from conditional knockout mice exhibited comparable expression of macrophage surface markers F4/80 and CD11b as those from wild-type mice.Therefore silencing of Mysm1 had no effect on macrophage differentiation.We observed that silencing of Mysm1 increased TLR3 and TLR7-triggered the induction of the pro-inflammatory cytokines such as tumor necrosis factor-alpha,interleukin-1?,interleukin-6 and the type I interferon upon stimulation with defined agonists for different TLR pathways.Conditional silencing of Mysm1 resulted in elevated production of chemokines upon stimulation with LPS and poly(I:C)in macrophages.In the peritonitis model,Mysm1 deficiency resulted in an increase in the percentage and number of neutrophils in the peritoneal exudates without affecting macrophages,which indicated that MYSM1 specifically suppressed neutrophil recruitment in vivo.Mice were more sensitive to DSS-induced colitis than their counterparts,as indicated by a sharper and continued loss of body weight,exaggerated shorten and enlargement of colon,as well as amplified inflammation in terms of extensive infiltration of mononuclear cells.Together these datas suggested that Mysm1-silenced mice were more susceptible to DSS induction.We detected cytoplasmic and nuclear fractions of BMDMs upon stimulation with LPS or poly(I:C)and found MYSM1 accumulated rapidly in both cytoplasm and nucleus of macrophages.Wild-type and Mysm1 deficiency BMDMs exhibited comparable levels of transcripts for those signaling inhibitory regulators except for PPARg.PPARg expression was repressed significantly in Mysm1-silenced macrophages upon LPS and poly(I:C)stimulation as compared to WT macrophages.Furthermore,stimulated with different TLR agonist stimulation,PPARg was always downregulated.In consistent with the transcription,PPAR? was downregulated in the level of translation in Mysm1 deficiency macrophage.While overexpression of MYSM1 potently enhanced the expression of PPAR? relative to that in mock-transfected cells.Next we observed that enhanced and continuous phosphorylation of signal molecular in mitogen-activated protein kinase pathway for ERK and JNK in LPS or poly(I:C)-triggered Mysm1-silenced macrophages.CONCLUSION The H2AK119 deubiquitinase MYSM1 suppressed cytokines and chemokines production in Toll-like receptor(TLR)-triggered macrophages.We found that Mysm1 restricted neutrophil recruitment by controlling the production of macrophage-derived chemokines.MYSM1 deficient mice exhibited a hyper inflammatory response,susceptibility to DSS induced experimental bowel disease.Our datas also indicated that the localization and function of MYSM1 in the nucleus and cytoplasm was subject to strict regulation.MYSM1 suppressed cytokine and chemokine production via promoting PPAR?-mediated the inactivation of the major signaling components of MAPK signal,providing mechanistic insight into epigenetic modulation of immune responses and inflammation.