Inflammation is the biological stress response to harmful stimuli such as foreign pathogens as well as damaged cells or tissues,which is the protective procedure of host to eliminate harmful stimulus and initiate tissue healing.In terms of its characteristics and patterns,inflammation can be divided into acute inflammation and chronic inflammation.The former occurs in the early phase of tissue damage or pathogen infection.The latter refers to a kind of continuing and low-degree inflammatory responses at the inflammation site.A variety of immune cells participate in the development of chronic inflammation.It can follow an acute form of inflammation or be a prolonged low-grade form that is characterized by simultaneous destruction and repair of the tissue.Inflammation is the common pathological process of a multitude of infectious and autoimmune diseases,which may determine the deterioration or improvement of these diseases.Macrophages and dendritic cells are two main kinds of innate immune cells to initiate innate immune responses and mediate acute inflammation and chronic inflammation through secreting various cytokines.Underactive innate immune cells cannot resist foreign pathogen infection,while overactive ones often lead to a series of immunopathological injuries and autoimmune diseases.Therefore,the innate immune responses and inflammation mediated by macrophages and dendritic cells must be tightly controlled.Nevertheless,the sophisticated regulation mechanisms of inflammatory responses,especially on the transcriptional level,remain to be discovered.Hence we focus on two significant scientific problems,including the roles of macrophages and inflammation in infectious and inflammatory diseases and the way to preciously regulate the function and activation of innate immune cells.In this article,we investigate the roles and mechanisms of histone demethylase(HDM)Kdm5b and RNA-binding protein(RBP)Rbm25 in initiation and resolution of inflammation mediated by innate immune cells.Macrophages and dendritic cells trigger rapid innate immune responses to resist invading pathogens through the recognition of pathogen-associated molecular patterns(PAMPs)by a variety of pattern recognition receptors such as Toll-like receptors(TLRs).However,it remains to be further studied the way to effectively eliminate foreign pathogens while avoiding tissue damage caused by excesive inflammatory responses.In the first section of this thesis,we screened the Histone 3 lysine 4(H3K4)demethylase through a reporter gene assay and identified the indispensable role of histone demethylase Kdm5 b in TLR-triggered production of various proinflammatory cytokines,for example,interleukin 6(IL-6),tumor necrosis factor-α(TNF-α),interferon-β(IFN-β)and so on.By Kdm5 b specific small interfering RNA(siRNA)and Kdm5 b knockout mice,we found that Kdm5 b knock-down or knock-out impaired the expression of proinflammatory cytokines by macrophages and dendritic cells.In an endotoxin shock model,we found Kdm5 b KO mice produced less IL-6,TNF,IFN-β,IL-1β and IL-12p70 in sera,and KO mice exhibited a less severe inflammatory injury phenotype of their lungs.The activation of NF-κB pathways was impaired due to Kdm5 b deficiency,while overexpression of Kdm5 b could dose-dependently promote the transcription activation of NF-κB reporter gene activities.Kdm5 b selectively inhibited the induced transcription of NLRC5,which has been identified as a significant negative regulator of innate immune responses.In stimulated macrophages,Kdm5 b directly bound to the proximal promoter region of Nlrc5 and mediated the demethylation of H4K4me3 in a H3K4 demethylase activity-dependent manner.Thus Kdm5 b inhibited the transcription of NLRC5,then promoted the activation of NF-κB pathway,and eventually facilitated the proinflammatory cytokine production.Our study uncovers the indispensable role of Kdm5 b in inflammation initiation in macrophages and provides a novel target of endotoxin shock and other infectious diseases.Apart from PAMPs,pattern recognition receptors can also recognizes intracellular contents released from damaged and necrotic cells,which are known as damage-associated molecular patterns(DAMPs).The immoderate activation of innate immune cells triggered by endogenic stimuli often give rise to pathological endogenic inflammation and cause severe immunopathological injuries,bringing about many chronic inflammatory diseases including rheumatoid arthritis(RA).In the second section,we analyzed the gene microarray of joint tissue from RA patients and collagen-induced arthritis(CIA)mice,and found the expression of RNA-binding protein Rbm25 was significantly decreased in RA patients and CIA mice.The mRNA expression of Rbm25 was down-regulated in macrophages stimulated with LPS.And knock-down of Rbm25 in macrophages by siRNA promoted the mRNA and protein expression level of several proinflammatory cytokines including IL-1β,IL-6,IL-23 and GM-CSF that play important part in differentiation and function of helper T cells 17(Th17)and in proliferation and activation of fibroblast-like synoviocytes(FLSs).Then we found that fibroblast-like synoviocytes(FLS)proliferation was enhanced by cultured with the supernatant of stimulated macrophages.Knock-down of Rbm25 in FLSs also promoted the production and release of several inflammatory mediators as well as matrix metalloproteinases(MMPs),mainly including MMP3,MMP9 and MMP13.Mechanistically,we found that Rbm25 did not impair the activation of MAPK and NF-κB pathways.Rbm25 did not influence the mRNA stabilization of above proinflammatory cytokines,either.Through a whole transcriptome resequencing assay of Rbm25-knock down macrophages,we discovered that skipped exons(SE),an important kind of alternative splicing events,happened in the pre-mRNA processing of many immune genes and epigenetics modifying molecule genes.Bioinformatics analysis showed that SE genes were responsible for gene transcription or regulation of gene transcription.Above all,we arrive at the preliminary conclusion that Rbm25 controls the balanced activation of macrophages and inflammation by determining the alternative splicing isoforms of significant transcription regulators.The low expression of Rbm25 erects the barriers for inflammation termination and resolution of macrophages,which may lead to chronic inflammatory diseases.However,the underlying molecular mechanisms of Rbm25 in inflammatory responses remain to be further investigated,especially the potential target pre-mRNA of Rbm25-modulated alternative splicing events and the role of these protein isoforms in inflammation and immunity.In sum,the results of above two sections reveal the regulatory roles of histone demethylase Kdm5 b and RNA-binding protein Rbm25 in the innate immune response and inflammatory response,proposing the potential hypothesis of infectious diseases or inflammatory diseases and providing the novel clinical treatment targets. |