Spliceosomal Protein RBM25 And RBM22 Inhibits Inflammation And Autoimmunity

Inflammatory response is an efficient and rapid physiological response of the immune system to tissue injury and infection.This process involves a variety innate and adaptive immune cells.The former includes monocytes/macrophages,dendritic cells,neutrophils,natural killer（NK）cells,mast cells,innate lymphoid cells（ILCs）,etc.,and adaptive immune cells mainly include T cells and B cells.They cooperate together to resist the infection of pathogens such as viruses or bacteria,and restrict each other to repair and heal the damaged tissues.Chronic inflammation is the main cause of many diseases,especially autoimmune diseases,such as rheumatoid arthritis（RA）,multiple sclerosis（MS）,inflammatory bowel disease（IBD）,psoriasis,asthma,and so on.The common feature of these diseases is that the persistent inflammatory response of the lesion or the whole body is difficult to resolve and inhibit,which leads to a series of serious pathological damages.Therefore,to investigate the molecular mechanisms underlying the activation and function of specific immune cells will provide potential diagnostic indicators and novel drug targets for the remission and treatment of autoimmune diseases.In order to search for new susceptibility genes and related pathways,we analyzed the biological processes that were abnormal in RA,MS and IBD.Strikingly,the alteration in the“regulation of mRNA metabolic process”and the“regulation of mRNA splicing via spliceosome”have attracted our attention.Further studies found that the expression of conserved spliceosomal proteins RBM25 and RBM22 were significantly inhibited,which resulted in the abnormal activation of immune cells and the onset of autoimmune diseases through different mechanisms.Therefore,in this study,we conducted in-depth phenotypic and mechanistic studies on the different functions and regulatory networks of these two proteins.Part Ⅰ.Splicing factor RBM25 Restrain Macrophage Activation and Inflammatory Arthritis through RNA Splicing-mediated Metabolic RewiringRA is an autoimmune disease with synovial dysplasia and erosive arthritis as its clinical manifestations,and the immunological pathogeny underlying RA is the infiltration and accumulation of a large number of pathogenic pro-inflammatory immune cells in the joint cavity,mainly including macrophages,neutrophils,T cells and B cells.They interact with stromal cells in the arthritis lesion,resulting in malignant proliferation of synovial fibroblasts and disruption of the osteoblast-osteoclast balance,ultimately leading to irreversible joint deformity and dysfunction.Therefore,it is of great scientific research value and clinical significance to study the molecular mechanism underlying the abnormal activation of key immune cells in arthritis lesions,and to facilitate the translation of basic science to the clinical setting for blocking and intervening this target.Hence,we analyzed the gene expression profiles of synovial tissues from RA patients and healthy subjects,and focused on the biological function of differential expressed genes.The results showed that“regulation of mRNA metabolic process”and“regulation of mRNA splicing via spliceosome”were significantly down-regulated.Among them,the expression of RBM25,a key component of the U1 spliceosome,was significantly inhibited and negatively correlated with the malignancy of the disease.Subsequently,ChIP-Seq,ATAC-seq and GRO-seq demonstrated that inflammatory environment induced the inhibition of multiple transcriptional activation markers in the RBM25 gene promoter region in macrophages,indicating the transcriptional inhibition of RBM25 as well as the pathological down-regulation of its expression.Therefore,we constructed macrophage-conditional RBM25 knockout mice(Rbm25^fl/fl Lyz2-cre⁺,hereafter referred as cKO mice)to further analyze its physiological and pathological functions.Immunohistochemical and micro-CT results showed that joint inflammation and bone destruction in RBM25 cKO mice were significantly worse than that in wild-type control mice（hereafter referred to as WT mice）in an experimental arthritis model,along with the multiple pathogenic immune cell infiltration and increased production of pro-inflammatory cytokines including IL-1β.To further investigate the molecular mechanisms by which RBM25 deficiency promoted macrophage-mediated joint inflammation,bone marrow-derived macrophages（BMDMs）of WT and cKO mice were cultured in vitro and stimulated with lipopolysaccharide（LPS）for the different times for transcriptomic RNA-sequencing.It turned out that several key pathways of innate immune regulation,inflammatory responses and cytokine-mediated signal transduction were up-regulated,including inflammatory cytokines Il1b,Il6 and chemokine Cxcl10.We also verified these results in BMDMs and alveolar macrophages（AMs）,and additionally,WT and cKO BMDMs treated with double-stranded RNA poly（I:C）or Pam3CSK4 also showed similar phenotypes.In sepsis and peritonitis,two models of acute inflammation,RBM25deficiency in macrophages promoted IL-1βand IL-6 production in macrophages and resulted in immune cell infiltration,exacerbating the inflammatory response in vivo.In the mechanism study,we first found that RBM25 did not directly affect the signal transduction including NF-κB and MAPK pathways.Therefore,we speculated whether RBM25 might play an inhibitory role through regulating the metabolic reprogramming of macrophages.According to the targeted metabolomics and Seahorse cell energy metabolism analysis technique,we found that RBM25 deficiency led to the intracellular metabolism remodeling and enhanced glycolysis in macrophages,which was confirmed by the alternations in mitochondrial morphology observed by transmission electron microscopy（TEM）,indicating the mitochondrial lysis and impaired oxidative respiration.Next,we performed chromatin immunoprecipitation（ChIP）and CUT&Tag assays,and found that the metabolic remodeling and the changes in the abundance of several key metabolites in macrophages directly resulted in the enhancement of histone H3K9ac and H3K27ac modification,as well as the enrichment of hypoxia-inducing factor-1α（HIF-1α）in the pro-inflammatory gene promoter regions such as Il1b and they enhanced the transcription of Il1b gene.As a key element of U1 splicing spliceosome,RBM25 is involved in regulating a number of RNA alternative splicing（AS）events.Therefore,we analyzed RNA splicing events regulated by RBM25 using whole transcriptome sequencing and RNA immunoprecipitation-sequencing（RIP-seq）,which demonstrated skipped exon（SE）was the main AS event affected by RBM25 deficiency and its target genes were enriched in multiple cellular metabolic pathways.We further found that the 14th exon of ATP citrate lysis（Acly）was skipped in RBM25 cKO macrophages,producing a short Acly isoform（Acly-short,Acly-S）,which was dependent on the RRM domain.By small interfering RNAs,Splice-switching Antisense Oligonucleotides and lentivirus overexpression experiments,we showed that Acly-S promoted glycolysis and acetyl Co A production in macrophages,and enhanced the transcription of Il1b and other inflammatory genes in histone acetylation modification-and HIF-1α-dependent manner.Subsequently,we identified that Acly-L（Acly-long）,but not Acly-S,was subjected to protein lactate modification,at lysine 918 and995（K918 and K995）,which determined the difference in affinity to ATP and metabolic activity of two different Acly isoforms,thus affecting macrophage metabolic remodeling.Finally,we used SB204990,a specific inhibitor of Acly,to treat the mouse experimental arthritis,and found that SB204990 alleviated the deterioration of joint inflammation and pathological changes caused by RBM25 deficiency in macrophages.In addition,SB204990also inhibited the production of IL-1βand other pro-inflammatory cytokines in lung tissue and sera of RBM25 cKO mice in an aging-related auto-inflammatory model.In conclusion,in this part,we demonstrate that splicing factor RBM25 affects the metabolic enzyme activity of Acly by regulating its exon skipping,and controlls the production of inflammatory factors including IL-1βin macrophages in a glycolytic-epigenetic reprogramming-dependent manner,which elucidates a new pathogenesis hypothesis of RA.It also provides potential therapeutic targets and strategies for the treatment of joint inflammation and other inflammatory diseases.Part Ⅱ.Chromatin-associated RBM22 Controls Super Enhancer-induced Inflammation and Autoimmunity in Myeloid CellsBased on the patient data in the first part,we also found that the expression of RNA binding protein RBM22 was down-regulated in a variety of autoimmune diseases,among which the inhibition of RBM22 expression was the most obvious significant in multiple sclerosis（MS）.MS is a kind of immune demyelination disease of central nervous system（CNS）.Its main immunological cause was due to the infiltration of a large number of monocytes/macrophages,lymphocytes,plasma cells and other immune cells in the brain and spinal cord,which attacked the myelin,causing neuron death and demyelination,and eventually developing into movement disorders,ataxia,and even hemiplegia,paraplegia and quadriplegia in severe cases.Its pathogenesis remaines largely unclear.In recent years,more and more evidence showed that epigenetic factors and RNA-binding proteins played important roles in the pathogenesis of MS,which needed further elaboration.To clarify the relationship between down-regulation of RBM22 expression and MS disease,monocyte-conditional(Rbm22^fl/fl-Cx3CR1^Cre),macrophage-conditional(Rbm22^fl/fl-Lyz2^Cre)and dendritic cell-conditional(Rbm22^fl/fl-Itgax^Cre)RBM22 knockout mice were generated and used to construct MOG-induced experimental autoimmune encephalomyelitis（EAE）model.We found that deletion of RBM22 in myeloid cells significantly worsened the neuroinflammation and demyelination in CNS system.Subsequently,RBM22 deficiency also could promote the onset and development of other mouse models including colitis,psoriasis and arthritis.To investigate the biological function of RBM22 in myeloid cells,we obtained BMDMs and bone marrow-derived dendritic cells（BMDCs）from RBM22 knockout（cKO）and wild-type control（WT）mice and performed transcriptome sequencing.Gene expression profile analysis showed that RBM22 deficiency caused similar transcriptomic changes in these two myeloid cells.The up-regulated genes were mainly enriched in inflammatory responses,antigen presentation,T cell activation,differentiation,and chemotaxis pathways,including Il12b,Il23a,Tnfsf9,and H2-Dma.To further elucidate the molecular mechanisms by which RBM22 regulated myeloid cell activation,we investigated the subcellular localization of RBM22.LPS treatment induced the threonine phosphorylation of RBM22 and the phosphorylation sites might localized in its nuclear localization sequence（NLS）.RBM22could translocated into the nucleus and bound to chromatin in LPS-treated macrophages.Previous studies have reported that RBM22 functioned as a non-classical transcription factor to regulate gene transcription expression.Therefore,we used ChIP and CUT&Tag assays to study the genome-wide chromatin information that bound by RBM22.In activated macrophages and dendritic cells,the binding peak of RBM22 was highly correlated with histone H3K27ac and H3K4me1 as well as chromatin accessibility,suggesting that RBM22specifically bound to promoter and enhancer regions of Il12b,Il23a,Tnfsf9 and other pro-inflammatory genes,mediating the transcriptional inhibition of these target genes.CUT&Tag sequencing showed that the histone modification levels of H3K27ac and H3K4me1 in the RBM22-bound active enhancer were significantly up-regulated in cKO cells,but not in the active enhancer without RBM22 binding.Considering that RBM22 did not have the ability to catalyze histone modification,we performed co-immunoprecipitation（Co-IP）combined with mass spectrometry（MS）assay and found that RBM22 directly interacted with histone deacetylase HDAC3 and demethylase LSD1,which was dependent on its Pro-Rich domain.The lentivirus-mediated overexpression of wild-type RBM22 in cKO cells promoted the recruitment of HDAC3 and LSD1 to Il12b and other target gene locus,and inhibited the active histone modification and subsequent mRNA expression,whereas the Pro-Rich domain mutant of RBM22 had no such effect.The results of ATAC-seq showed the increased chromatin accessibility in the above enhancer region owing to RBM22 deficiency,which subsequently enhanced the binding and activity of transcription factor c-Jun.Reporter-gene experiments confirmed that RBM22 could inhibit the transcriptional activation of c-Jun on Il12b gene enhancers in a dose-dependent manner,thereby inhibiting promoter-enhancer interaction and gene transcription.Finally,we found that T-5224,a transcriptional activity inhibitor of c-Jun,suppressed the transcription of inflammatory genes downstream of RBM22 in vivo and in vitro,and had a therapeutic role in the neuroinflammation and paralytic phenotype in RBM22 conditional knockout mice.In this part,we found that RBM22 in myeloid cells underwent nuclear translocation in a MAPK-dependent manner,exerting the non-classical transcription factor function to inhibit the expression of inflammatory cytokines and co-stimulatory molecules,and control the activation of pathogenic T cells and central nervous system inflammation.Mechanistically,RBM22 selectively bound to the promoter and enhancer regions of its target genes,mediated the recruitment of HDAC3 and LSD1 for erasing the active histone modifications,and finally regulated chromatin binding and transcriptional activity of c-Jun.Our study identifies RBM22 as a critical controller of inflammation and autoimmunity,which sheds new light on the treatment strategy for the chronic inflammatory and autoimmune diseases.On the basis of the above two parts of our research,this study clarifies the functions and mechanisms of the spliceosomal RBM25 and RBM22 in controlling inflammatory responses and autoimmunity,and reveals their protective effects in autoimmune diseases（e.g.,RA and MS）.In addition,a series of advanced technologies such as transcriptomics,single-cell sequencing,metabolomics and epigenomics have been used to elucidate the metabolic and epigenetic mechanisms in regulating the immune system.The potential pathogenic factors for the pathogenesis of these inflammatory and autoimmune diseases have been proposed and the endogenous ingenious regulatory network of the host immune system has been enriched.