Transcriptomic Analysis Of Mice Spleen Infected With Extraintestinal Pathogenic Escherichia Coli And Studies On The Mechanism Of COX-2 In Inhibiting Escherichia Coli Infection

Extraintestinal Pathogenic Escherichia coli(ExPEC)can enter the bodily sites outside of the gastrointestinal tract and its infection can cause a variety of diseases.Based on its clinical symptoms and sources,ExPEC is mainly divided into human-source Uropathogenic Escherichia coli(UPEC),Neonatal Meningitis-associated Escherichia coli(NMEC),Sepsis-associated Escherichia coli(SEPEC),and animal-source Avian pathogenic Escherichia coli(APEC).However,further studies show that some APEC strains display closely relationship with UPEC and NMEC strains.Many virulence factors are shared by all ExPEC pathogenic types.In animal experiments using mice as a model,some APEC strains can cause meningitis,sepsis,and urinary tract infections in mice,suggesting that some APEC strains are potential zoonotic bacteria.ExPEC strains use virulence factors,including adhesions,toxins,capsules,lipopolysaccharides,iron intake systems,serum resistance factors,etc.,to avoid the host's immune system and cause diseases in humans and animals.However,in ExPEC mediated systemic infection,how the host onset the immune response to remove the invading pathogenic bacteria remains to be further investigated.In this study,with ExPEC of avian origin was taken as the research object,the transcriptome response of ExPEC-infected mouse spleen at different time points were firstly compared,which explained the immune response of host to ExPEC infection in genome-wide level.Additonaly,our transcriptome data were compared to those from avian models of avian E.coli-related sepsis and spleen transcriptome data of mice infected with different septic pathogens,the results provide a theoretical basis for explaining the differences in ExPEC cross-host infection and host septicemia caused by different pathogens.Finally,we explored the function of COX-2 molecular in ExPEC infection by using COX-2 specific inhibitor.1 Transcriptome analysis of mice spleen infected with ExPECCurrently,little is known about the potential mechanisms of host resistance to ExPEC-induced sepsis.Our research started with evaluating the transcriptome changes of mice spleen infected with ExPEC to screen the key molecules and pathways of the host against ExPEC infection.The bacterial load of ExPEC after infection reached 2.55�107 CFU/ml in the blood and 1.22� 107 CFU/g in the spleen.Histopathological analysis results show that ExPEC infection will lead to splenitis symptoms in mouse,and the spleen inflammation and pathological changes gradually aggravate in an time-dependent manner.Therefore,the global gene expression in spleen of ExPEC-infected BALB/c mice were analyzed by sequence total RNA collected from spleen of ExPEC infected mice at 3,6,and 12hpi using RNA-seq technology in the study.Compared with uninfected mice,there were 3723,4904 and 4816 differentially expressed genes(DEGs)at 3hpi,6hpi and 12hpi after ExPEC infection,of which 2484,3396 and 3136 down-regulated genes and 1239,1508,1680 up-regulated genes.GO functional analysis showed that immune-related DEGs were mainly categorized under regulation of immune system process,immune response,leukocyte migration,immune effector process,leukocyte activation,and immune system development.KEGG pathway analysis showed that toll-like receptor,NOD-like receptor,nucleic acid sensor,and interleukin-17 signaling pathways were activated after ExPEC infection and contributed to the inflammatory response.Further studies indicated that ExPEC infection may activate the caspase-1-mediated pyroptosis in vitro and in vivo.In addition,carbonic anhydrase IV was significantly up-regulated under ExPEC infection.Inhibition of carbonic anhydrase enhanced the survival of ExPEC in macrophages,but did not affect macrophage cytokine secretion.This study evaluated the gene expression changes of the host against ExPEC infection at the genome-wide level for the first time,these results will provide new insights into the immune response of host to ExPEC infection.2 Comparative analysis of spleen transcriptome data from different hosts infected with ExPEC and spleen transcriptome data of mice infected with different pathogensExPEC is an important potential zoonotic pathogen that can cause diseases in humans and animals,which seriously threat the human health and cause huge economic losses to the breeding industry.This study further compared the spleen transcriptome data of mice and chickens infected with ExPEC.Compared to the transcriptome response of chickens,ExPEC triggered a more robust immune response in mice;furthermore,lysosome pathways related genes were down-regulated in mouse,but up-regulated in chicken.In addition,the transcriptome response in mouse spleens to other bacterial sepsis-causing pathogens including Pseudomonas aeruginosa,Burkholderia pseudomallei,and PasIeurella multocida,as well as that of cecal ligation and puncture(CLP)treatment,were also compared.Only 53 up-regulated and 29 down-regulated genes were common differently expressed under these stimulus,and most of the common up-regulated expression genes are inflammation-related genes(casp4,1110,illa,illb,i16,trl7,ptgs2,cxcl5,and cxcl13).Notably,KEGG enrichment analysis indicated that ExPEC infection uniquely activated or repressed the expression of several metabolic pathways.Many genes related to the glycolysis pathway were up-regulated,while pathways relating to lipid,amino acid,and glycan metabolism were highly down-regulated.This study provides new insights into the immune response to sepsis in mice infected with different pathogens,and the immune response to ExPEC infection in different hosts(mouse and chicken).3 Studies on the mechanism of COX-2 in inhibiting ExPEC infectionCyclooxygenase-2(COX-2)is one of the key rate-limiting enzymes that convert arachidonic acid into active prostaglandins.Our transcriptome data shows that COX-2 is one of the top 10 DEGs in ExPEC-infected mouse spleen at 3hpi,6hpi,and 12hpi.In this study,we investigated the mechanisms of COX-2 upregulation in influencing the host defenses against ExPEC infection.Our results revealed that ExPEC infection leads to COX-2 upregulation in both mouse and macrophage cell models and COX-2 inhibition significantly enhances ExPEC infection.The upregulation of COX-2 in macrophages was mediated by Toll-like receptor 4(TLR4)through the activation of p38 and extracellular signal-regulated kinase/Mitogen-activated protein kinase(ERK/MAPK)pathways.Further studies showed that COX-2 inhibition significantly decreased autophagy in macrophages during ExPEC infection.Autophagy inhibition significantly enhanced while induction reduced ExPEC survival in macrophages.In addition,COX-2 inhibition significantly increased macrophage cell death during ExPEC infection and increased the expression of anti-inflammatory cytokine interleukin-10(IL-10).Our results indicate that COX-2 upregulation benefits host defense against ExPEC infection through increasing autophagy in macrophages,reducing IL-10 expression and macrophage cell death during ExPEC infection.