| Background:Although there are different classifications of peritonitis,secondary peritonitis,typically originating from a breach in the gastrointestinal tract,is a global problem as it may manifest as intra-abdominal sepsis（IAS）.The overall all-cause incidence of secondary peritonitis is difficult to gauge,but large-scale epidemiologic studies show secondary peritonitis accounts for 1%of all hospital visits and is the second leading cause of sepsis worldwide.Intrabdominal infection is the second most common cause of sepsis.Complicating the high incidence of IAS is high mortality estimated from 7.6%to 36.0%.Abnormal MRSA colonization in the gut markedly increases the risk of MRSA-associated enteritis,and the resistance gene can be transferred to commensal microbes.Paradoxically,combination antibiotic therapy for MRSA salvage treatment will aggravate intestinal disorders.Furthermore,antibiotics do not exert their effect in a vacuum but within a complex host.Thus,using novel host-based therapeutic strategies to remodel the health-promoting gut microbiota for MRSA-induced dysbacteriosis is a promising direction.The gut microbiota composition is subject to the host genome from birth and participates in multiple host metabolic pathway axes that regulate intestinal barrier function.Gut barrier integrity is seriously disrupted during severe peritonitis-induced sepsis.Thus,the abdominal sepsis-induced dysbiotic intestinal microenvironment leading to increased epithelial permeability and gut barrier disruption may facilitate pathogenic bacterial colonization.Specifically,as a gatekeeper against bacterial translocation,the intestinal epithelial barrier relies on enterocyte junctions to maintain integrity.However,a gap exists in understanding the host-microbiota interaction involved in regulating intestinal epithelial cell（IEC）junctions during abdominal sepsis.Notably,constitutive expression of cytochrome P4501A1（CYP1A1）throughout intestinal epithelial cells increases the susceptibility to enteric infection,and whether the host CYP1A1-gut microbiota axis affects IEC barrier disruption against abdominal sepsis remains unclear.Our previous study demonstrated that CYP1A1 m RNA expression levels are elevated in PBMCs from sepsis patients and strongly correlate with sequential organ failure assessment（SOFA）scores.Similarly,polymorphisms in the CYP1A1 gene partially contribute to the increased vulnerability to community-acquired pneumonia（CAP）and nosocomial pneumonia（NP）.Furthermore,germ-free mice（microbiota deficient from birth）or those treated with antibiotics demonstrated a lower level of CYP1A1 expression.Diurnal6-formylindolo[3,2-b]carbazole/aryl hydrocarbon receptor（AHR）/CYP1A1 feedback controls the intestinal immune system to coordinate the diurnal rhythmicity of gut microbiota.In this context,unsurprisingly,crosstalk between CYP1A1 and the resident gut microbiome may exist.Several studies have suggested that constitutive CYP1A1 expression rapidly metabolizes AHR ligands in the gut lumen,indirectly impairing AHR activation in the intestinal immune system following enteric infection.Intriguingly,we previously reported the possible AHR-independent regulation of CYP1A1 expression and CYP1A1-induced proinflammatory responses during the progression of inflammation and sepsis.However,we did not explore whether the interaction between CYP1A1 and microbe-mediated signaling is independent of AHR.Thus,the possibility of non-AHR-dependent action on intestinal barrier integrity in MRSA-induced abdominal sepsis remains to be determined.Methods:Part Ⅰ:Expression of CYP1A1 in ileal epithelium of MRSA abdominal sepsis mice and its effect on tight junction integrity in Enterocytes.Abdominal sepsis was induced by the intraperitoneal injection of MRSA in mice.Primary intestinal epithelial cells were carefully isolated from mice according to the established protocol provided by Dávalos-Salas et al.qRT-PCR,Western blot,IF technology and EROD enzyme activity detection were used to dynamically evaluate the changes of CYP1A1 expression and enzyme activity in ileal epithelial cells before and after MRSA infection.Intestinal permeability and the integrity of the ileal epithelium tight junction between Cyp1a1 knockout mice and their wild-type littermates before and after MRSA infection were assessed by FITC-dextran permeability assay,qRT-PCR,Western blot,IF technology,and transmission electron microscopy.Part Ⅱ:CYP1A1 mediates cadaverine metabolism and damages Enterocytes tight junctions during MRSA-induced AS.The effect of CYP1A1 deficiency on gut barrier integrity was investigated using RNA sequencing,targeted metabolomics and microbiome analyses.The microbiota-produced metabolites were validated in sepsis patients with persistent MRSA infection.To assess gut microbiota-mediated signaling in a non-AHR-dependent manner in Cyp1a1-/-mice,we performed a cohousing experiment.Ahr-/-mice were cohoused with Cyp1a1+/+mice and Cyp1a1-/-mice at a 1:1 ratio after weaning for 10 weeks.Other molecular biology experimental methods are the same as the first part.Part Ⅲ:CYP1A1 downregulates the expression of the ZO-1 in a hydroxylase-independent manner.Three in vitro models of intestinal epithelial injury induced by cadaverine（CAD）,mouse fecal supernatant extract（FS）and TNFαwere established.The MODE-K cell line with CYP1A1 overexpression,knockout and hydroxylase site mutation was constructed,and the CYP1A1 knockdown Caco-2 cell line was also established.The permeability of intestinal epithelial cells was detected by transepithelial electrical resistance TEER assay and fluorescein paracellular transport assay FITC assay.The binding site of the transcription factor RUNX1 and the tight junction molecule Tjp1 was predicted with the transcription factor public data platform and the JASPAR database.The EMSA method was used to detect the binding activity of RUNX1 to the Tjp1 promoter after Cyp1a1 knockout.CYP1A1knockdown model was established by injecting si RNA into the tail vein of mice.Other molecular biology experimental methods are the same as the previous two part.Results:Part Ⅰ:Expression of CYP1A1 in ileal epithelium of MRSA abdominal sepsis mice and its effect on tight junction integrity in Enterocytes.CYP1A1 m RNA expression was altered in the ileal epithelium of MRSA-induced septic mice,increased markedly within 6 hours,and then decreased(P MRSA-6h VS MRSA-0h<0.05,P MRSA-12h VS MRSA-6h<0.05).Additionally,a significant increase was found in the expression of the CYP1A1 protein at 12 hours post-MRSA infection(p-Mi,P MRSA-12h VS MRSA-0h<0.0001).Consistent with these findings,the level of CYP1A1 was upregulated by confocal imaging in IEC isolated from WT mouse ileum at 12 hours p-Mi relative to controls.Cyp1a1+/+littermates succumbed to sepsis,whereas Cyp1a1-/-mice showed markedly reduced mortality after MRSA challenge（hazard ratio=4.739;95%CI:1.23-18.25;P=0.003）.Furthermore,compared with Cyp1a1-/-mice at 12 hours p-Mi,Cyp1a1+/+mice had a significantly higher bacterial load in the peripheral blood（P=0.002）.The expression of ZO-1,occludin,and E-cadherin was significantly increased in the ileal epithelium from Cyp1a1-/-infected mice compared with that from Cyp1a1+/+littermates(P KO-MRSA VS WT-MRSA<0.05).A finding consistent with the decrease in FD-4 in Cyp1a1+/+-infected mice(P MRSA-12h VS MRSA-0h<0.01,P MRSA-24h VS MRSA-0h<0.05).Immunostaining also showed increased expression of ZO-1 in the ileum of Cyp1a1-/--infected mice.Part Ⅱ:CYP1A1 mediates cadaverine metabolism and damages Enterocytes tight junctions during MRSA-induced AS.The GO analysis of upregulated genes in the Cyp1a1-/-＿MRSA group revealed several DEGs encoding antibacterial proteins.KEGG pathway analysis illustrated that these downregulated genes in the Cyp1a1-/-＿MRSA group were primarily related to metabolic pathways,including lysine degradation,retinol metabolism,steroid hormone biosynthesis,and histidine metabolism.Mice lacking CYP1A1 exhibited an altered gut microbiome,a reduced metabolic shift from lysine to cadaverine in the caecal contents and antimicrobial molecule production（Retnlb,Gbp7,and Gbp3）,and they were protected against gut barrier disruption when subjected to MRSA challenge.Furthermore,increased cadaverine levels in feces and serum were detected in critically ill patients with gut leakiness during persistent MRSA infection,whereas cadaverine was not detected in healthy controls.Antibiotic administration-induced intestinal dysbacteriosis disrupts intestinal barrier integrity in Cyp1a1-KO mice.Enterococcus faecalis and cadaverine pretreatment counteracts the original intestinal barrier protection in Cyp1a1-KO mice with MRSA infection.These beneficial effects were validated in AHR-KO mice by cohousing with CYP1A1 KO mice and abrogated after supplementation with cadaverine or Enterococcus faecalis,the primary microbiota genus for cadaverine synthesis.Antibiotic-driven gut dysbacteriosis impaired the survival benefit and disrupted the intestinal barrier integrity in CYP1A1 KO mice after MRSA infection.Additionally,microbiota-derived cadaverine induced enterocyte junction disruption by activating the histamine H4 receptor/nuclear factor-κB/myosin light-chain kinase signaling pathway.Part Ⅲ:CYP1A1 downregulates the expression of the ZO-1 in a hydroxylase-independent manner.CAD,FS and TNFαmodel affected the CYP1A1 gene expression in the two types of intestinal epithelial cells MODE-K and Caco-2,but there was no significant difference in the changes of hydroxylase activity（P FS-6h VS Con<0.05,P CAD-12h VS Con<0.01,P CAD-24h VS Con<0.05,P FS-12h VS Con<0.01,P FS-24h VS Con<0.05,P TNF-12h VS Con<0.05,P TNF-24h VS Con<0.05）.Compared with CYP1A1 overexpression and hydroxylase site mutation groups,CYP1A1knockout cell line significantly improved the permeability and increased tight junction expression after CAD and TNFαtreatment.Independent of its hydroxylase activity,CYP1A1inhibits the binding of transcription factor RUNX1 to the tight junction molecule Tjp1promoter and down-regulates the expression of ZO-1.Inhibition of CYP1A1 by intraductal administration si RNA reduced mice mortality(P si RNA VS WT=0.011,P KO VS WT=0.002),histological score(P KO VS WT=0.021,P si RNA VS WT=0.024),cadaverine level and TJ disruption following MRSA infection.Conclusion:In conclusion,this study reveals an unexpected mechanism of host CYP1A1 in microbiota-mediated cadaverine metabolism during MRSA-induced abdominal sepsis,through which host-microbiota metabolic axes directly affect enterocyte junction disruption in an AHR-independent manner.Therefore,suppressing CYP1A1 or blocking intestinal cadaverine-HRH4 signaling by modulating host-microbiome crosstalk represents a promising strategy to maintain gut barrier homeostasis to treat abdominal sepsis.We also show that transcription factors RUNX1 regulate Tjp1 and that the RUNX1/Tjp1 signaling pathway is activated in Cyp1a1-deficient IECs.These results identify a non-enzymatic,regulatory function of CYP1A1 in tight junction ZO-1 transcription,possibly related to intestinal barrier disruption. |
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