| Objective:Superbug infections can lead to sepsis or septic shock and cause death in patients,posing a serious risk to human health.The clearance of bacteria by immune cells plays an important role in anti-infective immunity in sepsis,but the molecular mechanisms involved have not been investigated.CD74 is an immune molecule expressed mainly in MHC II positive antigen-presenting cells.On the one hand,as a chaperone of MHC II class II molecules,it binds to the antigenic peptide binding groove of class II molecules,thus preventing other peptides from binding to MHC II class II molecules before the antigenic protein reaches the endosomal compartment;on the other hand,CD74 is also associated with the development and poor prognosis of several diseases.Whether CD74 regulates sepsis due to bacterial infection,however,has not been clarified.In this study,a mouse sepsis model was constructed using methicillin-resistant staphylococcus aureus(MRSA),and the survival rate and sepsis-related phenotypes of C57BL/6(WT)and Cd74 knockout mice(Cd74-/-)were compared and analyzed to initially explore the regulatory role of Cd74 on the pathological process of sepsis,and to further sort out neutrophils from WT and Cd74-/-mice.The transcriptome sequencing(Bulk-RNAseq)technique was used to compare and analyze the transcript levels of different genes in these two types of neutrophils,and to explore the molecular mechanisms of Cd74 effects on neutrophils,thus providing new strategies for sepsis treatment.Methods:(1)WT mice(males,8~12 weeks)were divided into control,low-dose and high-dose groups,and a sepsis model was constructed using tail vein injection of MRSA.The transcript levels of Cd74 m RNA in liver,kidney,spleen and lung tissues of mice at different time points of infection were detected using real-time fluorescence quantitative PCR(q-PCR).(2)WT and Cd74-/-mice(males,8~12 weeks)were divided into low-dose and high-dose groups,and sepsis models were constructed using tail vein injection of MRSA to assess the survival rate of mice,respectively.At the same time,the body weight changes of mice in the low-dose group were compared and detected at 48 h of infection;the concentrations of interleukin-1β(IL-1β),interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α)and inducible nitric oxide synthase(i NOS)in serum were detected using enzyme-linked immunosorbent assay(ELISA);hematoxylin-eosin(HE)staining was performed on paraffin sections of liver to observe the pathological damage;the amount of charge in liver,kidney,spleen and lung tissues was also compared and detected.(3)A sepsis model was constructed using WT and Cd74-/-mice with tail vein injection of MRSA.At 48 h of infection,ELISA was performed to determine the serum concentrations of portal aminotransferase(AST)and glutamate-pyruvate transaminase(ALT);flow cytometry was performed to detect the percentage of liver macrophages,monocytes and neutrophils;immunohistochemical staining of liver paraffin sections was performed to observe neutrophils;Macrophages were cleared by tail vein injection of clodronate liposomes,and the clearance of macrophages was observed by immunofluorescence staining of frozen sections of liver after 48 h.MRSA was then injected into each mouse to compare and analyze the mortality of mice.(4)Using WT and Cd74-/-mice,flow cytometry was used to detect the development of neutrophils in mice as well as peripheral blood and spleen neutrophils.Mature neutrophils from mouse bone marrow were further sorted out for Bulk-RNA transcriptome sequencing using a magnetic bead negative selection method,and the expression of different genes in neutrophils derived from WT and Cd74-/-mice was comparatively analyzed by the BGI Genomics multi-omics system.Results:(1)MRSA infection elevated the expression of Cd74 in the liver,kidney and spleen of mice.Different concentration doses of MRSA promoted the m RNA transcription of Cd74 compared to the control group.For example,the relative expression of Cd74 m RNA in the liver was significantly increased by 72.90% and 84.25% at 24 h and 36 h of infection,respectively,in the low-dose group(p<0.05);in the kidney by 80.67% and 90.82%,respectively(p<0.05);The relative expression of Cd74 m RNA in spleen was significantly increased by 57.69%,82.87% and 85.26% at 12 h,24h and 36 h of infection,respectively(p<0.05).(2)Knockout of the Cd74 gene attenuates MRSA-induced sepsis symptoms and significantly prolongs survival in mice.In both the high-dose and low-dose groups,the survival rate of Cd74-/-mice was significantly higher compared to WT mice(p<0.05).In the low-dose group,at 48 h of infection,Cd74-/-mice showed less change in body weight compared to WT mice(p<0.05);liver HE staining showed smaller necrotic foci caused by infection and more inflammatory cell infiltration;serum concentrations of TNF-α,IFN-γ and i NOS decreased significantly by 12.22%,1.85% and 26.14%,respectively(p<0.05),and IL-1β significantly increased by 37.21%(p<0.05);and the amount of charge bacteria in the liver and spleen significantly decreased by 98.37% and 92.51%(p<0.05),respectively.(3)Knockout of the Cd74 gene significantly increases the number of liver neutrophils in MRSA-infected mice.At 48 h of infection,there was no significant change in AST and ALT concentrations in Cd74-/-mice compared to WT mice(p>0.05);no significant change in macrophages and monocytes in the liver(p>0.05),and a significant increase in neutrophils by 46.76%(p<0.05);liver Immunohistochemical staining showed a significant increase in neutrophils(p<0.05).Removal of macrophages in mice and reconstitution of sepsis model showed that knockout of Cd74 still significantly prolonged the survival of septic mice(p<0.05).(4)Knockout of the Cd74 gene resulted in enhanced neutrophil recruitment due to upregulation of IL-17 signaling pathway-related gene expression.There was no significant change in the level of CD74 on the surface of neutrophils at 48h in the infected mice compared to the control group(p>0.05).There was no significant change in neutrophil development in Cd74-/-mice compared to WT mice(p>0.05);neutrophils in peripheral blood and spleen were significantly increased by 53.17% and 33.79%,respectively(p<0.05).Transcriptome sequencing analysis of neutrophils from WT mice and Cd74-/-mice with a screening condition of |log2Fc|≥1.5 and FDR≤0.001 yielded 1499 significantly differentially expressed genes.Among them,1393 genes were significantly down-regulated and 106 genes were significantly up-regulated in neutrophils from Cd74-/-mice.GO enrichment analysis and KEGG signaling pathway enrichment analysis showed that the significantly up-regulated genes were significantly enriched mainly in IL-17 signaling pathway.Conclusion:In a mouse sepsis model constructed from methicillin-resistant staphylococcus aureus,knockout of Cd74 significantly attenuated the symptoms and improved the survival rate of septic mice.The possible mechanism of this effect is to promote the recruitment of neutrophils by upregulating the expression of Il-1β,Cxcl1 and other related genes in the IL-17 signaling pathway,thus improving the clearance efficiency of bacteria. |
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