| PART? EFFECTS OF ANTIBIOTICS PRETREATMENT INDUCED MICROBIOTA DYSBIOSIS ON ACUTE PSEUDOMONAS AERUGINOSA PNEUMONIAObjective:Antibiotics are the first-line drugs for the treatment of bacterial infection in clinical practice.However,clinical studies have found that antibiotics pretreatment are also an independent risk factor for multiple drug-resistant Pseudomonas aeruginosa infections.Recent studies have found that microbiota dysbiosis,in the respiratory tract and the gut,has been shown to induce dysfunctional immune modulation and susceptibility to infectious diseases.Therefore,we hypothesized that antibiotics pretreatment would lead to microbiota dysbiosis,thus increasing the susceptibility of subsequent Pseudomonas aeruginosa infection.Methods:Mice were randomly assigned to different cages,and were fed with ordinary drinking water(Water group)or antibiotic cocktail water(Abt group)for 3 weeks.The antibiotic pretreatment was stopped 3 days prior to establishment of acute Pseudomonas aeruginosa pneumonia model(PAO1 suspension 10~7 CFU/mouse).Bacterial burdens in lung tissues and spleen were detected by plate counting method.For mortality rates,mice were monitored for 7 days.Lung tissue HE staining and inflammatory pathological score were obtained.Expressions of inflammatory cytokines TNF-?,IL-6 and IL-10 in lung tissues were detected by ELISA and RT-PCR.Assay of commensal bacteria in the lungs and small intestine were performed by 16S rRNA.Results:1.All the mice in the PBS group survived.The spontaneous mortality was 46%in the Water+PAO1 group,however,all the mice in the Abt+PAO1group died within 2 days of infection(p<0.05).2.The bacterial burdens of the lungs and spleens in the Abt+PAO1group were significantly greater than those in the Water+PAO1 group(p<0.01 and p<0.05,respectively).3.HE staining and inflammatory pathological score:In the Water+PAO1 group,there was a large number of inflammatory cell infiltration dominated by neutrophils,and multiple abscess foci.Higher lung pathology scores were observed in the Water+PAO1 group compared with Abt+PAO1 group(p<0.05).4.The abundance of pulmonary cytokines TNF-?and IL-6 were significantly decreased(both p<0.01),whereas IL-10 was increased in the Abt+PAO1 group compared with the Water+PAO1 group(p<0.01).The mRNA expression level of these cytokines in the lung showed similar changes(all p<0.01).5.Compared with the non-antibiotic-treated group,the abundance of Proteobacteria in the intestine was significantly higher in the Abt group,while the proportion of Bacteroidetes,Firmicutes and Deferribacteres in the intestine was significantly lower.At the genus level of the gut microbiota,Klebsiella and Parasutterella were the most prominent genera in the Abt group,whereas the proportions of Bacteroidales were significantly decreased(p<0.001).However,we did not observe any changes at the phylum or genus level in the lung microbiota.Antibiotic exposure not only altered the composition of intestinal microbiota,but also decreased diversity of intestinal microbes.Conclusion:Antibiotics pretreatment induced inflammatory hypo-responsiveness and poor clearance of Pseudomonas aeruginosa by microbiota dysbiosis,resulting in decreased host survival.PART ? MICROBIOTA DYSBIOSIS AGGRAVATES ACUTE PSEUDOMONAS AERUGINOSA PNEUMONIA VIA GAMMA DELTA T CELL-NEUTROPHIL AXISObjective: We have confirmed in the PART ? that antibiotics pretreatment induced inflammatory hypo-responsiveness and poor clearance of Pseudomonas aeruginosa by microbiota dysbiosis.Neutrophils are the major orchestrators of the pulmonary innate defense against Pseudomonas aeruginosa infection and regulated by microbiota.In this part,we would discuss what are the effects of microbiota dysbiosis on antimicrobial immune response?Methods: The study was divided into 4 groups: Water+PBS group,Water+PAO1 group,Abt+PBS group,and Abt+PAO1 group.Humoral immune response Ig A,Ig M,and Ig G and the inflammatory cytokine IL-17 A were detected by ELISA;the expression of myeloperoxidase in the lungs was detected using the kit;neutrophils and ??T cells in the lungs were detected by flow cytometry;and the expression of chemokines Cxcl1 and Cxcl2 in the lungs were detected by RT-PCR.The anti-??TCR m Ab mouse model was constructed,on which the bacterial load of the lung and spleen and survival rate,as well as the proportion of neutrophils were detected to verify the effect of the microbiota on the ??T cells.Results: 1.There was no significant difference in the expression of IgA,IgM and IgG in lung tissues between Abt+PAO1 group and Water+PAO1 group.2.Compared with Water+PAO1 group,Abt+PAO1 group had lower myeloperoxidase(p<0.001),less infiltration of neutrophils in the lungs(p<0.001),and lower neutrophil chemokines Cxcl1,Cxcl2 and its upstream cytokine IL-17A(p<0.01,p<0.01,p<0.001,respectively).3.After 3 weeks of antibiotic pretreatment,the percentage and number of ?? T cells were significantly decreased(p<0.05).After the challenge with P.aeruginosa,?? T cell expansion was nearly completely abrogated in the Abt group.Up to 50% ?? T cells accumulated in the Water+PAO1 mice produced IL-17 A,however,?? T cells in the Abt+PAO1 mice exhibited dramatically reduced IL-17 A production(p<0.05).In addition,?? T cells of the Water+PAO1 mice exhibited an activated phenotype(defined as CD44hiCD62Llo)compared to the corresponding ?? T cells from Abt+PAO1 mice(p<0.001).4.Treatment of mice with anti-??TCR blocking antibody blocked the increase in pulmonary neutrophils.Compared with the Ig G-control mice,the anti-??TCR mice presented with a significantly increased bacterial load in lungs and spleens(p<0.001 and p<0.05,respectively),which nearly recapitulate the phenotype observed in antibiotic-treated mice.Furthermore,the anti-??TCR-treated mice had markedly decreased survival,though not to the same extent as Abt+PAO1 mice.Conclusion: The humoral immunity is not affected due to microbiota dysbiosis.Antibiotics pretreatment led to defective ?? T17-neutrophil axis,which resulted in the susceptibility of the mice to P.aeruginosa infection. |
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