| Klebsiella pneumoniae?K.p.?is one of the most concerned pathogens related to antibiotic resistance.The infections caused by multidrug-resistant?MDR?K.p.and XDR Enterobacteriaceae are increasing rapidly.The infections caused by these strains usually have high mortality,prolonged hospitalization and increased hospitalization costs.Recent studies have shown that K.p.have both virulence and multidrug resistance genes,which can transmit vertically and horizontally,causing the emergence of highly virulent and resistant Klebsiella pneumoniae clones in healthy individuals.At present,our independent and drug-resistant transmission path and pathogenesis of high virulence and drug-resistant K.p.are still unclear,which makes it necessary for us to better understand its infection microbiology and lay the foundation for developing new strategies for K.p.infection.There are a large number of microorganisms in the lumen of the gastrointestinal tract.It is estimated that there are 103 to 109 cells /m L in the lumen of the small intestine,while there are 1011 to 1012 cells / g in the large intestine.As a whole,it constitutes a dynamic and balanced ecosystem.Intestinal microorganisms can not only decompose food,but also promote the absorption of enteral nutrition and the enhancement of intestinal development,improving the diversity of diet structure and human evolution.In addition to digestion and metabolism,intestinal microflora also contribute to the development and maintenance of intestinal epithelial barrier,the development of immune system and the competition with pathogenic microorganisms,thus preventing their reproduction.With more and more studies on intestinal flora and lung diseases,the existence of enteropulmonary axis has been confirmed and the mechanism of intestinal flora affecting lung immune response is being revealed step by step.The relationship between intestinal microflora and K.p.pneumonia has been poorly studied and limited understanding.Based on intestinal microflora,this study explored how it regulates the pulmonary immunity of K.p.pneumonia and mediates the elimination of pathogenic bacteria.Recent studies have shown that SCFAs play an important role in the communication network between hosts and microbiota.SCFAs is the final product of intestinal bacteria fermenting dietary fiber,which is the energy source of epithelial cells,hepatocytes and peripheral tissues.With the development of research,SCFAs can also be directly involved in the regulation of immune system and immune cells.It is also a link between intestinal microflora and host organism and an important part of maintaining homeostasis.Previous studies have described the protective effect of SCFAs on respiratory tract infection in mice,and revealed that the main mechanism of SCFAs on immune regulation of neutrophils and macrophages is through binding and activating GPCR.Based on the previous studies,this study focuses on high-throughput methods,gene editing technology,the core technology of modern molecular biology,and metabonomics of intestinal contents,16 Sr RNA and transcriptome analysis of macrophages in the lung tissue,and studies the mechanism of intestinal flora in K.p.pneumonia from intestinal flora to metabolites in vivo and in vitro.It provides a new research strategy and theoretical support for controlling intractable bacterial pneumonia and other diseases in the future.OBJECTIVE: The aim of this study was to study the role of intestinal flora in K.p.pneumonia,and the mechanism of its action.Combined with gene editing technology,the regulatory network of intestinal flora metabolites which may regulate innate immunity of lung was discussed from the cell level,and the molecular mechanism of intestinal flora metabolites regulating alveolar macrophage sterilization was further clarified,which laid a theoretical foundation for finding a new clinical diagnosis and treatment method of antibiotic resistant pathogens infection.METHODS:?1?After the mice drank the mixed solution of antibiotics freely and exhausted the intestinal flora of mice,the K.p.pneumonia model was constructed by nose drop method: the lung tissue and blood K.p.colony forming unit?CFU?of the two groups were calculated by plate colony count method;Lung pathological changes of the two groups were analyzed by lung histopathology;Changes of lung tissue cytokines were detected by enzyme-linked immunosorbent assay;Survival difference between the two groups was compared by Mantel-Cox test.After the mice were supplemented with normal intestinal flora,the survival difference and lung histopathology of mice were analyzed by Mantel-Cox test to further clarify the protective effect of intestinal flora on K.p.pneumonia?2?The fresh feces of patients with bacterial pneumonia were collected and transplanted to mice with exhausted intestinal flora and the changes of pulmonary immune cells were analyzed by immunohistochemistry;?3?The phagocytosis and clearance ability of alveolar macrophages from different intestinal flora background were studied in vitro;?4?16Sr RNA and metabonomics of intestinal contents were used to analyze the status quo of intestinal flora and the changes of metabolic pathway after intestinal flora depletion.Further correlation analysis showed that the changes of metabolic product pathway projected to the corresponding changes of flora;?5?After SCFAs was supplemented in mice,the pneumonia model of mice was established.The effect of SCFAs supplementation on K.p.pneumonia was observed: the number of K.p.colonies in lung tissue and blood was counted by MHA plate;the pathological changes of lung tissue inflammation were observed by H & E staining;the survival difference between the two groups was compared by Mantel-Cox test.The alveolar macrophages were isolated from SCFAs group and control and their scavenging ability of K.p.was studied in vitro;?6?RNA-seq analysis of alveolar macrophages and bioinformatics analysis technology were used to screen out the differential expression of antimicrobial related genes in the background of intestinal flora depletion mice and normal intestinal flora.Furthermore,RT-q PCR was used to verify the accuracy of RNA-seq results.Combined with sh RNA interference technology,we further screened the expression of antimicrobial related genes in K.p.pneumonia;?7?Using CRISPR /cas9 technology to knock out lamtor2 and gpr43 genes on the DNA level of RAW264.7 cell line,using RT-q PCR to identify the mode of SCFAs regulating lamtor2 fund expression,Western blot to identify how SCFAs regulates LAMTOR2 and its downstream network signal,and further clarify how intestinal flora and its metabolites precisely regulate the killing innate immune cells in pulmonary of bacterial pneumonia.RESULTS:?1?A stable animal model of acute lung injury can be established by mouse nose drop method.The amount of bacteria in lung tissue and blood of mice with intestinal flora depletion was significantly higher than that of mice with normal intestinal flora 12 hours and 24 hours after infection with Klebsiella pneumoniae.The lung tissue of mice with intestinal flora depletion showed more extensive inflammatory cell infiltration,pulmonary vascular endothelial injury bleeding,pulmonary tissue hyperemia and alveolar space after infection with K.p.12 h,the liver and kidney tissues also showed increased pathological damage and shortened survival time.The results of ELISA showed that the levels of TNF-?,IL-1 ?,IL-6,CXCL1 and MCP-1 in lung tissue were significantly reduced of intestinal flora depletion mice after infected with K.p.24h;?2?The feces of the patients with bacterial pneumonia were transplanted to the mice with depleted intestinal flora,and the intestinal microflora of the mice were replanted.Immunohistochemistry showed that there were more macrophage infiltration in the lung tissue of the mice with intestinal microflora and inflammatory pathological changes in the normal lung tissue;?3?The phagocytosis of K.p.by alveolar macrophages from antibiotic depleted mice was lower than that of the control group.The clearance of intracellular K.p.was observed at 1h,6h and 12 h.It was found that the bacterial load of alveolar macrophages was increased at 6h after intestinal flora depletion;?4?A total of 1126 different metabolites were identified by non-target metabonomics analysis of cecal contents in mice with intestinal flora depletion and mice with normal controls.The metabolic pathways of SCFAs?acetic acid,propionic acid and butyric acid?also changed;?5?A total of 736749 high-quality 16 Sr RNA gene sequences were obtained by sequencing and analyzing the bacterial 16 Sr RNA variable region V4.These sequences aggregate into different operational taxon at 97% similarity level.The analysis of species diversity showed that there was no significant difference in species richness and diversity between the mice with intestinal flora depletion and the control mice.At the genus level,we found that the composition and structure of the community changed greatly,and the Paracharacteroids,Bifidobacterium,Clostridium,Coprococcus and Prevotella that produced SCFAs decreased significantly in mice with intestinal depletion;At the level of phylum,Bacteroides and Veronica decreased,but Protein phylum increased;?6?Spearman correlation analysis showed that the change of SCFAs metabolic pathway was corresponding to the change of short chain fatty acid producing intestinal flora;?7?When SCFAs was supplemented additionally,the mortality and survival time of mice with sepsis caused by Klebsiella pneumoniae were significantly reduced;Compared with the control group,the bacterial load of lung tissue and blood in SCFAs group was also significantly reduced;Meanwhile,the pathological changes and scores of lung tissue were also significantly reduced;The results of in vitro experiments showed that the alveolar macrophages in SCFAs group were significantly lower than those in the control group The phagocytosis of K.p.increased significantly,but also accompanied by the increase of clearance.It was found that the bacteria load was slightly higher in the SCFAs group at 1h because of the more phagocytosis in the initial stage,but at 6h and 12 h,the clearance capacity of alveolar macrophages in the SCFAs group was significantly stronger than that in the control group;?8?The results of transcriptome analysis of alveolar macrophages showed that there were significant differences in transcriptional level between the intestinal flora depletion group and the control group.The differential gene annotation showed that a large number of genes involved in antibacterial changed.The enrichment of the differential gene KEGG signal pathway showed that some signal pathways involved in the invasion of pathogenic microorganisms,such as the defense response to bacteria,MAPK / ROS signal;?9?The results showed that TNF-? and 1L-1 ? were down regulated in RAW264.7 cells when lamtor2 was knocked down by sh RNA;?10?The results of CRISPR / cas9 gene editing technology knocked out the lamtor2 and gpr43 genes of RAW264.7 strain of mice.It was observed that there was no difference in K.p.bacterial clearance in RAW264.7 cells of lamtor2-/-group at 1h,but after 6h and 12 h,there was a significant decrease in K.p.bacterial clearance,and no change in phagocytosis was observed;?11?Western blot showed that the expression of p-p38 and p-JNK in RAW264.7 cells of lamtor2-/-and lamtor2+/+ groups was not changed after K.p.infection;?12?RT-q PCR analysis showed that the expression of TNF-?,1L-1?,IL-6,CXCL1 and MCP-1 m RNA in RAW264.7 cells of lamtor2-/-and lamtor2+/+ groups decreased after infection with K.p.6h.CONCLUSION:?1?The depletion of intestinal flora can aggravate the lung injury of K.p.pneumonia in mice,aggravate the systemic inflammatory response and increase the mortality;?2?The depletion of intestinal flora can reduce the levels of pro-inflammatory factors and chemokines in lung tissue of K.p.pneumonia,and weaken the antibacterial response in body;?3?Supplementation of intestinal microorganisms could reverse the aggravation of K.p.pneumonia and lung injury caused by intestinal flora depletion,and prolong the survival time of mice with intestinal flora depletion;?4?The depletion of intestinal microflora resulted in the decrease of phagocytosis and clearance of K.p.of alveolar macrophages in vitro;?5?Free diet with antibiotic solution can deplete the normal intestinal flora of mice;?6?The exhausted intestinal flora changed the metabolism of cecum contents,and the disordered metabolism was mainly related to carbohydrate and amino acid metabolism;?7?Correlation analysis showed that the number of SCFAs producing bacteria decreased and the number of harmful metabolites increased;?8?The depletion of intestinal flora changed the transcription profile of macrophages,and affected the cell defense response,antibacterial effect and MAPK / ROS signal pathway;?9?When SCFAs was added to mice with depleted intestinal flora or normal intestinal flora,it was beneficial to the prognosis of K.p.pneumonia;?10?SCFAs promotes the antibacterial effect of i NOS by up regulating LAMTOR2 to activate ERK signal in MAPK instead of p-JNK or p-p38 pathway. |
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