The Roles Of IELs And IECs In Resistance To Salmonella Typhimurium Infection

Objects:The intestine is in touch with numerous food antigens and it is the main site of microorganisms settle. Intestinal mucosal immune system is the first line that host against intestinal pathogen, at the same time, it plays an important role in establishing and sustaining homeostasis between host and external environment.S. typhimurium is the major cause of diarrhea and illness due to food sourse. The pathogenic factor of S. typhimurium includes virulence island SPI, pSLT virulence plasmid, adhesin, flagella, etc. Intestinal immune cells play a vital role in defense of pathogen infection. Intestinal intraepithelia lymphocyte (IELs) were embedded in the intestinal epithelial cells (IECs), IELs and IECs collaborate to form the first defensive line against foreign infection.Intestinal epithelial cells play an important role in response to intestinal mucosal immune, and it is the key physical barrier to prevent excessive pathogens and other antigens to invade the intestine. Studies have shown that the tight junction of intestinal epithelial cells, the secretion of cytokines and antimicrobial peptides may play an important role in pathogen defensin. IELs is the unique and cytotoxicity-related lymphocytes and play an important role in resistant to pathogen infection through maintaining the epithelial barrier function and regulating native and adaptive immune. Thus, intestinal epithelial cells and intraepithelial lymphocytes can play an important role during pathogen infection. While epithelial cells produce cytokines such as IL-15, which have a significant impact on the proportion and function of CD8αα+IELs. At the same time, IL-15can promote the expression of apoptotic related molecules Bcl-2and Bcl-xl, which affect the function of intraepithelial lymphocytes. Intestinal intraepithelial lymphocytes can highly express IFN-y, TNF-a, which regulates the epithelial cell tight junction molecules. However, at present the mutual regulation of epithelial cells and intraepithelial lymphocytes is still less studied, In this study, according to the establishment of intestinal infection model, we observed the vital role of IELs and IECs after infected with S. typhimurium. Meanwhile, we researched on whether there is a mutual regulation in the course of pathogen defense, thereby provide a new direction for treatment of infectious intestinal diseases.Methods:1. C57BL/6mice were orally infected with S. typhimurium. We observed the change of weight, intestinal shape, HE stain to determine whether the model was established successfully.2. The expression of inflammasome (NLRP3, NLRC4) in IECs was detected by quantitative PCR and Western Blot.3. The change of defensins in IECs was measured by quantitative PCR and Western Blot. And the alteration of ROS was detected by Flow cytometry.4. Flow cytometry show the expression of activated molecules (CD44and CD69) and killing related molecules (NKG2D, CD107a, IFN-y) after S. typhimurium infection.5. Flow cytometry analysis the expression of cytotoxity-related molecules in IELs after IL-1β stimulation.6. The cytotoxicity of IL-1β stimulated IELs against infected IECs was measured by LDH assay at different E:T ratios.7. After adding anti-IL-1β blocking antibody, the killing efficiency of IELs against infected epithelial cell were measured by LDH at different E:T ratios.8. The influence of infected IECs on chemotaxis for IELs was determined by Transwell.Results:1. Successfully established intestinal infection model of S. typhimurium infection.2. The expression of inflammasome (NLRP3, NLRC4) in IECs increased during S. typhimurium infection, which promoted the activation of caspase-1and the secretion of IL-1β.3. The expression of bacteriocidal substances (ROS,β-defensin3,β-defensin4) were enhanced in response to S. Typhimurium infection.4. The population of CD8+TCRγδ+IELs were up-regulated, at the same time, the activation related molecules(CD44, CD69) and killing related molecules (NKG2D, CD107, IFN-y) were changed in resistance to S. Typhimurium infection.5. The activation related molecules (CD44, CD69) and killing related molicules (NKG2D, CD107a, IFN-y) was changed on CD8αα+IELs, CD8α+CD8p+IELs in resistance to S. Typhimurium infection.6. The cytotoxicity of S. typhimurium activated IELs against infected IECs was up-regulated.7. IL-1β strengthened the cytotoxicity of IELs against S. typhimurium-infected IECs, and the killing efficiency was weakened after adding anti-IL-1β antibody.8. Chemokines produced by infected IECs accelerated chemotaxis for IELs.Conclusion:1. Based on the activation of inflammasome and the secretion of bacteriocidal substances, IECs played an important role in resistance to S. typhimurium infection.2. IELs, particularly CD8a+TCRγδ+IELs, are critical for the clearance of pathogenic bacteria following S. typhimurium infection.3. IL-1β strengthened the cytotoxicity of IELs against S. typhimurium-infected IECs and promoted the clearance of pathogen in IECs.4. Chemokines facilitated the recruitment of IELs to IECs, and promoted cytotoxicity of IELs against S. typhimurium-infected IECs.