| Background Leptospirosis is a worldwide spread of animal-borne diseases, the causative agents of leptospirosis(leptospirefor short), belonging to the Leptospira genus.Almost all the continents are found the existence of the pathogen except for Antarctica, especially in tropical and subtropical countries. Rodents, pigs and dogs are the main reservoir hosts. Leptospires can be discharged to the outside world through the urine of these hosts.Human is infected mainly through contacting with contaminated water and soil with acute or chronic infection.The clinical symptoms varies from mild flu-like symptoms to severe pulmonary hemorrhage or renal failure, and even death. L.interrogans can be divided into 25 serogroups and over 260 serotypes at least, China has found 19 serogroups and 161 serotypes.During the early period of leptospiral infection, the innate immune system plays an important role to kill the leptospires,which can be recognized by the monocyte/macrophage in the blood or tissue. Studies show that scavenger receptor A(SR-A),a type of pattern recognition receptors of macrophage, plays a very important role in defencing againstmicrobial pathogens.LPS, lipoproteins,teichoic acid and other bacterial Component or intact bacteria can interact with SR-A. However, There is no reports of the interaction between leptospires and SR-A until now.Methods Primary murine macrophages, murine macrophage cell lines and L.interrogans strain 56606 v are as the main subjects of this project,which focuses on the interaction between L.interrogans strain 56606 v and macrophages.To detect whether macrophages recognise, adhere and phagocytose L.interrogans through SR-A,which Component of leptospires involve in the interaction with SR-A and how how the expression changes of SR-A and cytokine m RNA.To explore phagocytic pathways of the interaction between leptospires and SR-A. The steps are as follows: Firstly, SR-A, IL-6, TNF-α and IFN-β1 m RNA expression changes were detected in primary and passaged murine macrophages with leptospiral infection using Real-time PCR.Secondly, using poly I, a specific chemical inhibitor of SR-A,and monoclonal antibody(2F8) to inhibit the primary murine peritoneal macrophages and RAW264.7 cells, the ratio of adhesion and phagocytosis between macrophages and leptospires was detected by flow cytometry. Thirdly, mouse SR-AΙ and SR-A II were recombined into p DS-red2-N1 and transfected to non-phagocytic cells-Chinese hamster ovary cells(CHO-K1), then G418 was used to screen the stably transfected cell lines of high expression SR-A, and detect its adhesion and phagocytosis to leptospires. Fourthly, to confirm SR-A-mediated leptospires by macrophages activatingthe natural immune systems, peritoneal macrophages from SR-A gene knockout mouse were used to detect the ratio of adhesion and phagocytosis.According to the hot phenol-water method, we extracted and purified leptospiral LPS and quantified using the Limulus test, then CHO-K1 cells transfected SR-A were stimulated with different concentrations of leptospiral LPS, flow cytometry detected the phagocytosis ratio of FITC-labeled beads into p DS-Red2-N1-SR-AⅠ/Ⅱ-CHO-K1.At the same time, Real-time PCR detected the expression changes of SR-A, IL-6, TNF-α and IFN-β1 m RNA in primary and passaged murine macrophages after leptospiral LPS stimulation.Finally, Pull down detected the activity changes of GTPase Rac and GTPase Cdc42 in the p DS-Red2-N1-SR-AII-CHO-K1 after leptospiral infection to explore phagocytic pathway of leptospires, SR-A and Rac/cdc42.Results Real-time PCR detected that SR-A and IL-6 m RNA expression were significantly increased in RAW264.7 cells after L.interrogans strain 56606 v infection. When alternatively primary murine peritoneal macrophages could be obtained the same results, which suggest that SR-A may be involved in macrophage phagocytosis and the body’s inflammatory response during leptospiral infection. Results of specific chemical inhibitors Poly I and SR-A monoclonal antibody on macrophages showed that in the absence opsonization of serum complement, both of which can effectively inhibit the phagocytosis of the CFSE-labeled leptospires by murine macrophages, especially when the leptospires were inactivated by 4% paraformaldehyde, inbibitional effect was more significant. The inhibition rate was up to about 50%.Mouse SR-AΙ and SR-A II were recombined into p DS-red2-N1 and transfected to non-phagocytic cells-CHO-K1,the stably transfected cell lines of high expression SR-A were screened by G418. Flow cytometry results showed that compared with the p DS-red2-N1-CHO-K1, p DS-Red2-N1-SR-A-CHO-K1 could significantly increase the adhesion and phagocytosis ratio of leptospires(P<0.05). However,there was no significant difference between p DS-Red2-N1-SR-AⅠ-CHO-K1 and p DS-Red2-N1-SR-A II-CHO-K1(P>0.05). To confirm the above experimental results, we used peritoneal macrophages from the SR-A gene knockout mouse to interact with leptospires. Results showed that the adhesion and phagocytosis ratio was significantly lower than normal controls, which had a significant difference(P<0.05).Extracted and purified L.interrogans strain 56606 v LPS using hot phenol-water method was stained by Coomassie brilliant blue and silver staining, there was no obvious the bacterial miscellaneous protein,and with LPS size 15 k Da-25 k Da. Purified leptospiral LPS were used to stimulated p DS-Red2-N1-SR-A Ⅰ / Ⅱ-CHO-K1 with different concentrations(250ng/ml 500ng/ml) after quantitative determination.Results showed that phagocytosis capacity to Latex beads could besignificantly increased after leptospiral LPS stimulation. SR-A m RNA was significantly higher in either primary mouse peritoneal macrophages or RAW264.7 cells with dose-dependent after LPS stimulation. Meanwhile, the expression changes of IL-6, TNF-α and IFNβ1 m RNA were also significantly increased.Pull down results showed that GTPase Rac and GTPase Cdc42 in p DS-Red2-N1-SR-AⅡ-CHO-K1 increased after leptospiral infection,while there were no significant changes in control.Conclusions Murine macrophages phagocytose L.interrogans strain 56606 v through SR-A. SR-A plays an important role in macrophages during the process of phagocytosis. Leptospiral LPS can be recognized and bounded by SR-A, and be able to stimulate macrophages, resulting in increased expression for SR-A m RNA and different changes for IL-6, TNF-α and IFN-β1m RNA. Protein expression of GTPase Rac and GTPase Cdc42 increased after leptospiral infection. These results suggest that during the process of leptospiral infection, macrophages can adhere and phagocytose leptospires through SR-A and actived the downstream Rac and Cdc42 protein of the phagocytic pathway.The leptospiral LPS participates in the recognition and binding process of SR-A to activate the body’s natural immune system and inflammatory response. The experimental results will provide a reliable experimental evidence and theoretical basis for the in-depth study of leptospires after invasiving host,which defenses against leptospiral infection and plays natural immunity, increase our understanding of pathogenic mechanisms of leptospiral immune response(including innate immune response and adaptive immune response), and contribute to molecular vaccine research as well as drug targets screening. |
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