Researches On The Molecular Mechanisms By Which Streptococcus Pneumoniae Endopeptidase O Enhances The Phagocytosis By Macrophages Via SHIP1

Objective Increasing evidences demonstrate that microorganism and their metabolites protect against bacterial and viral pathogens through various mechanisms including immunomodulation.PepO is a virulence protein that is ubiquitous and conserved in different serotypes of Streptococcus pneumoniae capsular serotypes.Previous research demonstrated that PepO enhanced the phagocytosis by macrophages in a TLR2 and miR-155 dependent manner.However,the precise molecular mechanisms are still unclear,further research is still needed.We detected down-regulation of SHIP1 expression and up-regulation of CR3 expression in macrophages stimulated by PepO.In this proposal we will deeply explore the underlying mechanisms through investigating the role of SHIP1 and CR3 in modulating the PepO-induced phagocytosis by macrophages.Methods After transfection of SHIP1 over-expression plasmid or CR3 interfering RNA into PEMs in vitro,Western blot was used to detect the expression of SHIP1 and CR3 protein to verify transfection efficiency.Macrophages were stimulated with PepO after transfection,the phagocytosis ability of PEMs to S.aureus and S.pn was compared by phagocytosis experiments.After transfection of SHIP1 over-expression plasmid or SHIP1 interfering RNA into PEMs in vitro,the mRNA andprotein expression levels of SHIP1 and CR3 were detected by Q-PCR and Western blot,respectively.And flow cytometry was used to detect the expression of CR3 on the cell surface.Model of nasal infection in C57BL/6mice by nasal drip in vivo.Nasal instillation of PepO was followed by nasal infection with S.pn and S.aureus,and the differences in bacterial load in lung tissue and nasal lavage fluid were compared.Western blot was used to detect the expression of SHIP1 and CR3 in BALFs after intranasal instillation of PepO,and the expression of CR3 on the cell surface was detected by flow cytometry.After SHIP1 over-expression plasmid or CR3 interfering RNA was instilled into the nasal cavity of mice,Western blot was used to detect the expression of SHIP1 or CR3 in BALFs to verify the transfection efficiency.After transfection,PepO protein was instilled into the nasal cavity of mice and then infected with S.pn and S.aureus.The bacterial load in lung tissue and nasal lavage fluid was counted by plating.Results1.The SHIP1 overexpression plasmid up-regulated the expression of SHIP1 effectively in PEMs.Qualitative and quantitative results showed that the number of phagocytosed S.aureus and S.pn within PepO-treated macrophages was significantly smaller in the experimental group than in the control group.These results indicate that the down regulation of SHIP1 mediates the enhanced phagocytosis of S.aureus and S.pn by PepO-stimulated macrophages.2.The CR3 interfering RNA down-regulated the expression of CR3 effectively in PEMs.Qualitative and quantitative results showed that the number of phagocytosed S.aureus and S.pn within PepO-treated macrophages was significantly smaller in the experimental group than in the control group.These results indicate that the up regulation of CR3 mediates the enhanced phagocytosis of S.aureus and S.pn byPepO-stimulated macrophages.3.Both CR3 mRNA and protein were down regulated in the SHIP1 over expression group with PepO treatment.However,the expression of CR3 was up regulated in the SHIP1 interfering group with PepO treatment.Our researches show that the expression of CR3 is regulated by SHIP1 in the current system,and the down regulation of SHIP1 mediates the up regulation of CR3,thereby enhancing the phagocytosis ability to S.aureus and S.pn.4.The number of S.aureus and S.pn in nasal lavage fluids and lung tissues was significantly smaller in the PepO group than in the PBS group at each time point,which show that PepO promotes the clearance of S.aureus and S.pn and alleviates their infection in mice.Besides,the expression of SHIP1 decreased and CR3 increased in BALFs after PepO treatment.5.The expression of SHIP1 was up regulated after intranasal with SHIP1 over-expression plasmid,and bacterial load experiments confirmed that the ability to clear S.aureus and S.pn at various time points with PepO was weakened,which indicated that down-regulation of SHIP1 helps enhancing the elimination of S.aureus and S.pn in PepO-treated mice.6.The expression of CR3 was down regulated after intranasal with CR3 interfering RNA,and bacterial load experiments confirmed that the ability to clear S.aureus and S.pn at various time points with PepO was weakened,which indicated that up-regulation of CR3 helps enhancing the elimination of S.aureus and S.pn in PepO-treated mice.Conclusion Taken together,PepO enhances the phagocytosis of macrophages by down-regulating SHIP1 and up-regulating CR3.This study provides a new preventive and therapeutic option for respiratory infectious diseases and lays the theoretical basis for the development ofPepO as an immunomodulation agent.