| Background Insights into the host-pathogenic virulence factors interaction, especially the immune-signaling mechanisms, can provide novel prevention and treatment options for pneumococcal diseases.Streptococcus pneumoniae endopeptidase O(PepO) is a newly discovered and ubiquitously expressed pneumococcal virulence protein. Compared with their isogenic wild-type strains,PepO-mutant strain impaired the ability of adherence and invasion to host cells. Our study showed PepO has a strong inflammatory effector in mice lung. It is still unknown whether PepO is involved in host defense response against pneumococcal infection.Alveolar macrophages paly a important role in defensing against bacterial infection of respiratory tract. The removal of the S. pneumoniae began in macrophages. Hence this study intended to macrophages as the research object, to explore PepO effect on macrophages and the related mechanism..Methods Classic macrophage phagocytosis observation ability change of peritoneal macrophages stimulated by PepO; Q- PCR detect miR- 155 and SHIP1 mRNA expression in macrophage induced by PepO;Western blot detect SHIP1, AKT, NF- kB protein expression in macrophage induced by PepO; Overexpression or down-expression miR-155 test the phagocytosis of macrophages and SHIP1 mRNA expression;The signaling pathway inhibitor combined PepO processes macrophages, Q- PCR detect the expression of miR- 155;The luciferase report gene detects the luciferase activity that miR-155 targeted SHIP1; Immunofluorescencetests the phagocytosis of macrophage against bacteria,the expression of surface receptor CR3, and the change of NF- kB nuclear transfer.Results Our studies showed that phagocytosis against pneumococci and Staphylococcus aureus was enhanced in macrophages treated by PepO,and the ability was in a time and dose dependent manner. Q-PCR shows that PepO increased the expression of micro RNA-155(mir-155) in PEMs in a time and dose dependent manner. PepO- induced enhanced phagocytosis was decreased in cells transfected with inhibitor of mir-155,while increased in cells transfected with mimic of mir-155. It suggested that PepO- induced enhanced phagocytosis via increasing the expression of miR-155. Luciferase Reporter Assay SHIP1, luciferase activity as significantly decreased in cells transformed with miR-155, it can confirm miR-155 targets SHIP1. Q-PCR and Western blot showed PepO decreased the expression of SHIP1 in PEMs in a time and dose dependent manner.Overexpression or down-expression miR- 155, the mRNA expression of SHIP1 was decreased or increased. This suggested miR-155 induced by PepO could target SHIP1. CR3, as one receptor of macrophages to identify bacterial, some literature reported that SHIP1 negatively regulates CR3, our immunofluorescence test result shows that CR3 significantly increased in PepO group compared with negative control group.These results indicate that PepO may induce miR-155 to down-regulate SHIP1 expression,thereby promoting CR3 expression to increase and improve the phagocytosis of macrophage.Separately handle TLR2 KO and WT mice peritoneal macrophages with PepO, the results show that expression of miR-155 was decreased significantly in TLR2 KO macrophages compared with WT, the expression of SHIP1 did not reduced along with PepO, and the expression level of CR3 was not up-expression like WT. PepO-induced upregulation ofmir-155 was blocked by PI3K inhibitor and I κ B- α phosphorylation inhibitor. The phosphorylation Akt was notely actived by PepO,phosphorylation NF-κB and nuclear translocation are increased.Nevertheless PepO failed to stimulate the phosphorylation of p65 and still can activate p-Akt in TLR2 deficient PEMs, which proves that PepO induces the expression of mir-155 via TLR2- NF-κB signaling pathway.Conclusions Taken together, PepO induces up-regulation of mir-155 to down-regulate SHIP1 in PEMs through TLR2- NF- κB signaling pathway, contributing to enhanced the expression of CR3 and phagocytosis. |
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