Investigation Of Molecular Mechanisms Involved In Pneumococcal DnaJ Regulates Dendritic Cells Inducing T Cell Immune Response

ObjectiveStreptococcus pneumoniae is a gram-positive conditioned pathogen that causes many invasive diseases, including meningitis, sepsis and community-acquired pneumonia. The occurrence of drug-resistance bacteria made vaccination the effective approaches to control S.pn infections. Pneumococcal DnaJ is a virulence related heat shock protein, it can induce protective immune response against S.pn when used for intranasal or intraperitoneal immunizations in mice. Studying the host immune mechanism induced by DnaJ contributes to the development of vaccine.Dendritic cell is known to be the most effective professional antigen presenting cell that plays an important role in innate immunity and adaptive immunity to pathogen. Various Hsps of other bacteria could activate DC maturation. Recently, pneumococcal Hsp100/C1pP was found to be recognized by TLR4, and trigger a Thl immune response through activate DC depend on MAPK and NF-κB pathways. Howerver, as a kind of Hsp, whether DnaJ can induce DC maturation, what the mechanism involved in DC maturation and the effects on T cell differentiation has not been reported. In this study, we aim to investigate the efficiency of DnaJ on BMDC maturation and the mechanism of activated BMDC inducing T cell immune response.Methods1. After recombinant DnaJ protein(rDnaJ) expressed in Escherichia coli underwent purification, lipopolysaccharide (LPS) was removed from it.2. To investigate the mechanisms involved in BMDC activation that induced by rDnaJ protein.(1) After BMDCs were stimulated by rDnaJ protein, the expressions of MHCII, CD86 and CD40 on BMDC were measured by FCM; TNF-a, IL-6 and IL-12p40 levels were detected by ELISA.(2) BMDCs derived from TLR2KO, TLR4 KO and WT mice were stimulated by rDnaJ protein, then the expressions of MHCII, CD86 and CD40 were measured by FCM; the secretion of TNF-α, IL-6 and IL-12p40 levels were detected by ELISA.(3) The effects of pretreatment of mitogen-activated protein kinase(MAPK) inhibitors, nuclear factor-kappa B(NF-κB) inhibitor and phosphatidylinositol-3-kinase (PI3K) inhibitor on the secretion of TNF-a and IL-6 induced by rDnaJ protein were also evaluated by ELISA in BMDC; The phosphorylation levels of MAPK, NF-κB and Akt were determined by Western blot.3. To investigate the mechanism of T cell response induced by rDnaJ protein(1) To detect the effect of rDnaJ-stimulated BMDC on T cell differentiation Co-cluture rDnaJ-stimulated BMDC with CD4+ T cells for 36h, the expressions of IFN-y, IL-4 and IL-17A in the culture supernatant were detected by ELISA; percentages of IFN-y+-producing CD4+ T cells, IL-4+-producing CD4+ T cells and IL-17A+-producing CD4+T cells.(2) To evaluate the effect of rDnaJ-stimulated BMDC on T cell differentiation through adoptive transfer test WT mice were transferred intraperitoneally with rDnaJ-treated BMDCs on dayO,14 and 28. The levels of IFN-y, IL-4 and IL-17A in the supernatant were measured by ELISA after splenocytes removed from the transferred mice re-stimulated by rDnaJ protein for 36h.(3) To detect the roles of TLR2 and TLR4 in induction T cell response in vivo WT, TLR2 KO and TLR4 KO mice were devided into 3 groups respectively:CT+rDnaJ group, CT group and PBS group. Mice were immunized on dayO,7 and 14, then the splenocytes of immunized mice were stimulated with rDnaJ protein for 3d and IFN-y, IL-4 and IL-17A levels were detected by ELISA.Results1. Recombinant plasmid pET-28a(+)-dnaJ/was successfully constructed, the rDnaJ protein reached a high purity after induction and expression; the residual LPS would not affect the subsequent experiment after LPS removed from protein solution with PmB-agarose.2. The murine BMDCs were successfully cultured and reached a purity of more than 80%.3. rDnaJ protein could induce murine BMDC maturation, and promoted high expression of surface markers(MHC-II, CD86 and CD40)as well as secretion of cytokines(TNF-a, IL-6 and IL-12p40).4. rDnaJ protein induced murine BMDC maturation in a TLR4-dependent manner, but this process did not depend on TLR2.5. p38, ERK, JNK, AKT and NF-κB pathways were involved in murine BMDC maturation induced by rDnaJ protein.6. The mature murine BMDC that stimulated by rDnaJ protein promoted naive CD4+T cells to differentiate into Th1 and Th17 cells, induce the host Thl and Th17 immune response.ConclusionsThis study indicated that rDnaJ protein could induce strong BMDC immune response, which depended on TLR4 rather than TLR2, and regulated by MAPKs, AKTå'ŒNF-κB pathways. Mature BMDC stimulated naive T cells to differentiate into Thl and Th17, induce host Th1 and Th17 immune response. In this study, we illustrated the mechanism of host immune response induced by pneumococcal DnaJ protein, thus lay the foundation for developing pneumococcal DnaJ protein as a protein vaccine.