YghJ (SslE), a cell associated and secreted lipoprotein of neonatal septicemic E. coli induces TLR2 dependent macrophage activation and proinflammation through NFκB and MAP kinase signaling

Abstract

Background: YghJ (SslE), a cell surface associated and secreted lipoprotein was identified as a potential vaccine candidate for extraintestinal pathogenic E. coli. We showed YghJ from neonatal septicemic E. coli could trigger secretion of various proinflammatory cytokines in murine macrophages, the signaling pathway of which is still obscure. Methods & Materials: Cell surface receptor and down-stream adaptors of YghJ in murine macrophages (RAW264.7 cells) were identified by western blot and immunoprecipitation. Involvement of NFκB and MAP kinase in YghJ mediated cytokine secretion was determined using specific inhibitor against each signaling cascade and subsequent ELISA, immunoblot and immunofluorescence. Overexpression of iNOS was detected by RT-PCR. Production of chemokines was detected by RT-PCR and ELISA. Production of nitric oxide (NO) and reactive oxygen species (ROS) were detected by Griess assay and DCFDA staining respectively. Engagement of TLR2 was established using TLR2 siRNA knocked down RAW264.7. Flow cytometry was performed to detect overexpression of MHC II and co-stimulatory molecules on macrophages. Results: We found that YghJ specifically binds to TLR2/TLR1 heterodimer on RAW264.7 and follows MyD88 dependent pathway. Pretreatment of macrophages with specific inhibitors against each signal molecule showed the involvement of ERK1/2, JNK1/2 and NFκB in secretion of IL-1 (IL-1α and IL-1β) and involvement of p38 and ERK1/2 in secretion of TNF-α in a TLR2 dependent manner as confirmed by using TLR2 knocked down cells. Moreover, our study unveils that YghJ can stimulate TLR2 dependent production of NO and ROS, which contribute to the anti-infection immune response of the host. Furthermore, YghJ produced M1 proinflammatory chemokines such as RANTES, MIP-1α and MIP-1β and increased expression of MHC-II and other co-stimulatory molecules (CD80 and CD86) on macrophages. Conclusion: YghJ induces proinflammatory cytokine secretion in RAW via canonical pathway of TLR2 through NFκB and MAP kinase. The production of M1 chemokines, ROS and NO reflects that YghJ promotes activation and M1 polarization of macrophages, crucial in framing host's innate immune response to this protein. Furthermore, over-expression of MHC-II, CD80 and CD86 hints that YghJ may skew the innate response towards Type 1 adaptive response supporting previous observation that YghJ can be a potential vaccine candidate in neonatal sepsis.