Abstract
We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.
Highlights
OmpA activates innate immunity through TLR2, but its function and mechanism of targeting adaptive immunity remains unexplored
TLR2-dependent Transcriptional Activation of NF-B and Cytokine Production by OmpA of S. flexneri 2a—The observation that TLR2 mediates cellular responses to structures from numerous microbial cell wall constituents and may be central in host recognition of diverse bacterial pathogens [27] prompted us to investigate whether recognition of OmpA by TLR2 is critical for initiation of the innate immune response
Because transcription factor NF-B has been implicated in the expression of numerous cytokines and chemokines, we investigated the role of TLR2 in mediating NF-B activation in HEK 293 cells treated with OmpA
Summary
OmpA activates innate immunity through TLR2, but its function and mechanism of targeting adaptive immunity remains unexplored. The present study has been undertaken to elucidate whether OmpA of S. flexneri 2a is recognized by TLR2 at the onset of the innate immune response and mold it toward the adaptive immune system through activation of CD4ϩ T cells as well as the involvement of IL-12p70 and NO in the mechanism of action. The cells act by promoting adaptive responses in type I and type II directions, as well as by expressing specialized and polarized effector functions [12, 13] This is initiated by innate recognition of pathogen-associated molecular patterns by the pattern recognition receptors of the host, such as Tolllike receptors (TLRs), on macrophages or other mononuclear phagocytes. It has been observed that when OmpA is presented by TLR2 knockdown macrophages, macrophage surface expression of MHCII and CD80 as well as CD4ϩ T cell production of IFN-␥ in the macrophages:CD4ϩ T cells co-culture was noticeably reduced
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