Role of Hsp90 in CpG ODN mediated immunostimulation in avian macrophages

In mammals, CpG mediated immune activation is initiated through toll-like receptor (TLR) 9 and Hsp90 via activation of MAPK/ERK and PI3K/AKT pathways. However, in the absence of TLR9 ortholog in chicken genome, the role of Hsp90 and kinase (MAPK/ERK and PI3K/AKT) pathways in initiating CpG ODN(2007) induced immune activation in chicken is not clear. Using electrophoretic mobility shift assay (EMSA) and selective inhibitors of signal transduction pathways, we determined the role of these pathways in the production of Th1 cytokines/chemokines and nitric oxide (NO) in CpG ODN(2007) treated avian macrophage cells. Hsp90alpha but not Hsp90beta is bound to CpG ODN(2007). Inhibition of Hsp90 with geldanamycin resulted in the inactivation of MAPK/ERK and PI3K/AKT pathways leading to significantly reduced levels of IFN-gamma, IL-6 and NO mRNAs in CpG ODN(2007) stimulated cells. Moreover, inhibition of ERK1/2 and PI3/AKT kinase pathways with PD985009 and LY294002, respectively, suppresses the phosphorylation of ERK2 and AKT leading to the production of decreased amounts of IFN-gamma, IL-6 and NO mRNAs in CpG ODN(2007) stimulated cells. Our results demonstrate that binding of CpG ODN(2007) to Hsp90 induces activation of ERK2 and AKT phosphorylation leading to the production of high levels of IFN-gamma, IL-6, MIP-3alpha and nitric oxide (NO). In contrast to mammals, our results suggest that Hsp90alpha but not Hsp90beta binds with the CpG ODN(2007) and may play a major role in CpG ODN(2007) induced immunoactivation in avian macrophage cells. To our knowledge, this is the first report evaluating the involvement of Hsp90 and kinase (MAPK/ERK and PI3K/AKT) pathways in CpG mediated immunostimulation in avian macrophage cells.