Secretion of TNF-α induced by heat shock protein 10 of Chlamydophila pneumoniae were mediated by TLR2 and TLR4 in THP-1

Abstract

Objective To investigate the effect of heat shock protein 10 (HSP1O) of Chlamydophila pneumoniae in inducing TNF-α on THP-1 cells and the roles of TLR4 and TLR2 involved in it.Methods Purified native recombinant HSP10 from Cpn(CHSP10) were produced and inactivated the endotoxin contamination,then different concentration (0.5,1,5,10,20,30 μg/ml) of CHSP10 were used to stimulate THP-1 for different time (0,6,12,24,36,48,60 h).TNF-α were measured by using human TNF-α ELISA kit and compared among different groups.THP-1 were collected and analyzed for TLR2 and TLR4 mRNA levels and protein expression by RT-PCR and immunofluorescence.Peritoneal macrophages isolated from wide-type (C3 H/HeN) and TLR4-deficient mice (C3H/HeJ) were stimulated with endotoxin-free proteins respectively,and the TNF-α were measured.Furthermore,neutralizing anti-human TLR2/TLR4 McAb as a blocking Ab was preincubated with THP-1,after stimulation with CHSP10,ELISA was used to detect the concentration of TNF-α.Results TNF-α can be induced with CHSP10 in THP-1,while it significantly decreased with heated or deproteinized CHSP10.Both TLR2 and TLR4 mRNA and protein were detected in THP-1.Macrophages from C3H/HeN mice displayed higher TNF-α compared with it from C3H/HeJ mice after stimulation with CHSP10.The CHSP10-induced TNF-α would obviously decline when treated with antiTLR2/TLR4 McAb.Conclusion As a potential inflammation related protein,CHSP10 are involved in the pathogenesis of Cpn inducing inflammation cytokine TNF-α.TLR2 and TLR4 appear to be involved in CHSP10-mediated expression of TNF-α. Key words: Chlamydophila pneumoniae(Cpn) ; Heat shock protein 10; Toll like receptor; Tumor necrosis factor-α