The effect of exogenous HSP70 on expression kinetics of HSP70, TLR2, and TLR4 in peripheral blood mononuclear cells and possible crosstalk between them in Black Bengal goat

Abstract

ABSTRACT The present study aims to investigate the modulatory effect of eHSP70 on major heat stressed genes in in-vitro cell culture system. HSP70 (eHSP70) on mRNA expression profile of heat-shock protein 70 (HSP70), toll-like receptor 2 (TLR2) and TLR4 in peripheral blood mononuclear cells (PBMCs) in cell culture model of Black Bengal goat. After thermal challenge of cultured PBMCs were treated with a specific dose of eHSP70 (50 ng/ml) at 37°C for 0, 1, 2, 4, 8, 12, 16, 20, and 24 h. The mRNA expressions of the above-mentioned genes were determined by quantitative real-time PCR (qRT-PCR). The study showed that on administration of eHSP70 to the PBMCs cell culture system, the relative mRNA expression of iHSP70 and HSP90 increased significantly (p < 0.05) at 4, 20 and 24 h compared with control, and and TLR2 and TLR4 showed significantly higher expression (p < 0.05) at 2, 4, 8 and 24 h compared with control. Immunoreactivity revealed that all genes under study were localized in cytoplasm as well as in nucleus. The present findings specify that eHSP70 is probably involved in heat stress adaptive response in Black Bengal goat and the biphasic expression pattern may be providing a second window of protection during heat stress. Abbreviations: ANOVA: analysis of variance; GAPDH: gleceraldehyde 3-Phosphate dehydrogenase; bp: base pairs; cDNA: complementary DNA; Ct value: cycle threshold values; DEPC: diethyl pyrocarbonate; dNTP’s: deoxynucleoside triphosphate; DW: distilled water; DPBS: dulbecco’s phosphate buffer saline; FBS: fetal bovine serum; HSP: heat-shock protein; HSF: Heat shock factors; IG: immunoglobulin; KDa: kilo dalton; LN2: liquid nitrogen; NF-κB: nuclear factor kappa beta; TLR: toll-like receptor; PBMC: peripheral blood mononuclear cell; eHSP70: exogenous heat-shock protein 70.