Plasmids expressing interleukin-10 short hairpin RNA mediate IL-10 knockdown and enhance tumor necrosis factor alpha and interferon gamma expressions in response to porcine reproductive and respiratory syndrome virus

摘要

Porcine reproductive and respiratory syndrome virus (PRRSV) has been suggested to exploit interleukin-10 (IL-10) to suppress immune defense of infected pigs. The present study constructed plasmids encoding selected short hairpin RNA specific to porcine IL-10 mRNA (pIL-10sh) to knockdown IL-10 transcription and investigated the suppressive effect of PRRSV-induced IL-10 on various immune marker expressions. Naive blood monocytes from eight PRRSV-seronegative pigs were transfected with pIL-10sh and pNeg (plasmid vector) prior to PRRSV inoculation and subsequent lipopolysaccharide (LPS) stimulation. The mRNA expressions of IL-10, IL-1 beta, IL-12p40, tumor necrosis factor alpha (TNF alpha), interferon gamma (IFN gamma), transforming growth factor beta (TGF beta), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNF alpha, and IFN gamma protein productions were determined by ELISA. Compared with non-transfected monocyte control, transfection with selected pIL-10sh (pIL-10sh1), but not other pIL-10sh nor pNeg, significantly reduced IL-10 expression and significantly enhanced TNF alpha and IFN gamma expressions. Slight increases in IL-1 beta, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGF beta expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNF alpha and IFN gamma, and co-stimulatory molecules, CD80 and CD86. (C) 2012 Elsevier B.V