title: 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.
abstract: 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. Naïve 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-1β, IL-12p40, tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), transforming growth factor beta (TGFβ), CD80, and CD86 were evaluated by real-time PCR. The IL-10, TNFα, and IFNγ 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 TNFα and IFNγ expressions. Slight increases in IL-1β, IL-12p40, CD80, and CD86 expressions were also observed. Neither pIL-10sh1 nor pNeg transfection affected TGFβ expression. Our results indicate that PRRSV does exploit IL-10 to suppress the expressions of pro-inflammatory cytokines, mainly TNFα and IFNγ, and co-stimulatory molecules, CD80 and CD86.
authors.affiliation: Program of Biotechnology, Faculty of Science, Maejo University, Chiang Mai 50290, Thailand. wasin@mju.ac.th
authors.forename: Wasin
authors.initials: W
authors.lastname: Charerntantanakul
authors.forename: Watchara
authors.initials: W
authors.lastname: Kasinrerk
mesh_terms: D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D015967:Gene Expression Regulation, Viral; D007371:Interferon-gamma / Q000235:genetics / Q000276:immunology*; D016753:Interleukin-10 / Q000235:genetics / Q000276:immunology*; D009000:Monocytes; D010957:Plasmids / Q000235:genetics / Q000276:immunology; D019318:Porcine Reproductive and Respiratory Syndrome / Q000276:immunology / Q000821:virology*; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D013552:Swine; D014162:Transfection / Q000662:veterinary; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology*
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D007371:Interferon-gamma