title: Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages.
abstract: The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.
authors.affiliation: Institute of Animal Science, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany. misl@itw.uni-bonn.de
authors.forename: Mohammad Ariful
authors.initials: MA
authors.lastname: Islam
authors.forename: Mehmet Ulas
authors.initials: MU
authors.lastname: Cinar
authors.forename: Muhammad Jasim
authors.initials: MJ
authors.lastname: Uddin
authors.forename: Ernst
authors.initials: E
authors.lastname: Tholen
authors.forename: Dawit
authors.initials: D
authors.lastname: Tesfaye
authors.forename: Christian
authors.initials: C
authors.lastname: Looft
authors.forename: Karl
authors.initials: K
authors.lastname: Schellander
mesh_terms: D000367:Age Factors; D000818:Animals; D000831:Animals, Newborn; D005786:Gene Expression Regulation; D015850:Interleukin-6 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D016676:Macrophages, Alveolar / Q000187:drug effects* / Q000378:metabolism; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D051193:Toll-Like Receptors / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D012333:RNA, Messenger; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha