The Polymorphisms Of TLR4 Gene And Function Analysis Of C1027A In Suzhong Pig

Toll-like receptors (TLRs) are among the molecules called pattern-recognition receptors (PRRs), which specifically recognize a broad class of pathogen-associated molecular patterns (PAMPs) and initiate the innate as well as the adaptive immune defense. As one member of TLRs, TLR4 is type I transmembrane protein with an extracellular domain containing leucine-rich repeats and a cytoplasmic Toll/IL-1 receptor (TIR) domain homologous to that of the interleukin 1 (IL-1) receptor. TLR4 plays a crucial role in triggering the innate response to lipopolysaccharide (LPS), which is a component of Gram-negative bacterial cell wall and probably the most powerful microbial stimulant of innate immune, by acting as a signal-transducing receptor. TLR4 specifically recognizes lipopolysaccharide and mediates the activation of nuclear factor-κB (NF-κB) and proinflammatory cytokines, by MyD88-dependent and MyD88-independent pathways.Objective:TLR4 is an important immune-associated gene, and its polymorphism has a profound influence on responses to a wide range of pathogens and underlies different resistance/susceptibility patterns to infectious diseases. This study speculates on the polymorphism of TLR4 and function analysis of the mutation (C1027A) in its coding sequence in Suzhong pig.Method:PCR-SSCP was used to search the mutations of TLR4 gene in 127 samples of Suzhong pig to approach the distribution of SNPs in this population. The expression levels of TLR2,4 and proinflammatory cytokines TNF-α, IL-1βwere detected by real-time PCR in porcine alveolar macrophages (PAMs) which were induced by LPS. The PAMs were grouped by their different genotypes (CC and AC) in 1027bp of coding sequence(CDS) to discuss whether this mutation of TLR4 influenced its sensitivity to LPS.Results:1 Three nonsynonymous SNPs were found in the leucine-rich repeats domain of TLR4 gene:T611A, G962A and C1027A. In those SNPs,611bp (Leu204His) and 1027bp (Gln343Lys) changed the character of amino acid and caused acquisitions of charge. In the whole population, only C1027A was moderate polymorphism and others were low polymorphism.2 LPS(200 ng/ml) can markedly increased TLR4 mRNA expression (P<0.01) in 20 min in porcine alveolar macrophages(PAMs) and maintained a high level in 48 h. The expression levels of proinflammatory cytokines TNF-α, IL-1βwere also significantly enhanced by LPS, but TNF-αmRNA elevated faster than IL-1β.3 PAMs had different sensitivity and response to LPS between genotype CC and AC. A significant difference of TLR4 expression appeared at 20 min in the two groups(P<0.001), and mRNA level of genotype CC was higher than that of genotype AC before 12 h. And differences of TNF-αmRNA expression were highly significant at 20 min(P<0.001), and in the same period(1h), when the TNF-αexpression level of group CC began to upregulate markedly, the level in group AC has been its 5 times(P<0.05). However, differences of IL-1βexpression in the two genotypes began at 12 h (P<0.05), and continued a long time. IL-1βmRNA of genotype AC was higher than that of genotype CC, and this marked difference lasted until 48 h(P<0.01).Conclusions:1 The coding sequence of TLR4 was relative conservation in Suzhong pig. Population genetics analyses showed that this population had low polymorphisms and genetic variation.2 The mutation in 1027bp in coding sequence of TLR4 can affect the binding ability to LPS. PAMs with CC allelotype had more significant ability in regulation of TLR4 than allelotype AC and showed high sensitivity to LPS. However, the lower mRNA expression of proinflammatory cytokines demonstrated there was a lighter inflammatory reaction in allelotype CC. So, alleles C in 1027bp of TLR4 may be the resistance gene to Gram-negative bacterial infections in Suzhong pig.