The Effect Of Recombinant Staphylococcal Enterotoxins On Porcine Innate Immunity

Staphylococcal Enterotoxins(SEs) function as a superantigen to promoteactivation of T cells and overproduction of cytokines and result in extensive systemicinflammation and shock. The rapid effects and strong pro-inflammatory ability ofSEs imply their impacts on the mechanism of innate immune response. Toll-likereceptor belonging to pattern recognition receptor has led an important role in earlyinnate anti-infection to later modulate acquire immunity, but its response to SEsstimulation is still unclear. Thus, we studied the function of SEs on inducing thetranscription and expression of porcine TLRs and inflammatory cytokines so that toexplore the molecular mechanisms in innate immune response caused by enterotoxins.RT-PCR assay was applied to clone eight Toll-like receptors (TLR1,2,4,5,6,8,9,10) from porcine alveolar macrophages. By using bioinformatics, weanalyzed the single nucleotide acid polymorphisms (SNPs) and amino functionaldomains of TLRs, predicted their structures and ligand recognition domains, exploredthe impact of mutations on TLR function. The results showed an individual differenceand species specificity in TLR genes with non-synonymous mutations located inextracellular region, suggesting their pathogen recognition diversity. The predictedTLRs are type I transmembrane proteins with extracellular leucine-rich repeat regions(LRRs); secondary structures are mainly composed of α-helix, β-strands; TLRmonomer and dimer are respectively horseshoe and "M" shape.An relative fluorenscence quantitative PCR assay was established to detect genetranscription.By analyze its specificity, sensibility and repeatability to optimize PCRcondition, we get50℃and2times dilution of primary cDNA for PCR reaction.Thenthe transcription and expression of TLR1-10and IL-1β、MCP-1、MIP-1β、GM-CSF、TNF-α、IFN-γ、IL-2、IL-4were detected by this assay and some by ELISA kits.Enterotoxins can cause a significant increase in TLR1,2,6,10mRNA transcription,resulting in a large number of pro-inflammatory cytokines, such as IL-1β, TNF-α,GM-CSF, chemokines MCP-1and MIP-1β,thus lead enhanced cytokine storm. MTTassay showed SEs can induce PBMCs or T cells proliferation. Intestinal ligationexperiments showed that five kinds of enterotoxins(except SEU) can cause varyingdegree of porcine intestinal edema, inflammatory cells increase and intercellular spaces dilatation. TLR6transcripts in different SEs-treated intestines were allup-regulated, while the influences of different enterotoxins in intestine were distinctfor a certain TLR or cytokine and different TLRs or cytokines were in variouschanges by the same enterotoxin.In summary, SEs can activate the innate immune molecules TLR1,2,6,10andtheir signal transduction pathways, stimulate inflammatory cytokine to product，TLR6might be the PRR of SEs; SEs can stimulate lymphocyte proliferation or leadmacrophages immune responses that lead to cytokine storm, causing multi-systeminflammation or pig intestine local inflammation.