Study Of Regulation Of TLRs Expression And Molecular Mechanism Of Resistance To Salmonella In Chickens

Toll-like receptors (TLRs) signaling pathways are the first lines in defense against Salmonellainfection. Ordinarily, TLRs signaling is activated immediately when chickens infected with Salmonella,but the molecular mechanism underlying susceptibility to S. enteritidis infection in chickens remainsunclear. This research included three sections:Section1: The transcriptional regulation of TLRs in peripheral blood mononuclear cells (PBMCs) ofchickens infected in vitro with LPS or Salmonella. PBMCs were segregated from20specific-pathogen-free (SPF) chickens with30d old. Three treatments including LPS treatment,Salmonella enteritidis (S. enteritidis) infection and control groups were designed. The expression of9TLRs at1h,6h and24h post infection in PBMCs were investigated. In order to verify the effect of S.enteritidis infection dose on the expression of TLR4, TLR2-1and TLR21, PBMCs infected with S.enteritidis with1×10~9to1×10~4CFU with10folds dilution for6h. The results are as follow:(1)Expression of9TLRs including TLR4, TLR2-1and TLR21in PBMCs were upregulated by LPS orSalmonella.(2) At6h, expression of TLR4and TLR2were significantly upregulated in LPS treatmentgroup (P <0.05), but expression of TLR4was significantly downregulated compared with that ofcontrol (P <0.05), there was not significant change of expression of TLR21and TLR2(P>0.05). At24h, the upregulation of TLR21and TLR1-2was greater in Salmonella-treated group while thetranscription of TLR4, TLR1-1, TLR2(TLR2-1and TLR2-2) increased more significantly in LPS group(P <0.05).(4) The expression of TLR4, TLR21and TLR2-1decreased with different concentration ofSalmonella treatment. The depression of TLRs enhanced with the increase of concentration in the caseof low concentration (0～1×10~6CFU/mL) of Salmonella but go to the opposite direction in highconcentration (1×10~6～1×10~9CFU/mL).Section2: The effect of transcriptional regulation of TLRs, pro-inflammatory genes and negativeregulators of TLRs on the susceptibility to Salmonella in Chickens. This section aimed to demonstratethe molecular mechanism underlying susceptibility to S. enteritidis infection. SPF chickens injectedwith8.7×108CFU of S. enteritidis were partitioned into two groups, one consisted of those fromSalmonella-susceptible chickens (died within5d after injection, n=6), the other consisted of sixSalmonella-resistant chickens that survived for15d after injection. The results showed that:(1) Thepresent study showed that the bacterial load1.1×10~7CFU in susceptible chickens was significantlyhigher than that in resistant chickens at16h post infection (P <0.05).(2) Temperature of resistantchickens with42.6℃at16h was significantly higher than41.9℃at0h (P <0.05), but the increase oftemperature in susceptible chickens was not significant (P>0.05); The temperature of susceptiblechickens with41.1℃on3d was significantly lower than that of resistant chickens with42.3℃(P <0.05). Body weight of susceptible chickens decreased after16h but that of resistant chickens increased;Body weight of susceptible chickens with212.7g was significantly lower than that of resistant chickenswith252.9g (P <0.05).(4) Expression of TLR4, TLR2-1and TLR21was strongly diminished in theleukocytes of susceptible chickens compared with those of resistant chickens (P <0.05).(5) The induction of expression of IFN-β and pro-inflammatory cytokine genes like IL-6, was greatly enhancedin the resistant chickens but not in susceptible chickens (P <0.05).(6) Contrasting with the reducedexpression of TLRs genes, which of the zinc finger protein493(ZNF493) gene and Toll-interactingprotein (TOLLIP) gene were enhanced in the susceptible chickens.Section3: The role of epigenetic regulation in the depressed transcription of TLRs in susceptiblechickens. The main task of this section was to confirm whether ZNF493-related epigenetic modificationaccounted for the diminished expression of TLRs in susceptible chickens. The results demonstrated that:(1) The expression of TLR4in peripheral blood mononuclear cells (PBMCs) infected in vitro with S.enteritidis increased significantly as a result of treatment with5-Aza-2-deoxycytidine (5-Aza-dc) ortrichostatin A (TSA) while either5-Aza-dc or TSA was effective in up-regulating the expression ofTLR21and TLR2-1.(2) DNA methylation, in the predicted promoter and proximal promoter region ofTLR4and TLR21genes, and an exonic CpG island of the TLR2-1gene was significantly higher in thesusceptible chickens than that in resistant chickens. Taken together, the results demonstrate thatZNF493-related epigenetic modification in leukocytes probably accounts for increased susceptibility toS. enteritidis in chickens by diminishing the expression and response of TLR4, TLR21and TLR2-1.