The Effect Of TLR4 On The Functional Activity Of Treg In Sepsis Mice And Its Mechanism

Objective:Study on the expression and role of toll like receptor 4 in septic mice,and effect of TLR4 on treg functional activity in sepsis.Methods:1?Effect of TLR4 on inflammatory response in sepsis:40 SPF grade healthy male C57/BL rats?wild type,WT?,40 SPF grade healthy male TLR4 knockout rat(TLR4^-/-),were selected and divided into SHAM operation group?WTSHAM group,n=20?,WTCLP group?n=20?,and TLR4^-/-Sham group?n=20?,TLR4^-/-CLP group?n=20?by random number table method.In CLP group,the rats were cacal ligation puncture?CLP?,in SHAM group,the rats were sham-operation.After 24h CLP,the serum levels of IL-2?IL-4?IL-10?TNF-?.2.Effect of TLR4 on treg functional activity in sepsis:40 SPF grade healthy male C57/BL rats?wild type,WT?,40 SPF grade healthy male TLR4 knockout rat(TLR4^-/-),were selected and divided into Sham operation group WT Sham group?n=20?,WT CLP group?n=20?,and TLR4^-/-Sham group?n=20?,TLR4^-/-CLP group?n=20?by random number table method.In CLP group,the rats were cacal ligation puncture?CLP?,in SHAM group,the rats were sham-operation.After 24h CLP,Treg counts,apoptsis rate,expression of Foxp3 mRNA?TLR4mRNA?Foxp3?TLR4?NF-?B?P65 were determined.Co-culture CD4⁺CD25⁺Foxp3⁺Treg and CD4⁺CD25^-T lymphocyte,after culturing and stimulation,cell culture supernatants were collected to determine the levels of TNF-??IL-2?IL-4 and IL-10 with ELISA.Result:1 Effect of TLR4 on inflammatory response in sepsis:1.1 Postoperative Sham group(include WT Sham group and TLR4^-/-Sham group)them act as usual,breathing as usual,eating and drinking water are normal,relieve themselves as normal,and reaction to the outside stimulation as usual.WT CLP group of mice activities are significantly lower than before,breathing deepening and quickening,their hair was messy and dull,pilo-erection,,drowsiness,shiver,eating and drinking water were decreased,and slow down to external stimuli,most of them have pyoperitoneum.TLR4^-/-CLP group of mice activities are significantly lower than before,the hair was slightly messy but shiny,only part of them occur pilo-erection and shiver,breathing deepening and quickening,eating was decreased,drinking water as normal,slow down to external stimuli.In WT mice,time to 50%survival rate was 48 h and to 40%survival rate was 72 h after CLP.In striking contrast,the survival rate of TLR4^-/-mice was 70%and 60%respectively.1.2 Postoperative 24 hours,Compared with TLR4^-/-CLP group and Sham group,the lung interstitium of WT CLP group was significantly enlarged,accompanied by congestion,bleeding and infiltration of large numbers of neutrophils,and the perivascular space increased.And compare to the TLR4^-/-CLP group,the injury of WT CLP group liver was hydropic and fatty degeneration heavier.1.3 Postoperative 24 hours,WT CLP group was higher in the levels ofIL-2?IL-4?IL-10?TNF-?than Sham group?P<0.05?;TLR4^-/-CLP group was lower in the levels of IL-4 and IL-10 and higher in the level of IL-2 and TNF-?than WTCLP group?P<0.05?.2 Effect of tlr4 on treg function and activity in sepsis:2.1 The expression of CTLA-4 on the surface of mouse splenic Tregs and intranuclear Foxp3 was markedly increased in WT CLP mice in comparison to the TLR4^-/-CLP and Sham mice?P<0.05?,the expression of TLR4 was also significantly increased in WT CLP mice compared to the others?P<0.05?,whereas in TLR4^-/-CLP mice,the expression only slightly increased compared to Sham mice?P>0.05?.2.2 The expression of Foxp3 mRNA and TLR4 mRNA in splenic Tregs was markedly increased in WT CLP mice in comparison to Sham control?P<0.05?,whereas in the TLR4^-/-CLP mice,neither Foxp3 nor TLR4 mRNA remained slightly elevated compared with sham?P>0.05?.In contrast,expression of both factors was higher in WT CLP than in TLR4^-/-CLP?P<0.05?.2.3 The percentage of Tregs in CD4+T lymphocyte increase both in WT CLP and TLR4^-/-CLP mice?P<0.05?,although the level in WT CLP mice was significantly higher compared with that in TLR4^-/-CLPmice?P<0.05?.In contrast,the number of Tregs was decreased both in WT CLP and TLR4^-/-CLP compared with Sham mice?P<0.05?.The apoptosis of Tregs in CLP(both WT CLP and TLR4^-/-CLP)groups was apparently lower than that in Sham?P<0.05?,although that in the TLR4^-/-CLP group was apparently higher than in WT CLP?P<0.05?.2.4 Compared with the WT CLP group,the levels of secreted IL-2 and TNF-?of TLR4^-/-CLP were higher?P<0.05?.Conversely,the secretion of IL-4 and IL-10 was significantly decreased compared with the WT CLP group following co-culture with stimulated Tregs?P<0.05?.2.5 The expression of NF-?B and p65 in splenic Tregs was markedly increased in WT CLP mice in comparison to TLR4^-/-CLP mice?P<0.05?.Conclusion1.1 Lacking of TLR4 can reduce the secretion of inflammatory factors,and alleviate the immune damage caused by hyperproinflammatory response,improve the survival rate in experimental sepsis.1.2 The effect of a lack of TLR4 in experimental sepsis aligns closely with the suppressive function of Tregs.1.3 TLR4 in experimental sepsis aligns closely with the suppressive function of Tregs may via the TLR4/NF-?B signaling pathway.