The Roles And Mechanisms Of TLR2 And NLRP3 In Leptospira Infection

Leptospires are divided into two types: pathogenicity leptospires and non-pathogenic leptospires.Leptospirosis,caused by pathogenicity leptospires,is a wide spread zoonosis worldwide.Pigs,horses,cows,sheep,and humans can all be infected by leptispira to induce fever,jaundice,miscarriage and even death,which is harmful to our husbandry production and people's health.Until now,the detailed pathogenic mechanism of leptospira is still dimness.After leptospira infection,leptospira can rapidly spread to all tissues and organs throughout the body.The innate immune system is the first line of defense against pathogen identification.It will be significant to prevention and treatment of leptospirosis with studying the immunological recognition between the innate immune system and leptospira.It is reported that leptospira was recognized by TLR2 and TLR4.Both TLR2 and TLR4 are on the cell surface.The intracellular pattern recognition receptor NLRP3,also recognize the leptospira.After activation of TLR2 and TLR4,the cytokines IL-1,IL-6,TNF-a and so on will be expressed via nuclear transcription factor Nf-?B and MAPK signaling pathway.The activation of NLRP3 inflammasome needs two signals.One signal is the transcription of inflammatory cytokines via TLRs.Another signal is forms of inflammasome complex by ASC oligomerization and caspase-1 activation.Then the Pro-IL-1? will turn into mature IL-1?,which will be released to the outside of the cell.It is reported that TLR4-deficient mice and TLR2,TLR4-double deficient mice will died rapidly after leptospira infection.As susceptible animals of leptospira,hamsters also possess TLR2 and TLR4.What are the functions of TLR2 and TLR4 in susceptible animals after leptospira infection? Because leptospira can penetrate the cell membrane and pass into the cell,except NLRP3,whether will other inflammasome recognize leotospira or not? Are there differences of the roles of TLR2,TLR4 and NLRP3 between resistant and susceptible animals? The answer to these issues will contribute to further reveal the pathogenesis of leotospira,lay the theoretical and experimental foundation for the development of new targeted biological agents.In this thesis,the roles and mechanisms of TLR2,TLR4,NLRP3 and NLRC4 were studied by comparing between susceptible hamsters and resistant mice.The application value of new targeted biological agents was also discussed.The results indicated that TLR2 but not TLR4 transcript levels in BALB/c mice contrast with delayed induction in hamsters.So,In consideration of the early regulation of TLR2 in mice but not hamsters,TLR2 might play an important role in the development of leptospirosis.To verify this hypothesis,TLR2-deficient C57 mice were used for leptospira infection.And TLR2 agonist,Pam3CSK4,was used to treat leptospira infected hamsters.It was found that TLR2 deficiency aggravates the pathological changes of mice after leptsopira infection.As expected,treatment with Pam3CSK4 improved survival rate of hamsters.This indicated that the induction of TLR2 in early phase of leptospirosis would control leptospira infection.To further explore the molecular mechanism of TLR2,the downstream cytokines were analyzed in leptospirosis compared between mice and hamsters.And the IL-10/TNF-? ratio was studied in wild-type and TLR2-deficient mice and hamsters treated with or without Pam3CSK4.It was found that the expression of TLR2 was synergistic with IL-10.Then,through cells experiments,it indicated that the leptospira-induced IL-10 expression was TLR2 dependent.These results indicated that the activation of TLR2 contributed to control leptospira infection.This process might be achieved by raising the IL-10 or the IL-10/TNF-? ratio.The expressions of NLRP3 and NLRC4 were analyzed between C57 mice and hamsters with leptospira infection.It was found that NLRP3 but NLRC4 was delayed induction in hamsters compared with mice.This indicated that the different expressions of NLRP3 might be responsible for the different outcomes of leptospirosis in mice and hamsters.Then,using NLRP3-deficient C57 mice,the suevival and leptospira burden in organs were analyzed.Although the survival rates were not changed between wild-type and NLRP3-deficient mice,the leptospira burdens in organs were lesser and the histopathological changes were mild in NLRP3-deficient mice than wild-type mice.The IL-1? level was not correlative with the leptospira burden.These results indicated that the enhancement of leptospira scavenging in NLRP3-deficient mice was not dependent on the downstream IL-1?.To verify this point,macrophages of NLRP3-? NLRC4-? Caspase-11-?Caspase-1-11-deficient C57 mice were extracted for leptospita infection.It was found that leptospira-induced IL-1? was depended on NLRP3.Then,hamsters and C57 mice infected with leptospira were treated with diacerein,the inhibitor of IL-1?.The result indicated that diacerein had no effect on survival and leptospira burden in organs.These results indicated that the enhancement of leptospira scavenging in NLRP3-deficient mice was really not dependent on the downstream IL-1?.In addition to promote the IL-1? maturity,NLRP3 can also regulate the autophagy level.Whether was the enhancement of leptospira scavenging in NLRP3-deficient mice depended on the improvement of autophagy level or not? The result indicated that the deficiency of NLRP3 significantly increased the autophagy level of kidney in C57 mice infected with leptospira.Although the expression of NLRP3 was delayed,the autophagy level did not increase in hamsters infected with leptospira.These results indicated that the deficiency of NLRP3 promoted to clear leptospira in organs.This process might be achieved by raising the level of autophagy.In order to further verify the role of TLR2 and NLRP3 in leptospira infection,the animals and cells were treated with doxycycline,which was the preferred drug for the treatment of leptospirosis.It was found that doxycycline also modulated immune response in controlling leptospira infection in addition to its antibacterial action.Treatment with the doxycycline,the IL-1? level induced by leptospira was decreasing in both animals and cells.The TLR2 expression was increased compared with the decreasing expression of NLRP3 with the treatment of doxycycline.Moreover,doxycycline suppressed the NF-?B and MAPK signal pathway in leptospira infection.And doxycycline attenuated leptospira-induced IL-1? by suppressing NLRP3 inflammasome Priming.These results indirectly proved by doxycycline that increasing TLR2 and inhibiting NLRP3 were beneficial to the treatment of leptospirosis.In conclusion,TLR2 and NLRP3 played an important role in leptospira infection.The activation of TLR2 could effectively control leptospira infection.This process might be achieved by raising the IL-10 or the IL-10/TNF-? ratio.The deficiency of NLRP3 promoted to clear leptospira in organs.This process might be achieved by raising the level of autophagy.Doxycycline increased the expression of TLR2,suppressed the NLRP3 expression and NLRP3 inflammasome Priming.It indicated that increasing TLR2 and inhibiting NLRP3 were beneficial to the treatment of leptospirosis.