Research On The Effect And Mechanism Of IL-17A In Streptococcus Suis Infection

Streptococcus suis（S.suis）is an emerging zoonotic pathogen,which not only cause huge economic losses in porcine industry of the world,but also represents a significant threat the public health of humans.S.suis can cause multiple diseases,including streptococcal toxic shock-like syndrome（STSLS）,meningitis and sepsis.The infections unusually caused two large-scale human S.suis epidemics,in which 240 humans were infected and 53 died in China.And 97.4%fatal cases were observed with the development of STSLS.Moreover,meningitis is the most common pathogenic feature of S.suis infection.Therefore,it is vital to clarify the mechanism of STSLS and meningitis caused by S.suis.The high expression of suilysin（SLY）was required for the highly pathogenic S.suis strain[sequence type（ST）7]SC-19 to cause NLRP3 hyperactivation,which is essential for the induction of cytokines storm,dysfunction of multiple organs,and a high incidence of mortality,the characters of STSLS.And the highly pathogenic S.suis strain SC-19 was gradually evolved from ST1 strain（P1/7）and got the ability for high expressing of SLY.However,it remains to be elucidated whether acquiring high hemolysis activity is the vital reason for the SC-19 strain to cause STSLS.IL-1βand IL-18 could be activated after NLRP3 hyperactivation,further induced high level of IL-17A,but how NLRP3 activation causing STSLS through IL-1β、IL-18、IL-17A remain to be analyzed.In addition,IL-17A was also highly expressed in S.suis meningitis,and IL-17A contributed to the disruption of tissue barriers.Therefore,the role and mechanism of IL-17A in the S.suis meningitis are also worthy of in-depth exploration.Aiming at the above scientific problems,this study carried out the research on the effect and mechanism of STSLS caused by the strain P1/7 overexpressing SLY,to analyze whether the high level of hemolysin activity is the key reason of SC-19 leading to STSLS.And studying on the role of IL-1β,IL-18 and IL-17A in STSLS,to analyze which cytokines contribute to STSLS after NLRP3inflammasome activation.Considering the potential role of IL-17A on the disruption of tissue barriers,the role and mechanism of IL-17A in S.suis meningitis were studied.The main results are as follows:1.High expression of SLY in ST1 strain P1/7 could cause STSLS,by activating NLRP3 signal pathway.To analyze whether high hemolytic activity is the key reason for SC-19 suddenly leading to STSLS,we constructed the strain P1/7-SLY overexpressing the SLY by transferring SLY expression plasmid to S.suis serotype 2 strain P1/7.Besides the strain P1/7-m SLY overexpressing the mutant SLY（protein without hemolytic activity）and the control strain P1/7-Vec were also constructed.Subsequently,we evaluated its hemolytic activity,founding that compared with P1/7-Vec and P1/7-m SLY,P1/7-SLY showed higher level of hemolytic activity,which was similar to the highly pathogenic strain SC-19.We also evaluated the activation of NLRP3 inflammasome based on THP-1 cell model.We found that P1/7-SLY could obviously increase the inflammasome activation,including high level of cleavage of pro-caspase-1,pro-IL-1βand GSDMD,and high level of IL-1βsecretion and cell death,which was dependent on NLRP3 signal.In contrast,P1/7-m SLY overexpressing the mutant SLY and P1/7-Vec could not significantly increase the inflammasome activation.Furthermore,similar to the highly pathogenic strain SC-19,P1/7-SLY could cause STSLS in wild type mice but not in nlrp3^-/-mice,which is characterized by cytokines storm,dysfunction of multiple organs,and a high incidence of mortality.In contrast,P1/7-m SLY could not cause STSLS in wild type mice and nlrp3^-/-mice.In summary,we demonstrated that overexpression SLY could enable ST1 strain（P1/7）to cause NLRP3 inflammasome hyperactivation,which could further cause cytokines storm and STSLS.Noting that acquiring high expression of SLY was the key factor of SC-19 leading to STSLS.2.IL-18 played a vital role in STSLS after NLRP3 inflammasome hyperactivation.NLRP3 inflammasome hyperactivation cause high level of IL-18 during STSLS development.To further investigate the role of IL-18 in STSLS caused by S.suis,we used the highly pathogenic strain SC-19 to infect the il18r1 knockout mice(il18r1^-/-)and wild type mice.We found that the deficiency of IL-18 receptor could alleviate the clinical signs,tissue damages and acute death.In addition,we found that the bacterial loads of il18r1^-/-mice tissues were similar to wild type mice,but the levels of blood cytokines such as IL-17A and IFN-γwere significantly reduced in S.suis-infected il18r1^-/-mice.The data indicated that the deficiency of IL-18 signals could effectively inhibit the inflammatory cytokines storm,multi-organ injury,and reduced acute death in mice caused by S.suis.Therefore,we concluded that IL-18 was responsible for the STSLS development after NLRP3 inflammasome hyperactivation.3.IL-17A contributed to bacterial clearance at the early stage of S.suis serotype2 infection,but did not play a main role in STSLS development.S.suis infection regulated the expression of IL-17A through IL-18,after the NLRP3inflammasome activation.However,the role of IL-17A during STSLS remains to be elucidated.To further elucidate the function of IL-17A in S.suis infection,the study used SC-19 to infect the il17a knockout mice(il17a^-/-).And we found that compared to wild type mice,S.suis-infected il17a^-/-mice could also cause multi-organ dysfunction,and acute host death,which were the characterized of STSLS.However,the level of cytokines storm in blood and bacterial loads in different tissues did not decrease but increase.These results suggested IL-17A was not a key effect factor of STSLS,and even played a role in controlling infection at the early stage of infection.To further analyze the effects of IL-17A in controlling infection,this study evaluated the levels of blood neutrophils and antibacterial peptides in infected mice.And the results showed the lower level of activation of neutrophil and expression of antimicrobial peptides in il17a^-/-mice The data indicated that IL-17A contributed to clear the bacteria at the early stage of S.suis infection by mediating the activation of immunocytes and the production of antibacterial substances,but did not play main role in STSLS development.4.IL-17A could downregulate the transcription and expression of TJs,which undoubtedly facilitated the leakage of blood-CNS barriers for bacterial invasion and then caused meningitis during S.suis serotype 9 infection.IL-17A was highly expressed in S.suis meningitis,and IL-17A contributed to the disruption of tissue barriers.However,the role of IL-17A during S.suis meningitis remains to be elucidated.This study conducted a mouse infection experiment with a S.suis serotype 9 strain S29.And we found that 50%of mice died within 48 h post-infection.Besides,the rest 50%mice exhibited obvious nervous signs,meningitis,and high level of IL-17A after 60 h post-infection,and gradually died.To further evaluated the function of IL-17A in S.suis meningitis,we used S29 to infect the il17a^-/-mice and wild type mice.In contrast,the infection also caused 50%of il17a^-/-mice dead within 48 h,but the rest of il17a^-/-mice did not present any obvious nervous clinical signs,no damage of meninges and finally survived during the trial.These results indicated that IL-17A might not contribute to acute death but contribute to the nervous clinical signs and death caused by bacterial invasion during the meningitic S.suis infection.To further analyze the mechanism of the IL-17A promotion bacteria through Blood-CNS barriers into the brain,the study evaluated the expression of TJs in S29-infected mice.And we found that the transcription and expression of TJs（e.g.,ZO-1,Occludin and Claudin-5）were significantly decreased in the brains of S29-infected wild type mice.However,the decreased transcription and expression levels of TJs were significantly alleviated in the brains of S29-infected il17a^-/-mice,suggesting that IL-17A could inhibit the transcription and expression of TJs（e.g.,ZO-1,Occludin and Claudin-5）,and facilitated bacteria through blood-CNS barriers into the brain during S29 infection.The data indicated that IL-17A could downregulate TJ proteins,which undoubtedly facilitated the leakage of blood-CNS barriers for bacterial invasion and then caused S.suis meningitis.In summary,the study found that high expression of SLY in the ST1 strain could cause cytokines storm and STSLS through NLRP3 inflammasome hyperactivation.Indicating that acquiring high SLY expression was the key factor for the highly pathogenic S.suis strain SC-19 to cause STSLS.We also indicated that S.suis serotype 2could through NLRP3 inflammasome activation mediated high level of IL-18 to cause STSLS.Although IL-17A played a minor for bacteria clearance at the early stage of S.suis infection,it did not contribute to STSLS development.In addition,the present study indicated that IL-17A could downregulate TJs,undoubtedly facilitated the leakage of blood-CNS barriers for bacterial invasion,and then caused S.suis serotype 9 meningitis,providing a potential target for future prevention and treatment of this disease.