The Differences Of IL-1β Secretion Induced By High And Low Virulence Pasteurella Multocida Serotype A Infection

Pasteurella multocida（P.multocida,Pm）is a gram-negative bacterial pathogen,which causes a large number of diseases in mammals,birds and human.P.multocida is classified into five capsular serotypes A,B,D,E,or F and 16 Heddleston serotypes based on lipopolysaccharide（LPS）antigens.Different isolates of P.multocida have huge differences in virulence,even though they are the same serotype.P.multocida serotype A isolates are common residence in the upper respiratory tract and associated with respiratory diseases in animals.At present,the pathogenic mechanism of Pasteurella multocida and interaction with the immune response mechanisms of the host are still not fully understood.Macrophages,a member of the innate immune system,play an important role in host defense.The NLRP3 inflammasome,a multiprotein complex,is one of the well-studied players in the innate immunity,NLRP3 receptor can respond to diverse stimuli and form the inflammasome complex with pro-caspase-1 and apoptosis-associated speck-like protein containing CARD（ASC）.Activation of caspase-1 enables cleavage of interleukin-1βprecursor（pro-IL-1β）and subsequent secretion of the mature IL-1β.To date,the pathogenic mechanism of bovine P.multocida still remained unknown.By studying the inflammatory response of macrophages induced by P.multocida,we can give a further study of pathogenic mechanism of P.multocida.In this study,mouse peritoneal macrophages were chosen as the target cell to research cell inflammatory signaling pathways induced by two bovine P.multocida setotype A strains,including high virulence（PmCQ2）and low virulence（PmCQ6）.We used ELISA,RT-PCR,Western blot to detect the transcription,maturation and secretion of IL-1β,and transmission electron microscope（TEM）to detect capsular thickness of P.multocida.The main objectives of this research were as follows:to investigate whether（1）differences of inflammatory responses of mouse peritoneal macrophages were induced by PmCQ2（high virulence）and PmCQ6（low virulence）in vitro;（2）the cytokine IL-1βtranscriptional expression was different and which signal pathway was involved in this;（3）which inflammasomes were involved in the regulation of IL-1βproduction;（4）IL-1βproduction were affected by the capsular thickness of the two strains;（5）differences of inflammatory responses were induced by PmCQ2and PmCQ6 in vivo.The results are as follows:1.The differences of IL-1βsecretion of mouse peritoneal macrophages were induced by PmCQ2 and PmCQ6In this study,the morphology of bacteria and survival rates of mice intranasally inoculated with 1×10⁵ CFU PmCQ2 or PmCQ6,were determined.The results showed that the morphology of PmCQ2 and PmCQ6 were very different.Mice was all died at 6d post PmCQ2 infection.However,there were all survival in PBS and PmCQ6 infection.Next,to detect inflammatory responses induced by the two P.multocida strains,mouse peritoneal macrophages were infected with 1 multiplicity of infection（MOI）of PmCQ2and PmCQ6,respectively.The cell lysates and supernatants were collected at 24h post infection.The secretion of inflammatory cytokines IL-1β,IL-6,TNF-αand IL-12p40were detected by ELISA.The results showed that the secretion level of IL-1βinduced by PmCQ6 was significantly higher than PmCQ2,while the secretion of IL-6,TNF-αand IL-12p40 were not significantly different.This indicated that the mechanism of inflammatory reaction,especially IL-1βsecretion induced by those two strains,might be different.2.TLR2 were involved in IL-1βsynthesisIn order to investigate whether TLR2 and TLR4 were involved in the IL-1βproduction,the expression level of TLR2 and TLR4 were detected at 9h post infection.RT-PCR results showed that the level of TLR2 but not TLR4 increased significantly after macrophage were infected by PmCQ2 and PmCQ6.This indicated that TLR2 were involved in signal pathway activation related with pro-IL-1βsynthesis.3.p38 signaling pathways play different roles in PmCQ2 and PmCQ6 infectionTo further illuminate whether downstream signaling pathways,such as NF-κB,PI3K,Syk signaling pathways,were involved in IL-1βsecretion,macrophages were treated with some signaling pathway inhibitors for 1h respectively and then were treated with 1 MOI of PmCQ2 and PmCQ6.The secrection of IL-1βwere measured by ELISA and western blot.The results showed that the secretion of IL-1βwere decreased after the macrophage were incubated by NF-κB,PI3K,Syk signaling pathways inhibitors and caspase-1 inhibitor respectively.The secretion of IL-1βwere decreased significantly during macrophage infected with PmCQ6 after p38 signaling pathways inhibitor.However,p38 signaling pathways inhibitor have no effects on the secretion of IL-1βinfected by PmCQ2.The results indicated that p38 signaling pathways were involved in IL-1βsecretion during PmCQ6 instead of PmCQ2 infection.4.PmCQ2 and PmCQ6 induced the same extent of the gene expression of IL-1βIn order to explore the mRNA level of IL-1β,the expressions of IL-1βwas detected by real-time RT-PCR.The results indicated that the relative gene expression of IL-1βwere increased significantly to the same extent after PmCQ2 and PmCQ6 infection.This would suggest that gene expression of IL-1βwas not affected by PmCQ2 and PmCQ6 infection5.NLRP3 inflammasome was involved in the regulation of IL-1βsecretionIn order to investigate which inflammasomes were involved in host innate resistance to PmCQ2 and PmCQ6 infection,macrophages from C57BL/6 WT,caspase1^-/-,Asc^-/-,Nlrp3^-/-,Nlrc4^-/-,Aim2^-/-mice were infected with 1 MOI of each bacterium respectively.The secretion of IL-1βand IL-6 was detected by ELISA.The results showed that the secretion of IL-1βwas significantly decreased in caspase1^-/-,Asc^-/-,Nlrp3^-/-mice,while levels of IL-6 showed no differences among 6 kinds of mice after infection with PmCQ2 and PmCQ6.This indicated that NLRP3 inflammasome was involved in IL-1βsecretion.In order to explore the mRNA level of Nlrp3,the expressions of Nlrp3 was detected by real-time RT-PCR.At the same time,caspase-1 was detected by western blot.The results indicated that the Nlrp3 expression were increased significantly after PmCQ2 and PmCQ6 infection.However,compared with the PmCQ2 infection,Nlrp3expression level of PmCQ6 infection were much more higher.This would suggest that the extent of NLRP3 inflammasome activation were attached importance with IL-1βsecretion.6.Phagocytosis involved in IL-1βsecretion of inflammatory signaling pathwaysNLRP3 receptor is a intracellular PRR,how Pm activates it to induce the production of IL-1β.In order to investigate the relationship between phagocytosis and IL-1βsecretion,mouse peritoneal macrophages were treated with 1 MOI of PmCQ2 and PmCQ6.The number of bacteria adherent and invading macrophages were counted by plating on agar plates.The results showed that the number of PmCQ6phagocytosed/associated with macrophages was significantly more than PmCQ2,indicating that more PmCQ6 bacteria invaded into macrophages and PmCQ2,PmCQ6may cause the assembly of NLRP3 inflammatory corpuscles by entering cells.At the same time,after treating cells with Cyto.B,the secretion level of IL-1βinduced by PmCQ2 and PmCQ6 were reduced.In addition,to further verify these results,macrophages were infected with inactive PmCQ2 and inactive PmCQ6 respectively,and then supernatants were collected and tested for IL-1β,TNF-αand IL-6 by ELISA.The results indicated that the secretion level of those cytokines,whether IL-1β,TNF-αor IL-6,were all the same.However,according to the above study,the secretion of IL-1βfrom macrophages infected by active PmCQ6 was significantly higher than active PmCQ2.It indicated that phagocytosis might be involved in activation of inflammatory signaling pathways.7.The capsular thickness of PmCQ2 and PmCQ6 were involved in IL-1βsecretionMost importantly,the bacterial structure of PmCQ2 and PmCQ6 were examined by a transmission electron microscope.The results showed that the capsule of PmCQ2 was thicker than that of PmCQ6.Besides,the secretion of IL-1β,TNF-αand IL-6 were decreased significantly after treatment of PmCQ2 and PmCQ6 with 100 U hyaluronidase,which digests the main component of capsule of P.multocida.The above results suggested that the ability of PmCQ2 and PmCQ6 in adhesion and invasion of macrophages to the inflammatory response,especially IL-1βsecretion was affected by their capsular thickness.8.Differences of host inflammatory responses induced by PmCQ2 and PmCQ6Mice was intranasally inoculated with 1×10⁵ CFU bacteria PmCQ2 and PmCQ6,respectively.The pathological changes of lung tissue were determined.The results showed that there was extensive inflammatory cell infiltration in the lung after PmCQ2infection.However,there was only a slight inflammatory response in the lungs of PmCQ6 infection.Next,to detect inflammatory responses induced by the two P.multocida strains,mice was infected with 1×10⁵ CFU PmCQ2 and PmCQ6,respectively.The BALF and peripheral serum was collected.The secretion of inflammatory cytokines IL-1β,IL-6,and TNF-αwere detected by ELISA.The results showed that the secretion level of IL-1β,IL-6 and TNF-αin BALF induced by PmCQ2 was significantly higher than PmCQ6.However,the serum levels of those cytokines in the peripheral serum infected with PmCQ2 and PmCQ6 were very low,and there was no significant difference compared with the control group.This indicated that the mechanism of inflammatory reaction might be different.To the further study,mice was infected by PmCQ2 and PmCQ6,respectively.By counting the colonies of the mice lung tissue,the colonization of the bacteria in lungs was detected.The results showed that PmCQ2 was proliferating in lungs,while the colonization of the bacteria was not detected in the lungs of the mice infected by PmCQ6.The results suggest that the difference in bacterial colonization may lead to a difference in the inflammatory response.In vitro,the PmCQ6 was more likely to be phagocytosed by macrophages due to its thinner capsule,resulting in higher levels of inflammatory cytokines and the further analysis of related signaling pathways.This phenomenon might be explained that the host clears the low virulence strain-PmCQ6;At the time of host infection,high virulent strain-PmCQ2 due to thicker capsule and other reasons,colonization and anti-phagocytic ability were stronger,causing a strong inflammatory reaction,and high mortality of mice.