The Mechanism Of IL-1α Maturation And Secretion In Macrophages Infected With Corynebacterium Pseudotuberculosis

Corynebacterium pseudotuberculosis can infect a variety of animals and humans,including goats,sheep,horses,and cattle.The infection of C.pseudotuberculosis in goats and sheep mainly causes abscesses and caseous lesions in superficial lymph nodes or internal organs,and results in caseous lymphadenitis（CLA）,which is characterized by caseous lymph nodes lesions.In recent years,reports of the disease have been increasing in China,with up to 53%of goat farms exist this pathogen infection in some areas,and flock prevalence rates as high as 30-80%,once the goats or sheep are infected with C.pseudotuberculosis,it is extremely difficult to eradicate it,and making it one of the most important diseases seriously affecting the healthy and rapid development of the sheep industry in China.Therefore,it is urgent to clarify the pathogenicity of C.pseudotuberculosis infection and the host immune response mechanism,and provide guidance for effective prevention and control of this disease.C.pseudotuberculosis infection caseous comprehensive inflammatory response,characterized by a massive increase in inflammatory cytokines at the infected site.Therefore,it is important to clarify the mechanism of inflammation caused by C.pseudotuberculosis infection.IL-1αis considered to be an extremely special multifunctional protein type molecule among many cytokines.It can be used as a nuclear transcription factor to regulate the expression of related genes in the cell;It can also be used as an"alarmin"factor by binding to IL-1R1 receptor and promotes the production of inflammatory responses,and plays a key role in the occurrence and development of pathogenic microbial infections,spontaneous inflammation,and tumors.Therefore,IL-1αcan be used as an important target for the prevention and treatment of related diseases.Our previous study found that C.pseudotuberculosis infection mediated the mature secretion of IL-1βthrough the NLRP3 inflammasome-dependent pathway.Although both belong to the IL-1 family cytokines,there are obvious differences between the mature secretion process of IL-1αand IL-1β.At present,the detailed mechanism of the maturation and secretion of IL-1αinduced by C.pseudotuberculosis infection is still relatively vague.Therefore,in this experiment,we explore the mechanism by which C.pseudotuberculosis induces IL-1αmaturation and secretion in macrophages.1.The effect of C.pseudotuberculosis infection on IL-1αmaturation and secretion in macrophagesMouse peritoneal macrophages were infected with C.pseudotuberculosis Xuanhan strain（XH02）at different multiplicity of infection（MOI=1,10,50,100）,and the secretion of IL-1αwere detect by ELISA.Further,peritoneal macrophages were infected with C.pseudotuberculosis at MOI of 10,and the m RNA of IL-1αwas detected by real-time fluorescent quantitative PCR（q PCR）,the levels of IL-1αin supernatant were detected by ELISA,the mature form of IL-1α,and precursor form of IL-1α（pro-IL-1α）were detected by Western blotting.The results showed that at MOI=1,10,and 50,the IL-1αsecretion of macrophages infected by C.pseudotuberculosis increased with the increasing of the MOI.Repeated experiments confirmed that the IL-1αm RNA expression level,pro-IL-1αexpression level and mature form of IL-1αsecretion in C.pseudotuberculosis infected macrophages were significantly increased.The above results indicate that C.pseudotuberculosis infection can activate the expression,the maturation and of secretion IL-1αin macrophages.2.The role of phospholipase D in the maturation and secretion of IL-1αinduced by C.pseudotuberculosis macrophagesPhospholipase D（PLD）is one of the most important virulence factors of C.pseudotuberculosis,which is closely related to the spread and pathogenicity of the pathogen.To evaluate the role of PLD in the inflammatory response of this pathogen infection,the peritoneal macrophages were infected with international standard strain of C.pseudotuberculosis ATCC19410 and its pld gene-deficient strain（ATCC19410Δpld）,clinical isolate strain XH02 and its pld gene-deficient strain（XH02Δpld）,the levels of secreted cytokines including IL-1β,IL-1α,IL-6 and TNF-αin culture supernatants were detected by ELISA,the m RNA expression of IL-1β,IL-1α,TNF-αand IL-6 were detected by q PCR;At the same time,the mature form of IL-1αin supernatant,and precursor form of IL-1α（pro-IL-1α）in cell lysates were detected by Western blotting.The results showed that,the m RNA expression and secretion of IL-1α,IL-1β,TNF-αand IL-6 in ATCC19410Δpld-infected and XH02Δpld-infected macrophages were significantly decreased compared with those of ATCC19410-infected and XH02-infected macrophages,except for the decreasing of TNF-αsecretion in XH02Δpld-infected macrophages,which did not reach a significant level.However,there was no significant difference on the expression of pro-IL-1αin the cell lysis in macrorphages infected with ATCC19410 and ATCC19410Δpld,or infected with XH02 and XH02Δpld.The above results indicated that C.pseudotuberculosis PLD was involved in the maturation and secretion of IL-1αin macrophages infected by this pathogen.3.The role of NLRP3 inflammasome in the maturation and secretion of IL-1αin macrophages infected by C.pseudotuberculosisPeritoneal macrophages from C57BL/6 wild-type（WT）and NLRP3 inflammasome components knockout mice(Asc^-/-,Caspase1^-/-and Nlrp3^-/-)were respectively infected with C.pseudotuberculosis XH02.ELISA and Western blotting were used to detect IL-1αmaturation and secretion.The results showed that the secretion of IL-1αin macrophages from Asc^-/-and Caspase1^-/-mice were significantly decreased compared with those in macrophages from WT mice after C.pseudotuberculosis infection.However,there was no significant difference on the secretion of IL-1αbetween C.pseudotuberculosis-infected Nlrp3^-/-macrophages and WT macrophages.In addition,no significant difference were found in the expression level of pro-IL-1αin lysates of macrophages from WT,Asc^-/-,Caspase1^-/-and Nlrp3^-/-mice.The above results indicate that during the process of C.pseudotuberculosis infection,ASC and Capase 1,but not NLRP3 molecules were involved in the maturation and secretion of IL-1α.4.The role of Ca²⁺and calpain on the maturation and secretion of IL-1αin macrophages infected by C.pseudotuberculosisTo investigate whether intracellular Ca²⁺affects the maturation and secretion of IL-1αmediated by C.pseudotuberculosis infection,the macrophages from mice were infected with C.pseudotuberculosis XH02 and its pld gene-deficient strain（XH02Δpld）,the changes of intracellular Ca²⁺concentration were detected by Fluo-4 NW kit.In addition,the secretion of IL-1αin supernatant of C.pseudotuberculosis-infected macrophages were detected by ELISA after adding of Ca²⁺chelating agents EDTA and EGTA+Mg²⁺.The results showed that the intracellular Ca²⁺concentration of the infected cells increased gradually with the extension of infection time and reached the peak at 20hours after infection.The intracellular Ca²⁺concentration of XH02Δpld-infected macrophages was significantly reduced compared with XH02-infected macrophages,after adding EDTA and EGTA+Mg²⁺to XH02-infected macrophages,it was found that the secretion level of IL-1αin the supernatant decreased significantly.Compared with infected cells in the presence of Ca²⁺,the secretion of IL-1αin macrophages in the absence of Ca²⁺was significantly reduced.Similarly,the secretion level of IL-1αin XH02Δpld-infected macrophages after adding of Ca²⁺vector A23187 was significantly increased.To evaluate whether calpain is involved in IL-1αmaturation and secretion in macrophages infected by C.pseudotuberculosis,macrophages were firstly infected with ATCC19410 and XH02,and the content ofα-fodrin degradation fragment（α-fodrin is the endogenous substrate of calpain,the molecular of degradation fragment is 145 k Da）in the infected supernatant,and the expression of full-α-fodrin（240 kDa）in cell lysis precipitates were detected by Western blotting.The results showed thatα-fodrin degraded fragments from supernatant were significantly increased in macrophages infection with C.pseudotuberculosis,whileα-fodrin was not detected in the supernatant of uninfected control cells.However,no significant difference were observed on the expression of full-α-fodrin in the cell lysis of the C.pseudotuberculosis-infected macrophages and uninfected macrophages;The mouse macrophages were further infected with ATCC19410 or XH02,in the presence or absence of calpain inhibitorⅢ,calpain inhibitorⅣ,EST and Ca²⁺chelating agent BAPTA-AM,the level of IL-1αsecretion were detected by ELISA.The level of IL-1αin supernatant and pro-IL-1αin cell lysates were determined by Western blotting.The results showed that after adding calpain inhibitorⅢ,calpain inhibitorⅣand BAPTA-AM,the secretion level of IL-1αand the protein expression of IL-1αwere significantly reduced,while there was no significant change on IL-1αsecretion in C.pseudotuberculosis-infected macrophages after adding EST.Finally,macrophages were infected with ATCC19410,XH02,ATCC19410Δpld and XH02Δpld,respectively,and the m RNA and protein expression levels of calpain-1,calpain-2,and calcineurin were detected.The results showed that there were no significant difference on the expression of calpain-1,calpain-2,calcineurinm in C.pseudotuberculosis-infected macrophages compared with the uninfected macrophages.The above results indicated that C.pseudotuberculosis infection in macrophages causes a significant increase in intracellular Ca²⁺concentration,and then activates calpain,and mediates the maturation and secretion of IL-1α.Calpain-1,calpain-2 and calcineurin may not be involved in the maturation and secretion of IL-1αin macrophages infected by C.pseudotuberculosis.Based on the above results,this study showed that C.pseudotuberculosis infection of macrophages could activate the maturation and secretion of IL-1α.This process is related to the increase in intracellular Ca²⁺concentration and the activation of calpain caused by the pathogen infection,and PLD was partly involved the maturation and secretion of IL-1αin macrophages infected by C.pseudotuberculosis.