Role Of STLR9~+ PMNs In TLR9 Mediated Lethal Shock Systemic Inflammatory Response In Mice

Polymorphonuclear neutrophils(PMNs)are the first line of defense against pathogenic microbial invasion,involved in the removal of pathogenic microorganisms.However,PMNs can be considered as a "double-edged sword",since they also contribute to the significant tissue damage that occurs as a result of extravagant inflammation,which result in an imbalance between the pro-inflammatory response and anti-inflammatory response.PMNs have been classified into distinct subsets on the basis of their morphology,cytokine production,surface antigens,and expressed TLRs.Moreover,PMNs with distinct phenotypes show different susceptibilities to a specific pathogen and can adjust to the stressor by changing their phenotypes in the course of inflammation.Recently,surface TLR9(sTLR9)has been detected on the surface of human and mouse PMNs and can sense mt DNA and initiate signaling pathways that lead to acute inflammation.The expression of sTLR9 on PMNs can be upregulated in response to stimulation.This type of PMNs were designated as sTLR9~+ PMNs.However,so far it remains elusive whether and how the sTLR9~+ PMNs are involved in the development of systemic inflammatory response syndrome(SIRS).In previous study we investigated the number and expression of inflammatory cytokines of sTLR9~+ PMNs in the peritoneal lavage cells(PLCs)of mice,a lethal shock SIRS mouse model with by intraperitoneal(i.p.)injection of Cp G ODN into D-Gal N-sensitized mice based on our previous research,and observed their effects on the SIRS mice through adoptive therapy with PMN-rich PLCs collected from SIRS model mice at 1 h or 6 h post induction and pretreatment with CCT ODN,an ODN with CCT repeats,which we have previously shown to be capable of inhibiting the TLR9 trafficking and rescuing mice from lethal shock.Our observations would provide insights into the protective roles of sTLR9~+ PMNs in the development of SIRS and suggest a new way rescue severe SIRS patients by up-regulating this population of the neutrophils.1.D-Gal N pre-sensitized BALB/c mice followed by Cp G ODN induction developed lethal shock systemic inflammatory responseIn order to study the role of PMNs in TLR9-induced systemic inflammation,we firstly established a SIRS mouse model by intraperitoneal(i.p.)injection of Cp G ODN into D-Gal N-sensitized mice based on our previous research.In order to observe the systemic inflammatory response in mice,we recorded the general conditions and survivals of the mice,and analyzed the pathological changes of the main organs in the model mice.It was found that all of the model mice were sacrificed within 48 h post induction in accompany with weight loss of about 2-3 g,the temperature drops 5-10 ?,multiple neurological symptoms,including dispirited behavior,staggered gait,and trembling in the model mice.By histopathological analysis severe hemorrhage and necrosis was found in the livers of the dead mice.To observe the local inflamamtion,we collected PLCs and analyzed the type of inflammatory cells and the production of inflammatory cytokines in the model mice.The results showed that there were a large amount of PMNs(about 60-70%)in the PLCs in conjunction with a significant increase of TNF? and IL-10 levels compared to the control group.The further analysis on cytokine production levels of PMNs and non-PMNs suggested that PMNs is the main inflammatory cells in PLCs.2.sTLR9~+ PMN plays a protective role in SIRS model miceSince the rapid accumulation of a mass of PMNs in the peritoneal cavity is a typical feature of Cp G ODN-induced model mice,the role of PMNs in the lethal shock SIRS mouse model is focus of our works.2.1 Effect of early recruited peritoneal PMNs on survivals of SIRS model miceTo ascertain the role of large amounts of PMNs in the SIRS model mice,we investigated the effect of removal and transfer of the PMN-rich PLCs on the survival of the SIRS model mice.The results showed that the removal of PMNs by intraperitoneal lavage could not improve the survival of mice.On the contrary,the transfer with PMN-rich PLCs collected from model mice at 1 h after induction could rescue the mice.However,the transfer with PMN-rich PLCs collected at 6 h after induction can not.Further we transferred the PMNs(92.6% purity)isolated from the protective PLCs to the model mice and found that the transfer protected the mice from lethal shock.All these data confirmed that the early recruited PMNs play protective role in the SIRS model mice.2.2 Expression of TNF? and IL-10 in peritoneal PMNs collected from the model miceTo explain the different function of PLCs containing large amounts of PMNs collected at 1 h and 6 h after SIRS induction,first of all,we collected PLCs from the model mice and the control mice at 1 or 6 h post induction and tested their expression of TNF? and IL-10.Obviously,the expression of TNF? and IL-10 in the PLCs collected from SIRS model mice was elevated remarkably compared to that in control mice.The results obtained at 6 h post induction indicated a significant increase in the level of TNF? and a clear decline in the level of IL-10 compared to that at the 1 h post induction in the SIRS model mice.Our further study was to verify the correlation between the change in the cytokine levels and the accumulation of PMNs.We examined the changes of TNF? and IL-10 mRNA level in sorted PMNs and B cells that accounted for the major proportion of PLCs after 1-h or 6-h SIRS induction.After 6-h SIRS induction,we observed an obvious increase in the level of TNF? mRNA in PMNs and no significant change was detected in B cells.The mRNA level of IL-10 in both PMNs and B cells decreased sharply at 6 h after inducing the model.In the protein levels we also confirmed an increase in the proportion and quantity of TNF? produced by PMNs in PLCs at 6 h after SIRS induction compared to that observed after the first hour whereas the level of IL-10 showed the opposite trend.However,the expression of TNF? and IL-10 in PLCs apart from PMNs were much lower than that in PMNs and remained unchanged post induction.Consequently,we found that PMNs are the main PLCs that produce inflammatory cytokines in the SIRS model mice and their function is correlated with their cytokine levels.2.3 Expression of sTLR9 and production of TNF? and IL-10 in sTLR9~+ PMNs in SIRS model miceSince PMNs in peripheral blood can express sTLR9 which is able to sense TLR9 agonist and initiate pro-inflammatory responses,to confirm whether peritoneal PMNs in SIRS model mice also expressed sTLR9,we collected PLCs and tested the ratios of sTLR9~+ PMNs by flow cytometry and found that 10% of the peritoneal PMNs were sTLR9~+ PMNs.After inducing for either 1 h or 6 h,this percentage remained approximately the same,but the expression level of sTLR9 represented by mean fluorescence intensity(MFI)decreased at the 6-h time point.The proportion of sTLR9-high-expressing neutrophils(sTLR9 high neutrophils)was nearly 20% in the sTLR9~+Ly6G+ PMNs after the 1-h induction,which was relatively higher than that after 6-h induction.In order to examine the difference between sTLR9~+ PMNs and sTLR9– PMNs in terms of their function,we further evaluated their expression of TNFa and IL-10,respectively,and found that sTLR9~+ PMNs produced much more TNFa and IL-10 than sTLR9– PMNs at both 1-h and 6-h time point.In sTLR9~+ PMNs at 6 h compared to that observed after 1 h of SIRS induction there is a clear increase in the level of TNF? and a significant drop in level of IL-10.Thus,the data demonstrated the prominent role of sTLR9~+ PMNs in peritoneal inflammation,and sTLR9~+ PMNs generated higher levels anti-inflammation cytokines than pro-inflammation cytokines at early stages of SIRS induction.3.The role and possible mechanism of sTLR9~+ PMNs in SIRS model mice based on CCT ODNTo further confirm the protective role of sTLR9~+ PMNs in the SIRS model mice,we selected an oligodeoxynucleotide with CCT repeats(CCT ODN)to treat the model mice aiming to determine whether CCT ODN can regulate the levels of sTLR9 on sTLR9~+ PMNs,since CCT ODN was shown to be capable of rescuing mice from the development of SIRS in our previous work.At 30 min before Cp G ODN injection,CCT ODN was injected i.p.followed by the monitoring of mouse survival and collection of PLCs from mice 1 h post-SIRS induction.The results revealed that CCT ODN could significantly prolong survival in the model mice.After collecting PLCs from model mice treated with or without CCT ODN at 1 h post-SIRS induction and detecting sTLR9~+ PMNs by flow cytometry,we found that 70-85% of PLCs in CCT ODN-treated model mice were PMNs at 1 h post-SIRS induction,which was significantly higher than that detected in the model mice.For sTLR9~+ PMNs,their sTLR9 levels were also clearly up-regulated by CCT ODN treatment.These results suggest that the protective role of CCT ODN on SIRS development may be due to the up-regulation of sTLR9 on sTLR9~+ PMNs,which increases the ratio of sTLR9 high PMNs in the local inflammatory area at an early stage of the inflammatory response.To confirm this,in vitro we collected PLCs and peripheral white blood cells(PBWCs)and co-cultured with either Cp G ODN or CCT ODN for 1 h followed by determination of sTLR9 levels in sTLR9~+ PMNs by flow cytometry.We found that Cp G ODN treatment down-regulated the level of sTLR9 on sTLR9~+ PMNs whereas CCT ODN treatment significantly inhibited the downregulation of sTLR9.Together,the rescue effect of CCT ODN treatment could be,at least partially,attribute to an increase in the expression of sTLR9 on sTLR9~+ PMNs at early stages of inflammation.Since the Cp G ODN recognition by endosomal TLR9 requires the cleavage of TLR9 in endosomes with acidic atmosphere and chloroquine(CQ),a TLR9 antagonist,interferes the acidification of endosomes,furthermore,we tried to find the correlation between sTLR9 expression of PMNs and endosomal TLR9.We co-cultured CQ at 20?g/ml with PBWCs,2h later,added Cp G ODN and/or CCT ODN,1h later,harvested the PBWCs and measured the MFI-sTLR9 on the sTLR9~+ PMNs.The results show that CQ treatment significantly prevented the decrease of sTLR9 on sTLR9~+ PMNs induced by Cp G ODN,implying that the sTLR9 turnover on PLCs was dependent on the activation of endosomal TLR9 by internalized Cp G ODN.In conclusion,early recruited PMNs play a protective role in model mice.sTLR9~+ PMNs may be an important sub-population of protective PMNs in SIRS,and regulation of the sTLR9 expression in these populations may also be a new way to treat severe SIRS patients.