| TLR9(Toll-like receptor 9), an intracellular pathogen associated pattern recognition receptor(PRR), can sense exogenous DNA to mediate innate immune responses. Recent studies have found that primary blood neutrophils express a functional cell surface Toll-like receptor 9 and the precise role of surface TLR9 positive PMN(s TLR9+PMN) in the pathogen-mediated innate immune response is not yet clear. As a kind of inflammatory cytokine which is more concerned in recent years, IL-17 can promote the generation of granular cells and the aggregation of neutrophils(PMN) to inflammatory sites by inducing chemokines and other cytokines. Although IL-17 is mainly produced by T cells, there are studies show that PMN is also a resource of IL-17. However, whether IL-17~+ PMN and s TLR9+ PMN are correlated or not in the pathogenesis of sepsis is still not clear. Sepsis is one of the leading causes of death in critically ill patients and normally infects the lungs and abdomen. As the first line of defense against infection, PMN plays an important role in the occurrence and development of sepsis. However, the roles of PMN subsets, s TLR9+ PMN and IL-17 in the development of sepsis, especially in the inflammatory response of the infection site are unclear. Therefore, the study built a mouse model by intraperitoneal injection of Escherichia coli(E.coli), chose the peritoneal lavage cell(PLC) as the research object and focused on the role of PMN in the inflammatory response, the expression of s TLR9 and IL-17,as well as their impact on the disease, thus it provides valuable experimental data for the diagnosis and treatment of sepsis.1. Induction of intraperitoneal injection of E.coli on septic peritonitis in miceThe septic peritonitis mouse model is established by intraperitoneal injection of E.coli. The completion of the model is evaluated by the clinical score, survival period, the quantity and the morphology and inflammatory response of PLC.The results were given as follows: 1) Intraperitoneal injection of E.coli could cause some systemic symptoms of severe infections in mice, including lethargy, piloerection, tremors, periorbital exudates and respiratory distress; 2) Intraperitoneal injection of E.coli could cause the death of mice. The mortality rate of mice was positively correlated with the amount of bacteria; 3) The amount of PMN in PLC was significantly increased, about 40-60%; 4) After infection, the inflammatory cytokines(IL-17 and IL-10), TLR9 and its downstream intermediates TRAF6, inflammation-related transcription factor IRF5 and NF-kB were significantly increased in PLC on the m RNA level. Therefore, intraperitoneal injection of E.coli could induce the occurrence of septic peritonitis in mice.2. Analysis of PMN and its subsets in PLC of mice with peritonitisIn order to study the roles of PLC and its PMN subsets in the development of peritonitis, flow cytometry was used to analyze the PMN and its subsets in PLC. Results showed that: 1) After intraperitoneal injection of E.coli, the proportion of PMN in each group was significantly increased at each time point, but the differences were not significant; 2) In the PLC of mice with peritonitis, the proportion of CD11b~+ PMN and CD11b- PMN were different. In the early stage of infection, the proportion of CD11b~+ PMN decreased and the proportion of CD11b- PMN increased. As the course of the disease was prolonged, the proportions of CD11b~+ PMN was increased significantly. It could be seen that the changes in the proportion of PMN subsets in the local infection might be related to the evolution of the disease.3. The proportion of s TLR9+ PMN during the development of peritonitisWe investigated the proportional changes of s TLR9 in PLC and its PMN subgroups. Results showed that: 1) After infection, the proportion of s TLR9+ cells in CD11b~+ PMN increased significantly in 18 hours, decreased significantly in 48 hours, still stayed at a low level in 72 hours and there was no significant differences among all the groups; 2) After infection, the proportion of s TLR9+ cells in CD11b- PMN increased significantly in 18 hours, then restored to normal quickly and there was no significant differences among the groups. Consequently, s TLR9+ PMN was involved in the inflammatory response of septic peritonitis. But the proportional changes of s TLR9+ cells in the PMN subgroup appeared to have little relationship with the severity of the disease.4. Analysis of IL-17 in PLC and its PMN of mice with peritonitisThe proportions of IL-17~+ cells in PLC and its PMN were analyzed by flow cytometry. Results showed that: 1) There were significant differences in the proportions of IL-17~+ cells between two PMN subsets of PLC in the mice with peritonitis; the proportion of IL-17~+CD11b~+ PMN was significantly decreased at every time point of infection and was not related to the amount of bacteria; the proportion of IL-17~+CD11b- PMN had not changed; 2) The experiment that PLC were co-cultured with E.coli in vitro and cytokine secretion was blocked showed that: Bacterial infection could stimulate the PLC to produce IL-17; 3) Gene expression levels of the sorted CD3-CD14-CD11b~+ PMN from PLC showed that it was IL-17-producing cell. These results indicated that the expression of IL-17 in PLC was increased after bacterial infection, CD11b~+ PMN could produce IL-17.5. The expression levels of IL-17 in different cell lines of PLC in mice with peritonitis during the earlier period of infectionWe examined the expression of IL-17 at the earlier time point(8h) after infection. Results showed that: 1) The expression levels of IL-17 in CD11b- PMN and CD11b~+ PMN were significantly increased(p<0.05), the differences were not significant among the different bacteria dosage groups. These results showed that intraperitoneal injection of E.coli could induce two PMN subgroups of PLC to produce IL-17.6. Expression and correlation of s TLR9 and IL-17 in PMN of PLC in mice with peritonitisWe used flow cytometry to detect the expression of s TLR9 in PLC in mice with peritonitis within 8 hours after infection and analyzed the relationship of expression between s TLR9 and IL-17 in PMN. Results showed that: 1) The expression levels of s TLR9 in the two PMN subgroups in PLC differs from each other. The expression level of s TLR9 on CD11b~+ PMN was increased, but it was not related to the amount of bacteria; the s TLR9 expression level of CD11b- PMN was significantly decreased, especially in the high bacteria dosage group; 2) In CD11b~+ PMN, the cells with high expression of s TLR9 and IL-17 were not the same. So at least two groups of cells in CD11b~+ PMN expressed s TLR9 and IL-17 separately. Respectively, both were involved in the immune response against bacterial infection.7. Effects of TLR9 activator or inhibitor on the disease evolution and inflammatory response of mice with septic peritonitisWe chose TLR9 activator(Cp G1826) and inhibitory ODN(MS19) to intervent the mice with septic peritonitis. The survival rate and expression levels of s TLR9 and IL-17 in immune cells, such as PLC and PMN, were observed. Results showed that: 1) MS19 could significantly raise the survival rate of mice with peritonitis; 2) MS19 could inhibit the expression level of TLR9 m RNA in PLC of mice with peritonitis; 3) MS19 could not only down-regulate the s TLR9 expression level of PLC and its PMN subsets in mice with peritonitis, but also inhibit the expression level of IL-17 CD11b~+ PMN. These results indicated that the expression level of s TLR9 on PLC or its PMN was positively correlated with the severity of peritonitis in mice and IL-17 as an inflammatory cytokine played an important role in the local immune response.As suggested above, in this mice model inducing the lethal septic peritonitis by E.coli, peritoneal PMN played an important role in the inflammatory response. We observed the expression of s TLR9 and IL-17 in PMN from the mice with septic peritonitis. The expression of s TLR9 and IL-17 had a change during the course of infection. TLR9 inhibitor could inhibit the expression of s TLR9 and increase the survival rate of mice with peritonitis. These findings might be a cellular or molecular marker to determine the intensity of inflammatory response after infection and provide experimental basis for an appropriate therapeutic opportunity of sepsis. |
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