The Role Of Toll-like Receptor4in Pulmonary Infection Of Chlamydia In Mice

Chlamydia trachomatis (Chlamydia trachomatis, C.trachomatis) infection is one of the serious public health problem worldwide, but its mechanism of infection is still to be clarified. Toll-like receptor4, is one of the most important proteins in Toll-like receptor family. Currently, most of the research about TLR4is focused on its role in innate immunity. But in the course of chlamydial infection, the role of Toll-like receptor4was unclear. This study intends to establish Chlamydia trachomatis pulmonary respiratory infection in C3H/HeN mice, C3H/HeJ mice (TLR4locus mutation) and study the related inflammation mechanisms:(1) C3H/HeN, C3H/HeJ mice macrophages stimulation experiment in vitro.Splenic macrophages were derived from C3H/HeN and C3H/HeJ mice treated with LPS(100ng/ml).Supernatant was collected at24h,48h and assayed for IL-6by ELISA.We conclude from these in vitro studies that macrophages derived form C3H/HeJ mice are not hyperresponsive to LPS.(2) C3H/HeN, C3H/HeJ mice aged6-8weeks were inoculated intranasally with5×lO3IFU of Cm to set up the murine model of Chlamydial pneumonia.The body weight was monitored every day. The body weight changes were calculatde and expressed as percent change form the original bady weight.C3H/HeJ mice showed marginal bady weight loss starting on day3postinfection.C3H/HeJ mice had significantly more bady weight loss than that observed in C3H/HeN mice.(3) The survival rate in each mouse strain over the course of10days of infection was monitored,calculated,and expressed as a percentage of a total number of mice. Only40%of C3H/HeN mice reached the end point after this dose of infection.Consequently,all of C3H/HeJ mice reached the end point starting day10after infection.(4) Infected C3H/HeN and C3H/HeJ mice were sacrificed on days3,7and12postinfection,and the bacterial burden in each mouse lung was determined based on the copy number of16S rRNA by quantitative PCR.While we observed a statistically significant higher bacterial burden in C3H/HeJ mice on days3and12postinfection than that in C3H/HeN mice.(5) Lungs from C3H/HeN and C3H/HeJ mice at days0,3and7postinfection were stained with H&E.Remarkably,C3H/HeN mice displayed less acute inflammatory infiltration in the lung by days3,7postinfection.(6) The level of MPO activity was first used as an indirect measurement of neutrophil infiltration in C3H/HeN and C3H/HeJ mice on days0,3and7postinfection. C3H/HeJ mice had significantly higher MPO activities in lung homogenate samples than C3H/HeN mice did on days3and7postinfection.(7) The cells were derived form BAL fluid,form C3H/HeN and C3H/HeJ mice on day3and7postinfection.Cells were then surface labeled with anti-CD llb,anti-Grl monoclonal antibodies for FACS analysis.Remarkably, C3H/HeJ mice displayed more neutrophils infiltration in the BAL fluid by days3and7postinfection.(8) Dynamic change of the numbers of CD4+CD25+Foxp3+Treg in the spleen post Cm infection.The percentage of CD4+CD25+Foxp3+T cells in the spleen between C3H/HeN and C3H/HeJ mice were similar.But the absolute number of CD4+CD25+Foxp3+T cells in the spleen were significantly decreased than control C3H/HeJ mice on day3postinfection.(9) The percentage of CD4+CD44+IFN-y+T cells in the spleen between C3H/HeN and C3H/HeJ mice were similar..Lungs were collected and homogenized for the detection of the expression of IFN-y level by ELISA.We did not detect any difference in IL-17levels form C3H/HeN and C3H/HeJ mice on days3and7postinfection.(10) At different time points post Cm infection,mice were sacrificed.Lungs were collected and homogenized for the detection of the protein level of cytokines by ELISA.We did not detect any difference in IL-4levels form C3H/HeN and C3H/HeJ mice on days3and7postinfection.Similarly,IL-5production in the lung from the two strains was comparable.(11) At different time points post Cm infection,mice were sacrificed.Lungs were collected and homogenized for the detection of the expression of IL-17and IL-6mRNA by RT-PCR and IL-17level by ELISA.Splenocytes were isolated,stimulated with PMA(50ng/ml)and Ionomycin (1μg/ml) or unstimulated control buffer cultured in the presence of GolgiPlug for5h.Data from flow cytometric analyses shows the percentage of IL-17+cells and mean fluorescence intensities(MFI) of IL-17.All data show that C3H/HeN mice had higher than levels C3H/HeJ mice on day3and7postinfection.(12)Thl7-related chemokines KC and MIP-2mRNA expression in the local lung tissues post Cm infection.However,C3H/HeN mice had significantly higher levels than did C3H/HeJ mice.In summary,TLR4plays a protective role in the mice infected with Chlamydia trachomatis.