| Objectives:1) To observe the time courses of expression of AIM2 inflammasome in spleen macrophages and its downstream cytokines, IL-1βand IL-18, in sera from mice with acute disseminated murine cytomegalovirus (MCMV) infection in order to demonstrate whether AIM2 could recognize MCMV DNA and explore the role of AIM2 inflammasome pathway in innate and acquired immune responses against MCMV in vivo.2) To investigate the differential levels of T helper 17 (Th17) cells and expression of cytokine, IL-17A, in the different organs during the acute stage of disseminated MCMV infection and to analyze the relationship between expression of IL-17A and MCMV titers or pathological changes in the tissues for further understanding the role of Th17 cells in the pathogenesis of the acute disseminated MCMV infection in vivo.Methods:1) Development of the mouse model:Fory-five BALB/c mice aged of 4.5-week-old were inoculated intraperitoneally with salivary gland homogenates containing 5×103 PFU of MCMV Smith strain to establish the model with the disseminated MCMV infection, another 45 mice injected with normal salivary gland homogenates served as mock-infected controls. On the 3d,7d,14d,28d (the end of acute infection) and 45d of post infection (PI), nine mice were sacrificed and their venous blood, spleens, salivary glands, livers and lungs were obtained, respectively. In order to get enough volume of sera and number of macrophages from spleen, every 3 mice as one subgroup were randomly selected and their blood samples and the two to third of spleens in size were mixed up as one sample, and the rest spleens and other tissues were randomly selected from 3 mice, so that there were 3 samples at each time point.2) Expression of AIM2 inflammasome proteins:Macrophages were isolated from spleens and their proteins were extracted, then the expression of AIM2 inflammasome proteins including AIM2, ASC, pro-caspase-1 and caspase-1 were detected by using Western blot assay. GAPDH protein served as the internal control. The expressing levels of each protein were semi-quantitatively analyzed by the ratio (K value) of the integral density values of the desired and internal bands.3) Levels of IL-1βand IL-18 in sera:The levels of IL-1βand IL-18, downstream cytokines of AIM2 inflammasome pathway, in sera were measured by double antibody sandwich enzyme-linked immunosorbent assay (ELISA).4) The optimizing method for induction of Th17 cell differentiation:Spleen lymphocytes were isolated from 3 normal mice and then cultured in the four different conditions, including no stimulus, heat-inactivated MCMV, PMA/Ionomycin and heat-inactivated MCMV/PMA/Ionomycin. After preset culture time intervals, cells were harvested for detection of the percentages of CD3+CD4+IL-17A+cells by using flow cytometry. Moreover, levels of IL-17A in the culture supernatants were measured by ELISA. The induction effect of each group was analyzed for determining the optimizing method for induction of Th17 differentiation.5) Expression of Th17 cells and MCMV-specific IL-17A protein from spleens:On the 3d, 7d,14d, and 28d PI (acute stage of infection), isolated spleen lymphocytes were stimulated with heat-inactive MCMV/PMA/Ionomycin for 6h and then the percentages of CD3+CD4+IL-17A+cells in spleen lymphocytes were determined by flow cytometry. For measurement of viral specific IL-17A levels in culture supernatants by ELISA, isolated spleen lymphocytes were stimulated simultaneously with heat-inactive MCMV/PMA/Ionomycin and PMA/Ionomycin, respectively, and the difference of IL-17A levels between those with above two stimulated methods was considered as MCMV-specific IL-17A level.6) Expression of IL-17A protein in organs:Immunohistochemical stains were employed to detect the expression of IL-17A in the tissues of spleen, salivary gland, liver and lung from experimental mice during acute stage of infection, for analyzing the difference of IL-17A expression in the different organs.7) Pathological changes of spleen and salivary gland:The pathological changes of spleen and salivary gland during the acute infection were assessed by histological examination and a semi-quantitative evaluation method.8) Infective MCMV loads:Virus titers of tissue homogenates were determined using a standard plaque assay.9) Correlation analysis was performed between the levels of MCMV-specific IL-17A in spleen cell culture supernatants or expression of IL-17A in organs and the pathological changes of spleen and salivary gland or virus titers.Results:1) Expression of AIM2 inflammasome proteins:Expressing levels of all AIM2 inflammasome proteins (AIM2, ASC, pro-caspase-1 and caspase-1) in spleen macrophages were significantly increased and peaked on day 3 after MCMV infection, with obviously higher K values of AIM2, ASC, pro-caspase-1 and caspase-1 in MCMV-infected mice than those in mock-infected mice,1.120±0.243 versus 0.230±0.046,1.318±0.333 versus 0.248±0.090,2.049±0.401 versus 0.390±0.120, and 1.483±0.420 versus 0.176±0.045, respectively (P＜0.01). Afterwards, the expressing levels of the four proteins in MCMV-infected mice rapidly dropped to the control levels.2) Levels of IL-1βand IL-18 in sera:Serum levels of IL-1βand IL-18 in MCMV-infected mice were also significantly higher than those in mock-infected controls (111.050±28.500 versus 55.858±2.983 pg/ml, P＜0.05, and 99.911±2.222 versus 57.206±6.228 pg/ml, P＜0.01, respectively) on day 3 after infection.3) Optimizing method for Th17 differentiation:Percentages of Th17 cells in spleen lymphocytes stimulated with heat-inactivated MCMV/PMA/Ionomycin for 6 hours were significantly higher than those with the same stimulus for 1 day and 6 hours and those with PMA/Ionomycin for 6 hours (0.163±0.035 versus 0.083±0.032%, P＜0.05, and 0.163±0.035 versus 0.033±0.015%, P＜0.01, respectively), and levels of IL-17A in spleen lymphocytes culture supernatants showed the same findings (67.246±4.578 versus 45.317±8.793 pg/ml, P＜0.05, and 67.246±4.578 versus 24.474±5.134 pg/ml, P＜0.01, respectively). But no expression of Th17 cells and IL-17A was observed in spleen lymphocytes stimulated with no stimulus or heat-inactive MCMV.4) Percentages of Th17 cells and expression of IL-17Ain spleen lymphocytes:On the 3d, 7d,14d and 28d PI, percentages of Th17 in spleen lymphocytes stimulated with optimizing induction method were all increased in MCMV-infected mice compared with mock-infected mice (0.67±0.13 versus 0.22±0.02%, P=0.004,0.82±0.02 versus 0.18±0.06%, P=0.004,1.14±0.09 versus 0.19±0.04%,P=0.000, and 0.47±0.11 versus 0.20±0.06%, P=0.017, respectively). Levels of MCMV-specific IL-17A were markedly higher on the 14d and 28d after infection in MCMV-infected mice than those in mock-infected mice (81.98±12.37 versus 44.96±8.44 pg/ml, P＜0.01, and 57.58±8.14 versus 35.10±10.41 pg/ml, P＜0.05, respectively), but no significance was observed on the 3d and 7d (P＞0.05). Both of percentages of Th17 cells and levels of MCMV-specific IL-17A peaked on day 14 after MCMV infection. 5) Expression of IL-17A protein in organs:Expression of IL-17A showed a obvous organ disparity in MCMV-infected mice. IL-17A+cells significantly accumulated in spleen and salivary gland of MCMV infected mice. The number of IL-17A+cells reached the peak on day 14 and some positive staining could be observed in epithelial cells of secretory duct in salivary gland. Only fewer IL-17A+staining was found in livers and lungs on day 3 PI.6) Pathology of spleen and salivary gland:Histological evaluation showed that the most severe damages could be observed both in spleen and salivary gland on day 14 after infection.White pulp structures were extensively destructed and large numbers of phagocytic cells, dead cells and debris could be seen in spleens, and severe inflammatory infiltrates and loss of normal tissue architecture in salivary gland.7) MCMV titers:Viral titers gradually increased and peaked on day 14 in salivary gland and remained higher on day 28. Viral titers peaked on day 3 in liver and spleen and then quickly diminished and virus was not detected on day 14. Virus was undetectable in lung throughout the experiments.8) Correlation analysis:Levels of MCMV-specific IL-17A in spleen lymphocytes culture supernatants showed positive correlation with MCMV titers in salivary gland (r=0.74, P＜0.01) and injury degrees of spleen and salivary gland (r=0.78, P＜0.01). Expression of IL-17A also displayed positive correlation with MCMV titers in salivary gland (r=0.63, P＜0.05) and injury degrees of salivary gland (r=0.68, P＜0.01).Conclusions:1) AIM2 of spleen macrophages could recognize MCMV DNA in cytoplasm and then active AIM2 inflammasome pathway during early MCMV infection. AIM2 inflammasome was involved in innate immune response against MCMV at early stage of infection. Meanwhile, downstream cytokines of AIM2 inflammasome pathway, IL-1βand IL-18, play a major role in triggering the acquired immune responses against MCMV. 2) An organ disparity of IL-17A expression during the acute stage of MCMV infection was observed. The characteristic of local immune responses, IL-17A-positive cells markedly accumulated in the salivary glands, may play a key role in the persistent MCMV replication of the salivary glands, suggesting that Th17 cells may be involved in the pathogenesis of the persistent MCMV infection and the immune pathological damages by means of IL-17A expression.
|
PDF Full Text Request