The Studies On Immune Responses And Pathogenesis Of Mice With Disseminated Murine Cytomegalovirus Infection

Objectives:1) To observe the time courses of expression of AIM2inflammasome in spleenmacrophages and its downstream cytokines, IL-1β and IL-18, in sera from mice withacute disseminated murine cytomegalovirus (MCMV) infection in order todemonstrate whether AIM2could recognize MCMV DNA and explore the role ofAIM2inflammasome pathway in innate and acquired immune responses againstMCMV in vivo.2) To investigate the differential levels of T helper17(Th17) cells and expression ofcytokine, IL-17A, in the different organs during the acute stage of disseminatedMCMV infection and to analyze the relationship between expression of IL-17A andMCMV titers or pathological changes in the tissues for further understanding the roleof Th17cells in the pathogenesis of the acute disseminated MCMV infection in vivo.Methods:1) Development of the mouse model: Fory-five BALB/c mice aged of4.5-week-oldwere inoculated intraperitoneally with salivary gland homogenates containing5×103PFU of MCMV Smith strain to establish the model with the disseminated MCMV infection, another45mice injected with normal salivary gland homogenates served asmock-infected controls. On the3d,7d,14d,28d (the end of acute infection) and45dof post infection (PI), nine mice were sacrificed and their venous blood, spleens,salivary glands, livers and lungs were obtained, respectively. In order to get enoughvolume of sera and number of macrophages from spleen, every3mice as onesubgroup were randomly selected and their blood samples and the two to third ofspleens in size were mixed up as one sample, and the rest spleens and other tissueswere randomly selected from3mice, so that there were3samples at each time point.2) Expression of AIM2inflammasome proteins: Macrophages were isolated fromspleens and their proteins were extracted, then the expression of AIM2inflammasomeproteins including AIM2, ASC, pro-caspase-1and caspase-1were detected by usingWestern blot assay. GAPDH protein served as the internal control. The expressinglevels of each protein were semi-quantitatively analyzed by the ratio (K value) of theintegral density values of the desired and internal bands.3) Levels of IL-1β and IL-18in sera: The levels of IL-1β and IL-18, downstreamcytokines of AIM2inflammasome pathway, in sera were measured by doubleantibody sandwich enzyme-linked immunosorbent assay (ELISA).4) The optimizing method for induction of Th17cell differentiation: Spleen lymphocyteswere isolated from3normal mice and then cultured in the four different conditions,including no stimulus, heat-inactivated MCMV, PMA/Ionomycin and heat-inactivatedMCMV/PMA/Ionomycin. After preset culture time intervals, cells were harvested fordetection of the percentages of CD3+C D4+IL-17A+cells by using flow cytometry.Moreover, levels of IL-17A in the culture supernatants were measured by ELISA. Theinduction effect of each group was analyzed for determining the optimizing methodfor induction of Th17differentiation.5) Expression of Th17cells and MCMV-specific IL-17A protein from spleens: On the3d,7d,14d, and28d PI (acute stage of infection), isolated spleen lymphocytes were stimulated with heat-inactive MCMV/PMA/Ionomycin for6h and then thepercentages of CD3+CD4+IL-17A+cells in spleen lymphocytes were determined byflow cytometry. For measurement of viral specific IL-17A levels in culturesupernatants by ELISA, isolated spleen lymphocytes were stimulated simultaneouslywith heat-inactive MCMV/PMA/Ionomycin and PMA/Ionomycin, respectively, andthe difference of IL-17A levels between those with above two stimulated methods wasconsidered as MCMV-specific IL-17A level.6) Expression of IL-17A protein in organs: Immunohistochemical stains were employedto detect the expression of IL-17A in the tissues of spleen, salivary gland, liver andlung from experimental mice during acute stage of infection, for analyzing thedifference of IL-17A expression in the different organs.7) Pathological changes of spleen and salivary gland: The pathological changes of spleenand salivary gland during the acute infection were assessed by histologicalexamination and a semi-quantitative evaluation method.8) Infective MCMV loads: Virus titers of tissue homogenates were determined using astandard plaque assay.9) Correlation analysis was performed between the levels of MCMV-specific IL-17A inspleen cell culture supernatants or expression of IL-17A in organs and thepathological changes of spleen and salivary gland or virus titers.Results:1) Expression of AIM2inflammasome proteins: Expressing levels of all AIM2inflammasome proteins (AIM2, ASC, pro-caspase-1and caspase-1) in spleenmacrophages were significantly increased and peaked on day3after MCMV infection,with obviously higher K values of AIM2, ASC, pro-caspase-1and caspase-1inMCMV-infected mice than those in mock-infected mice,1.120±0.243versus0.230±0.046,1.318±0.333versus0.248±0.090,2.049±0.401versus0.390±0.120, and1.483±0.420versus0.176±0.045, respectively (P<0.01). Afterwards, the expressing levels of the four proteins in MCMV-infected mice rapidly dropped to the controllevels.2) Levels of IL-1β and IL-18in sera: Serum levels of IL-1β and IL-18inMCMV-infected mice were also significantly higher than those in mock-infectedcontrols (111.050±28.500versus55.858±2.983pg/ml, P<0.05, and99.911±2.222versus57.206±6.228pg/ml, P<0.01, respectively) on day3after infection.3) Optimizing method for Th17differentiation: Percentages of Th17cells in spleenlymphocytes stimulated with heat-inactivated MCMV/PMA/Ionomycin for6hourswere significantly higher than those with the same stimulus for1day and6hours andthose with PMA/Ionomycin for6hours (0.163±0.035versus0.083±0.032%, P<0.05,and0.163±0.035versus0.033±0.015%, P<0.01, respectively), and levels of IL-17Ain spleen lymphocytes culture supernatants showed the same findings (67.246±4.578versus45.317±8.793pg/ml, P<0.05, and67.246±4.578versus24.474±5.134pg/ml,P<0.01, respectively). But no expression of Th17cells and IL-17A was observed inspleen lymphocytes stimulated with no stimulus or heat-inactive MCMV.4) Percentages of Th17cells and expression of IL-17A in spleen lymphocytes: On the3d,7d,14d and28d PI, percentages of Th17in spleen lymphocytes stimulated withoptimizing induction method were all increased in MCMV-infected mice comparedwith mock-infected mice (0.67±0.13versus0.22±0.02%, P=0.004,0.82±0.02versus0.18±0.06%, P=0.004,1.14±0.09versus0.19±0.04%, P=0.000, and0.47±0.11versus0.20±0.06%, P=0.017, respectively). Levels of MCMV-specific IL-17Awere markedly higher on the14d and28d after infection in MCMV-infected micethan those in mock-infected mice (81.98±12.37versus44.96±8.44pg/ml, P<0.01,and57.58±8.14versus35.10±10.41pg/ml, P<0.05, respectively), but nosignificance was observed on the3d and7d （P>0.05）. Both of percentages of Th17cells and levels of MCMV-specific IL-17A peaked on day14after MCMV infection. 5) Expression of IL-17A protein in organs: Expression of IL-17A showed a obvousorgan disparity in MCMV-infected mice. IL-17A+cells significantly accumulated inspleen and salivary gland of MCMV infected mice. The number of IL-17A+cellsreached the peak on day14and some positive staining could be observed in epithelialcells of secretory duct in salivary gland. Only fewer IL-17A+staining was found inlivers and lungs on day3PI.6) Pathology of spleen and salivary gland: Histological evaluation showed that the mostsevere damages could be observed both in spleen and salivary gland on day14afterinfection.White pulp structures were extensively destructed and large numbers ofphagocytic cells, dead cells and debris could be seen in spleens, and severeinflammatory infiltrates and loss of normal tissue architecture in salivary gland.7) MCMV titers: Viral titers gradually increased and peaked on day14in salivary glandand remained higher on day28. Viral titers peaked on day3in liver and spleen andthen quickly diminished and virus was not detected on day14. Virus was undetectablein lung throughout the experiments.8) Correlation analysis: Levels of MCMV-specific IL-17A in spleen lymphocytes culturesupernatants 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). Expressionof 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) AIM2of spleen macrophages could recognize MCMV DNA in cytoplasm and thenactive AIM2inflammasome pathway during early MCMV infection. AIM2inflammasome was involved in innate immune response against MCMV at early stageof infection. Meanwhile, downstream cytokines of AIM2inflammasome pathway,IL-1β and IL-18, play a major role in triggering the acquired immune responsesagainst MCMV. 2) An organ disparity of IL-17A expression during the acute stage of MCMV infectionwas observed. The characteristic of local immune responses, IL-17A-positive cellsmarkedly accumulated in the salivary glands, may play a key role in the persistentMCMV replication of the salivary glands, suggesting that Th17cells may be involvedin the pathogenesis of the persistent MCMV infection and the immune pathologicaldamages by means of IL-17A expression.