Studing On The Immune Pathogenesis Of Murine Cytomegalovirus Infection

part Ⅰ The Actions of IL-17Involved in the Pathogenesis of Cytomegalovirus Infection.Objectives1) Establish an animal model of disseminated cytomegalovirus infection, to explore the effects of IL-17on murine cytomegalovirus (MCMV)-induced immunopathological liver and salivary gland damage in vivo;2) Blocked IL-17in vivo to further investigate the role of IL-17in the pathogenesis of murine cytomegalovirus infection.Methods1) To establish an animal model of disseminated cytomegalovirus infection: For all studies,4.5-week-old female BALB/c mice were used.32female BALB/c mice were randomly divided into two groups:control group and MCMV group. Experimental mice were sacrificed at3,7,14, and28days after infection. Blood serum samples were separated and livers and salivary glands were harvested, partially in4%paraformaldehyde embedded in paraffin, and the rest of what were saved in-70℃refrigerator;2) The viral titers in salivary glands and livers were determined with a standard plaque assay:Take mouse embryo-fibroblast cells as culture cells, viral titers of salivary glands and liver tissues were detected at different times after the MCMV infection;3) RT-PCR was used to detect the expression of IL-17and IL-17R mRNA in the salivary glands and liver tissues:Total RNA was extracted from liver and salivary gland tissues using the TRI REAGENT kit. Synthesis of cDNA was performed on5μg of total RNA using a reverse transcription system. IL-17and IL-17receptor (IL-17R) levels were expressed as relative copy numbers normalized to glyceraldehyde3-phosphate dehydrogenase (GAPDH) levels.4) The expression and distribution of IL-17in liver and salivary gland tissues was observed by immunohistochemical staining;5) Hematoxylin and eosin (H&E) staining was used to evaluate the pathology of the tissue sections:the pathology of the infected mice was assessed by observation of the liver and salivary glands histological examination in HE stained sections;6) Serum ALT levels were detected using a Roche DPPI biochemical analyzer;7) Blocked IL-17in vivo:Mice were randomly divided into4groups. Anti-mouse IL-17group, MCMV group, isotype control group and normal controls. All treatments were administered by intraperitoneal injection. Experimental and control mice were sacrificed on day7after infection. Blood serum was separated and livers and salivary glands were harvested, partially in4%paraformaldehyde embedded in paraffin, part of the organization saved in-70℃refrigerator. The viral titers in salivary glands and livers were determined with a standard plaque assay; the expression of IL-17were detected by western-blot; the expression of IL-17、IL-17R、IFN-γ、IL-10mRNA in the livers were measured by RT-PCR; Hematoxylin and eosin (H&E) staining was used to evaluate the pathology of the tissue sections; Serum ALT levels were detected using a Roche DPPI biochemical analyzer.Result1)Timing and extent of viral replication in tissues:In the salivary gland, viral titers peaked at104PFU/mg on day14and gradually declined on day28; MCMV titers in the livers quickly increased to peak levels of approximately10PFU/mg of tissue on day3post-infection but quickly declined and were not detectable on day14.2) Compared to control-infected mice, serious inflammatory cellular infiltrates in salivary glands were present on day7after MCMV infection. We observed organized into cellular foci and infiltration was constitutively located in secretory ducts (SD)(periductal) or perivascular areas. On day14, a lot of the cellular infiltrate changed larger to coalesce with neighboring foci, which made it difficult to determine the number of infiltrates foci as a measure of salivary gland lesions,. Compared to controls, the characteristic of liver pathological changes included hepatocyte ballooning, acidophilic changes, and intranuclear inclusions in hepatocytes on day3. Some sections showed mild piecemeal, spotted, and focal necroses. Inflammatory cell infiltrates were present in livers on day7after MCMV infection. Many cellular infiltrates coalesced with neighboring foci. Infiltrating inflammatory cells increased around the portal area on day7. Thereafter, the pathological injury of the liver gradually reduced;3) Alanine aminotransferase (ALT) activity:Significantly higher serum ALT levels were observed in MCMV-infected mice compared to controls [(144±11) vs (49.6±5), p<0.05],4) The expression of IL-17and IL-17R in tissues:①The expressions of IL-17mRNA in salivary glands tissues were gradually increased until day14, and then decreased. On day14, the mRNA expressions of the salivary gland IL-17in MCMV infected mice were markedly elevated compared to the normal control group([(0.4103±0.0636) vs (0.245±0.027),p<0.05]. There was significant correlation between the expression of IL-17and pathological damage of tissues (R=0.616; P<0.05); In MCMV-infected mice,the expressions of IL-17R mRNA in salivary glands tissues were gradually increased until day14[(0.4103±0.0636) vs (0.245±0.027),p<0.05] then decreased. There was significant correlation between the expression of IL-17R and pathological damage of salivary gland tissues (R=0.616; P<0.05).②Compared with the normals, the xpression of IL-17and IL-17R mRNA in the liver increased and achieved the highest level on day7([(0.67±0.104) vs (0.287±0.046),p<0.05,and[(0.7035±0.0901) vs (0.442±0.112), p<0.05)]), then gradually decreased on day14and28days, there were significant correlation between IL-17/IL-17R and the pathological damage of the liver, and the correlation coefficient was0.773and0.712respectively;5) The expression of IL-17:The IL-17positive cells in salivay gland were gradually increased until day14, and then decreased. They were mainly located around the duct or in the inflammatory cells site; while the IL-17positive cells from MCMV-infected mice in the liver tissue was significantly higher on7days than these from mock-infected group, they scattered in the liver tissues;6)①Neutralization of IL-17in vivo, the virus titers in liver significantly decreased comparing to the MCMV-infected mice and isotype controls on day7(p<0.05); but the virus titers in salivary gland tissues were found no significant different comaring those two groups②When neutralizing IL-17Ab was administered in vivo, the expression of IL-17protein decreased (p<0.05) in liver on day7, while the expressions in salivary gland were not blocked successfully;③blocked IL-17in vivo, the degree of liver damage was significantly lower than MCMV-infected mice and isotype controls; For the pathology salivary gland, compared with MCMV-infected mice and isotype controls, there was no improvement of IL-17Ab group after intraperitoneal injection of IL-17antibody;④The mRNA levels of IL-17, IL-17R, IFN-γ and IL-10in liver tissues: when blocked IL-17in vivo, the expression of interferon (IFN)-γ[(0.56±0.06) vs (0.55±0.13)vs (0.96±0.2), p<0.05] and IL-10[(0.55±0.073) vs (0.51±0.07) vs(0.903±0.18),p<0.05]relatively increased compared to isotype controls and the MCMV infected mice; the expression of IL-17decreased [(0.77±0.15) vs (0.80±0.14) vs (0.44±0.07), p<0.05]; IL-17R expression increased in MCMV-infected mice compared with normal controls, but there was no difference among the isotype control group and MCMV-infected mice and the IL-17mAb group,[(0.81±0.16) vs (0.89±0.38) vs (0.87±0.23), p>0.05]⑤Alanine aminotransferase (ALT) activity:when IL-17was blocked, ALT levels were reduced compared to MCMV-infected mice and isotype controls [(146±15) vs (102±11) vs (37±12),p<0.05]Conclusion1) The expression of IL-17and IL-17R were closely related with the pathological tissue damage. When IL-17was blocked in vivo, the IL-17mAb could alleviate the severity of MCMV-induced hepatitis, and promoted the recovery of liver function;2) IL-17-associated immune response in favor of the removal of virus in liver in early infection;3) Blocked IL-17in vivo through intraperitoneal injection can not effectively neutralize the inflammatory cytokine IL-17in salivary gland tissue, but can block the expression of IL17in liver tissues;4) The relative low expression of IL-17R and peak latency of IL-17and IL-17R in salivary gland may be conducive to the virus evade immune clearance;5) When IL-17was blocked in vivo, the expression of IFN-γ and IL-10relative increased, which suggested that these factors could regulate each other and play different functions during cytomegalovirus infections; IFN-y and IL-10expressed in liver tissue could accelerate viral clearance and reduce inflammation injury PARTⅡ MCMV Starts Caspase-1Signaling Pathways for Activating the Adaptive Immune Response[Objective]1) Establishing the mouse model with disseminated murine cytomegalovirus infection, to explore the effects of avtivated caspase-1signaling pathways on the pathogenesis of cytomegalovirus infection;2) The impact of caspase-1signaling pathway in the activating Th17cells responses of the adaptive immune.[Methods]1) To establish an animal model of disseminated cytomegalovirus infection: For all studies,4.5-week-old female BALB/c mice (8-10g) were used.32female BALB/c mice were randomly divided into two groups:controls and MCMV-infected mice. Experimental mice were sacrificed3,7,14, and28days after infection. Blood serum was separated and spleen tissues were harvested, partially in4%paraformaldehyde embedded in paraffin, part of the organization saved in-70℃refrigerator;2) A standard plaque assay was used to determine the viral titers in spleen tissues;3) The expression of caspase-1in the splenocyte was detected by western blot;4) The expressions of IL-1β and IL-18in spleen tissues were observed by immunohistochemical staining;5) Th17cells in the spleen were analyzed by flow cytometry.[Results]1) The virus titers in spleen tissues were the hightest on day3, then declined obviously on day7and cannot be detected by standard plaque assay on day14.2) Compared to the controls, the expressions of caspase-1[relative intensity:(1.483±0.420) vs (0.176±0.045), p<0.005)] in MCMV-infected mice were significantly increased on day3, then gradually relieved. 3) The expressions of IL-1β and IL-18in spleen tissues were gradually increased until day7, and then decreased on day14.4) The percentages of Th17cells were increased in MCMV-infected mice compared with controls, with the main increase of CD4+T cells being Th17cells on day14[(1.14±0.09) vs (0.19±0.04), p<0.05], then decreased on day28.5) There was no serious damage in spleen tissues on3days post-MCMV infection; Compared to control-infected mice, MCMV-infected mice exhibited splenomegaly and hyperplasia of white pulp in the spleen tissues on day7; and became much more serious on day14, macrophages, hemorrhagic necrosis and the proliferation of fiber cord were seen in the tissues and then improved obviously on day28.[Conclusion]Cytomegalovirus induced the activation of Caspase-1and promoted inflammatory cytokines (IL-1β,IL-18) to increase the synthesis and release, which promoted the activity of Th17cells not only had antiviral effect, and also caused the immuno-pathological damage of the spleen.