Effects Of Murine Cytomegalovirus Infected Mouse Astrocyte On The Fuctions Of Cocultured T Cells And The Expansion Of Regulatory T Cells

Paper one: Effects of murine cytomegalovirus infected mouse astrocytes on the functions of co-cultured T cells and the expansion ofregulatory T cellsObjectives: The purposes of the present study are: â‘ to study the T cell immune provoked by cytomegalovirus infected astrocytes; â‘¡ to explore the effect of cytomegalovirus on the expansion of regulatory T cells, and its possible mechanism; â‘¢to investigate whether allitridin can block the effects of cytomegalovirus on the expansion of regulatory T cells (Treg cells) and its mechanism.Methods:â‘  A cell model of MCMV infected astrocyte was established. Glial cells derived from BALB/c neonate mouse were primarily cultured and passaged to obtain pure astrocytes. The third generation astrocytes were used in following experiments. Immunohistochemistry method detecting the glial fibrillary acidic protein (GFAP), which is the characterized marker of astrocyte, was used to determine the purity of astrocytes. After attacked by MCMV, whether astrocyte permissive to MCMV proliferation was judged by the following methods: observing the cytopathic effect (CPE) under the inverted microscope, X-gal staining for the MCMV RM461 (a recombinant MCMV inserted a Lac-Z gene), and drawing the viral growth curve.â‘¡ A co-culture system of T cells and MCMV infected astrocytes was established: T cells were isolated from spleen and lymph nodes of BALB/c mouse, which was infected by MCMV two months ago. And then CD3+ T cells were isolated and purified by nylon fiber column method and confirmed by flow cytometry. T cells were co-cultured with astrocytes infected by MCMV (multiply of infection: 0.01).â‘¢ To observe the T cell immune in the co-culture system, the proliferation of T cells was evaluated by observing T cell clone formation and flow cytometry detecting with fluorochrome CFDA-SE staining; levels of Th1 cytokines, IFN-γ and TNF-α in co-culture supernatants were measured by double-antibody sandwich ELISA; suppression of co-cultured T cells on MCMV replication in astrocytes was determined by observing cytopathic effect (CPE) of astrocytes and measuring the load of MCMV DNA by PCR assay.â‘£ To investigate the effects of MCMV infection on expansion of regulatory T cells, levels of Foxp3 mRNA and proportion of CD4+CD25+ cells were measured at 24h, 48h, and 72h after co-culture by RT-PCR and flow cytometry, respectively. The ability of T cell proliferation at 72h after co-culture was assessed by mixed lymphocyte reaction.⑤ Whether MCMV infection of astrocytes influent the level of TGF-beta mRNA at 72h after co-culture was determined by RT-PCR assay.â‘¥ For investigation of allitridin's effects on regulatory T cells expansion, maximum tolerance concentration (MTC) of allitridin on astrocytes was firstly determined by MTT assay. And then MTC allitridin was added in the MCMV infected astrocyte and T cell co-culture system. After 72h, change of Foxp3 mRNA level and CD4+CD25+ T cells' proportion was measured by RT-PCR and flow cytometry methods, respectively.⑦ Influence of allitridin on the level of TGF-beta mRNA in MCMV infected astrocyte was also detected by using RT-PCR, to explore the mechanism of its effects on the expansion of regular T cells caused by MCMV.Results:â‘  We obtained pure astrocyte culture with GFAP positive cells more than 95%. After infected by MCMV, the astrocytes appeared typical cytopathic effects, expressed Lac-Z gene of the MCMV RM461, and released infective progeny virus. The viral growth curve showed that MCMV proliferated in astrocytes as quickly as in fibroblasts, indicating that astrocytes are fully permissive for productive infection of MCMV.â‘¡ CD3 positive cells reached 95% of the cells collected after nylon column treatment. And a co-culture system of T cell and MCMV infected astrocyte was successfully established.â‘¢After 3d co-cultures, in MCMV infection group, T cells aggregation and clone formation were observed, and flow cytometry assay showed that 6.68 ± 0.61% T cells had proliferated, while in uninfected controls, no clone formation was observed, and proliferated T cells was only 1.07 + 0.03% (P<0.01). The levels of IFN-γ in co-culture supematants of MCMV infected group were significantly elevated with 22.9 + 3.4 pg/mL (P<0.05 compared with uninfected control which level was below 8 pg/mL). While levels of TNF-α were under 8 pg/mL in both cytomegalovirus infected and uninfected groups. Compared with infected controls, 31.25%, 50% and 75% astrocyte CPE were diminished in T cell-to-infected astrocyte co-cultures with 0.25:1, 0.5:1 and 1:1 proportion, respectively. In 1:1 of T cells-to-infected astrocyte co-culture, MCMV DNA load was reduced by 75%.â‘£The supematants of T cell-to-infected astrocyte (1:1) 3d co-culture group also showed suppression on CPE (inhibiting rate: 81.25%) and viral DNA load (inhibiting rate: 70%) of infected astrocytes.⑤When T cells after 3d co-cultured with infected astrocytes were collected and co-cultured with fresh infected astrocytes again, thier antiviral effect was obviously diminished, the CPE score and MCMV DNA load of infected astrocytes were not different with those of infected controls (P>0.05), rate of proliferated T cells decreased (1.83 + 0.25%) and level of secreted IFN-γ was below 8 pg/mL in co-culture supematants, suggesting the inhibition of T cell functions.â‘¥Both the expression of Foxp3 mRNA and the proportion of CD4+CD25+ cells in T cell-to-infected astrocyte group were up-regulated, no mater compared with those of fresh T cells (before co-culture) or T cells co-cultured with uninfected astrocytes. The effects appeared time-dependently. The longer co-culture lasted, the more increase was found. The increases were already observed on 24h after co-culture, and dramatically up-regulated on 72h. At the same time, T cells after 72h co-cultured in MCMV infection group show lower response (simulation index: 1.18 + 0.06) to the mixed lymphocyte culture than those of the controls (simulation index: 1.78 ± 0.13), indicating a lower proliferating ability.⑦ Level of TGF-beta mRNA in MCMV infected astrocytes at 72h post infection was significantly up-regulated, reaching about two times of that of uninfected control (P<0.01⑧ MTC of allitridin on astrocytes was 9.6μg/mL (MTT assay). The treatment of 9.6μg/mL allitridin could obviously decrease the level of Foxp3 mRNA and the proportion of CD4+CD25+ T cell in MCMV infected co-culture system (P<0.01), although which still higher than those of normal control group (P<0.05). TGF-beta mRNA level in MCMV infected astrocytes was also significantly inhibited by allitridin treatment, compared with infected control (P < 0.05), but still higher than normal astrocyte control (P <0.01).Conclusion:â‘  AT cell and MCMV infected astrocyte co-culture system was established, providing a platform for investigation of T cell immune to central nervous system CMV infection.â‘¡ Cytomegalovius infected astrocytes could stimulate T cells proliferation and activation. Activated T cells had antiviral effect, which partly mediated by some secreting cytokines. The function of T cells was rapidly inhibited after activation, of which complicated mechanisms would underlie.â‘¢ CMV infected astrocytes could induce the expression of Foxp3 mRNA, and increase the CD4+CD25+ T cells' proportion of the co-cultured T cells. CD4+CD25+ regulatory T cells increased may contribute to secondary immune suppression in the co-culture system. TGF-beta mRNA level was significantly increased in astrocytes after CMV infection, which would be an important mechanism of expansion of Treg cells. The environment of central nervous system may favor Treg induction in affected region, which would benefit the host by limiting immune damage. On the other hand, CMV also might manipulate the functions of Treg cells to evade specific immune elimination so that to cause a persistent latent infection. â‘£ Allitridin could partly block Treg expansion causing by CMV infection, with a possible mechanism of inhibiting TGF-beta elevation. By using this strategy, allitridin could enhance the specific cellular immune reactions against CMV and be helpful for clearance of CMV viruses from host, suggesting another mechanism of allitridin against CMV.Paper two: Inhibition on immediate early gene expression, an importantmechanism of allitridin anti-HCMV effectsObjective: Allitridin (diallyl trisulfide), a main organic compound of garlic (Allium sativum), has been reported having anti-HCMV efficacy in experiments and in clinical. However, its mechanism of action is still poorly defined. Our previous study demonstrated that allitridin could markedly decrease the level of HCMV immediate early antigens (flow cytometry), which are considered as being the essential regulating proteins for HCMV replication and playing the important role in the pathogenesis of HCMV diseases as well, providing a clue for investigation of allitridin's action. This study was aimed to investigate the action of allitridin in the replication cycle of HCMV, especially to determine its effects on the transcription and expression of viral immediate early (ie) genes. Methods:â‘  The maximal tolerable concentration (MTC) of allitridin for HEL cells was determined based on the MTT assay.(2) The inhibition of allitridin on HCMV was measured by plaque-reduction assay.(3) For the determination when allitridin acts in the HCMV replication cycle, time-of-addition and removal studies in a single viral cycle format at MOI of 2.5 were performed. â‘£ And the effect of allitridin on the replication of HCMV genomic DNA was analyzed at 72 h post infection (after one round of viral replication) by Southern blot assay.⑤ The suppression on HCMV immediate early genes' transcription by allitridin was measured by Northern blot at 4 h post infection. And the suppression on the translation by allitridin was assayed by Western blot at 24 h post infection (at the first round of viral replication cycle) and 72 h post infection (at the second round of viral replication cycle), respectively.â‘¥ In all bloting tests, MTC and ED₅₀ concentrations of allitridin were used and corresponding concentrations of ganciclovir served as controls.Results:â‘  MTC of allitridin for HEL cells was 9.6μg/mL and the median cytotoxic concentration (IC₅₀) was 70μg/mL. â‘¡ In HCMV plaque-reduction assay, allitridin appeared a dose-dependent inhibitory ability with EC₅₀ value of 4.2 μg/mL (selective index, SI = 16.7).â‘¢ Time-of-addition and time-of-removal studies showed that allitridin inhibited HCMV replication only when the drug appeared in the period from 0 h to 8 h post infection.â‘£ Decrease of viral DNA load in infected cells was also detected under allitridin treatment. There was no significant difference between the suppressions on viral DNA load of allitridin and ganciclovir treatment, under the concentrations used in the experiments.⑤ Confirmed by Northern, immediate early gene transcription (iel) was suppressed by allitridin, but not by GCV, in a single HCMV cycle format. The inhibitory rate of allitridin on iel mRNA reached 30.38 %～46.61 % with EC₅₀ and MTC allitridin.â‘¥ Results of Western blot showed that both IE72 and IE86 level decreased after allitridin treatment. The expression of IE72 under allitridin treatment was suppressed by 42.6 %～64.9 % at 24h post infection, and the expression of IE86 by 50.7 %～70.6 %. And by 72h post infection, allitridin still obviously reduced the levels of IE72 and IE86 proteins. Furthermore, allitridin appeared stronger inhibition on IE86 than on IE72, with the inhibitory rate on IE86 (77.9 %～87.7 %) was almost twice of that of IE72 (36.4 %～49.3 %). Ganciclovir had no effect on IE protein expression at 24h post infection. Although inhibiting IE72 expression by 42.9%～64.3% at 72h post infection, Ganciclovir appeared weaker suppression on IE86 (inhibitory rate was 45.7%～79.2%) than allitridin.Conclusion:Allitridin could obviously suppress the transcription and expression of HCMV ie genes,by which it further effectively inhibits HCMV replication, indicating an importantmechanism of allitridin against HCMV.