| Part I Function and changes of natural killer cell in cryptococcal infection and its influence on the course of cryptococcal infectionSection I Establishment of mice model of pulmonary cryptococcal infection and natural killer ce-depletion mice modelObjectiveTo establish the mice model of pulmonary cryptococcal infection and natural killer-depletion mice modelMethodsC57BL/5mice were anesthetized by an intraperitoneal injection of2.5%Avertin and subsequently inoculated intranasally with1×106C.neoformans cells in40ul PBS. Control mice were inoculated intranasally with40ul PBS. The inocula used for nasal inhalation were verified by quantitative culture on YPD agar. At specific times following inoculation, mice were euthanized and observed the pathological and histological lesions in the lungs, determined the CFU in lung and brain tissue. C57BL/5mice were anesthetized by an intraperitoneal injection of anti-NK1.1monoclonal antibody peritoneal fluid (150μL/mice). At specific times following inoculation, mice were euthanized. The percentage of NK cells of splenocytes were analyzed through flow cytometric analysis.ResultsAt day7following infected with C.neoformans, there were pathological lesions in the lung. Sections of lung were analyzed by light microphotogragh and showed yeasts in the lung. At day3,7,10,14,28post infection, CFU of lungs were gradually increased and reached the peak at day10or day14post infection. Brain CFU were not positive until day10post infection.At day2after intraperitoneal injection of anti-NK1.1monoclonal antibody peritoneal fluid (150μL/mice), the percentage of NK cells in mice splenocytes were decreased92.33±0.15%compared with naive mice (2.74±0.22%vs0.21±0.03%), and the effect of depletion of anti-NK1.1mAb was lasted at least10days.Conclusions1. The mice model of lung cryptococcal infection were established through intranasal route, and this model can mimic the natural cryptococcal infection in human.2. Anti-NK1.1monoclonal antibody peritoneal fluid can effectively and efficiently deplete the NK cells in mice, and the NK cell-depletion mice model is a useful tool to study the function of NK cell in vivo. Section Ⅱ Time-phase characteristics of the number and function of NK cell during cryptococcal infection and its effects on the course of cryptococcal infectionObjectiveTo study the time-phase changes of the number and function of NK cell locally and systemically during cryptococcal infection, and to evaluate its effect on fungal growth in main organs and the survival rate of infected mice.MethodsUsing the mice model of lung cryptococcal infection and NK cell-depletion mice model, mice were grouped as anti-NK1.1treated and anti-NK1.1non-treated. At day3,7,14post infection, mice were euthanized. Absolute numbers, activation, de-granulation of NK cells in lung leukocytes and splenocytes were analyzed. At day1,3,7,10,14,28post infection, CFU of mice lung and brain were evaluated. At last, the survival rates of infected mice were observed.ResultsThe number of splenocytes, number of NK cells in splenocytes, number of lung leukocytes and number of NK cells in lung leukocytes were increased during the lung cryptococcal infection. After NK cells were depleted in vivo, the number of lung leukocytes were decreased significantly (P<0.05), but the number of splenocytes were not affected.The expression of CD69and CD107a and the excretion of IFNy and perforin of NK cell were increased at the early stage during cryptococcal infection, reached their peak at day3post infection, and decreased gradually thereafter.In the anti-NK1.1non-treated group, CFU of lung were below the inoculation at day3post infection and increased gradually, reached its peak at day10or day14and decreased thereafter. However, in the anti-NK1.1treated group, CFU of lung were increased during the infection course and there were more CFU of lung compared with anti-NK1.1non-treated group at day3, day14, day28(P<0.05).From about day7or day10post infection, the culture results of brain homogenates become positive, and there was no significant difference between two group (P>0.05).In anti-NK1.1non-treated group and anti-NK1.1treated group, deaths of mice were appeared at day54and day45post infection respectively. The survival rates of these two group were significantly different (P=0.0007).Conclusions1. During cryptococcal infection, the number of NK cells in lung leukocytes and splenocytes were increased, and depletion of NK cell in vivo can make the number of lung splenocytes decreased.2. After infected with Cryptococcus, NK cell can be activated and the ability of de-granulation was enhanced.3. With depletion of NK cell in vivo, CFU of mice lung were increased more than that of mice with intact NK cell at early stage (day3) and late stage(day14, day28) of infection.4. Depletion of NK cell can significantly influence the survival rate of lung cryptococcal infection mice. Part II Immune-regulatory function of NK cell and its possible mechanism in cryptococcal infectionSection I The influence of NK cell on adaptive immune response during cryptococcal infectionObjectiveTo evaluated the influence of NK cell on adaptive immune response and the balance of Th1/Th2response during cryptococcal infectionMethodsUsing the mice model of lung cryptococcal infection and NK cell-depletion mice model, mice were grouped as anti-NK1.1treated and anti-NK1.1non-treated. At day7,14post infection, mice were euthanized. The secretion of IFNy and IL-4of CD4+T cells in lung leukocytes were analyzed. The level of IFNy and IL-4of lung homogenates were assayed.ResultsAt day7,14post infection, secretion of IFNy of CD4+T cells in lung leukocytes were both increased significantly (P<0.05), and the percentage of IFNy+CD4+T cells at day14p.i. were greater than that at day7p.i.(P<0.05). Secretion of IL-4of CD4+T cells in lung leukocytes reached its peak at day7p.i, and the percentage of IL-4+CD4+T cells at day14p.i. were decreased compared to that at day7p.i.After depletion of NK cell in vivo, the percentage of IFNy+CD4+T cells in anti-NK1.1treated group were decreased compared to that in anti-NK1.1non-treated group at both time points, and in anti-NK1.1treated group, percentage of IFNy+CD4+T cells at day14p.i. were not greater than that at day7p.i.(P>0.05). However, percentage of IL-4+CD4+T cells in anti-NK1.1treated group increased gradually.At day7,14post infection, the level of IFNy of lung homogenates were increased significantly at both time points(P<0.05), and the level of IFNy of lung homogenates at day14p.i. were greater than that at day7p.i.(P<0.05). Level of IL-4of lung homogenates were increased at both time points, and reached its peak at day7post infection. After depletion of NK cell in vivo, level of IFNy of lung homogenates in anti-NK1.1non-treated group were greater than that in anti-NK1.1treated group at both time points(P<0.05). However, Level of IL-4of lung homogenates in anti-NK1.1treated group were increased gradually, and reached its peak at day14post infection.Conclusions1. Cryptococcal infected mice develop a protective, Th1-skewed immune reponse.2. NK cell depleted mice alter the balance of Th1/Th2response during cryptococcal infection and develop a non-protective, Th2-skewed immune response. Section Ⅱ The influence of NK cell on Dendritic Cells (DC) during cryptococcal infectionObjectiveTo investigate the influence of NK cell on the mature status and function of DC during cryptococcal infectionMethodsAfter heat-killed Cryptococcus co-cultured with BMDC for24hours, the expression of MHC Ⅱ, CD80, CD86were analyzed and supernatant levels of IL-12p40and TNFa were determined. Using the mice model of lung cryptococcal infection and NK cell-depletion mice model, mice were grouped as anti-NK1.1treated and anti-NK1.1non-treated. At day7post infection, mice were euthanized, expression of MHC II, CD80, CD86on splenic DC, percentage of DC in lung leukocytes, absolute number of DC in lung leukocytes, subset of DC, percentage of CD45+CD11b+Gr-1int in lung leukocytes and level of IL-12p40of lung homogenates were analyzed.ResultsAfter heat-killed Cryptococcus co-cultured with BMDC for24hours, expression of MHC Ⅱ, CD80, CD86on BMDC and supernatant levels of IL-12p40and TNFa were significantly increased (P<0.05). At day7post infection, expression of MHC Ⅱ, CD80, CD86on splenic DC were also significantly increased (P<0.05). Expression of MHC Ⅱ and CD86on splenic DC in anti-NK1.1treated group were significantly decreased compared to that in anti-NK1.1non-treated, and expression of CD86on splenic DC were not increased at day7post infection in anti-NK1.1treated group.At day7post infection, percentage and absolute number of DC in lung leukocytes, percentage of CD45+CD11b+Gr-1int in lung leukocytes were increased significantly(P<0.05). After NK cell depleted, although percentage and absolute number of DC in lung leukocytes, percentage of CD45+CD11b+Gr-1int in lung leukocytes were still more than that of un-infected mice(P<0.05), these were lower than that of anti-NK1.1non-treated mice (P<0.05). Percentage of mDC in lung leukocytes DC were not changed after cryptococcal infection and NK cell depletion(P>0.05).At day7post infection, level of IL-12p40of lung homogenates were elevated significantly (P<0.05). After NK cell depleted in vivo, level of IL-12p40of lung homogenates were lower in anti-NK1.1treated group than in anti-NK1.1non-treated mice (P<0.05).Conclusions1. During cryptococcal infection, percentage and absolute number of DC in lung leukocytes, level of IL-12p40of lung homogenates were increased and maturation of DC were induced, facilitate the development of Th1-skewed immune response. After NK cell depleted in vivo, number, maturation and function of DC were in injured.2. During cryptococcal infection, subset of DC was mainly mDC and not changed after NK cell depletion.3. During cryptococcal infection, percentage of CD45+CD11b+Gr-1int in lung leukocytes were increased. After NK cell depleted in vivo, percentage of CD45+CD11b+Gr-1int in lung leukocytes were decreased, and may induced the decreased the number of DC in lung leukocytes. |
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