Study On The Cellular Immunopathogenesis Of Candidiasis

Part One Expression of Chemokines and Chemokine Receptors during Experimental Vaginal Candidiasis【Objectives】To examine the local production of chemokines and chemokine receptors during an experimental Candida albicans vaginal infection, and to investigate the role of chemokines and chemokine receptors in immunopathogenesis of vulvovaginal candidiasis.【Methods】Mice were randomly divided into 4 groups: group A, group B, group C and naive mice. The former 3 groups were further divided into 5 subgroups: the 2nd-day group, the 4th -day group, the 7th-day group, the 14th-day group and the 21st-day group. Mice from group A were treated with estrogen every other day by injecting 0.1 mg of estradiol benzoate in 0.1 ml of sesame oil subcutaneously. Six days after the first estrogen treatment, mice were inoculated vaginally with 5×104 stationary-phase C. albicans blastoconidia in 20μl of PBS. Mice from group B were treated with estrogen as described above and given PBS intravaginally. Mice from group C were given a vaginal inoculum with C. albicans and every other day injections of sesame oil without estrogen. Infection was assessed by microbiological and histopathological evaluation. The expression of chemokines and chemokine receptors in vaginal tissue and in the lumbar(draining) lymph nodes were assessed by RT-PCR, Real-time RT-PCR, immunohistochemistry and ELISA.【Results】(1) Vaginal fungal burden: Mice from group A developed a high-titer vaginal infection, which persisted through the 3-week period. Mice from group C developed a low-titer vaginal infection with significantly fewer CFU of C. albicans compared with the Candida burden in mice from group A (days 2 to 7), the vaginal infection declined rapidly by day 14 and cleared by week 3. These results were supported by lavage fluid hyphal scores. Mice from group B did not acquire a vaginal Candida infection, evidenced both by the lack of growth on Sabouraud dextrose agar and the lack of yeast elements in wet-mount slide preparations. (2) Histological examination of viginal tissue: PAS stain of a vagina section from a mouse from group A shows a florid infection with pseudohyphae and blastoconidia in the luminal vagina as early as 2 days postinoculation. 4 days after inoculation, C. albicans organisms were plentiful in the vaginal lumen, and pseudohyphae were adhering to the surface of the epithelium. Pseudohyphae and blastoconidia present in mice 1 week after inoculation were observed penetrating the cornified epithelium, with a qualitative reduction of fungal luminal presence. At 2 weeks postinoculation, hyphae were appreciably more fragmented and hyphal fragmentation was increased during the third week. In mice from group C, there were only a few pseudohyphae in the vaginal lumen by day 14, and Candida organisms in histological sections were not detectable on day 21. In mice from group B, no Candida was observed in the lumen of the vagina. (3) RT-PCR: Estrogen had no significant effect on the expression of chemokines and chemokine receptors. Compared with the basal level of control mice, the mRNA expression of chemokines and chemokine receptors were significantly increased in the vaginal tissue of infected mice(P<0.05), vaginal chemokines production in mice from group C declined to basal levels as the infection resolved, while those from group A remained elevated. In contrast, in the lumber lymph nodes, levels of MCP-1, MCP-2, IP-10 and MIG were unaffected by infection(P<0.05), while levels of SDF-1 in mice from group A were significantly higher than those in mice from group B on days 2, 7~21(P<0.05), as for chemokine receptors, they were significantly increased in the lymph nodes of infected mice. (4) Real-time RT-PCR: Compared to levels in murine vaginae from group B, significant increases in MCP-1, CCR2 and IP-10 were detected at day 2 through 21, day 7 through 21, day 7 through 21 postinoculation, respectively (P<0.05) in murine vaginae from group A and peaked at week 2, week 1 and week 2. (5) Immunohistochemical study: In epithelial keratinocytes, MCP-1 protein was detected in the cytoplasm and nuclei, SDF-1 and its receptor CXCR4 protein was located in the cytoplasm and cell membrane. MCP-1, SDF-1 and CXCR4 were also detected in the cytoplasm of a proportion of infiltrating inflammatory cells and endothelial cells of the blood vessels in the subepithelial lamina propria. The results of the average OD of MCP-1, SDF-1 and CXCR4 in vaginal tissue showed significant increases at each time point postinoculation(P<0.05). (6) ELISA: Significant increases in MCP-1 were detected at day 2 through 21 and day 7(P<0.05) in vaginae from group A and C, respectively. In the lumber lymph nodes, MCP-1 levels did not significantly change in response to infection and/or estrogen (P<0.05). (7) The percentage of PMNs, macrophages, lymphocytes, and other leukocytes were not significantly altered in response to vaginal infection or pseudoestrus alone.【Conclusions】Chemokines and chemokine receptors may play a role in the local vaginal mucosal response to C. albicans. In addition, SDF-1 and chemokine receptors may involve in the lumbar(draining) lymph nodes response to C. albicans.Part Two The interaction of murine dendritic cells with Candida albicans【Objectives】To examine the functional outcome of the interaction of Candida albicans with immature murine dendritic cell line DC2.4.【Methods】(1) DC were incubated in suspension with Candida for 30min, 1h and 2h at 37℃, and phagocytosis was quantified. (2) Flow cytometric analysis of DCs were performed after 2h and 4h of incubation in the presence of Candida cells(1:1 ratio with respect to DCs). (3) DCs were electrophysiologically analysed using the whole cell configuration of the patch clamp technique. The effects of Candida albicans on delayed rectifier potassium current(Ik) of DC were evaluated. (4) The intracellular free calcium contration([Ca2+]i) was determined with the calcium-sensitive fluorescent dye Fluo-4 AM. The effects of Candida albicans on [Ca2+]i of DC were evaluated. 【Results】(1) DC2.4 phagocytosed Candida in a time-dependent manner. The percentages of DC-associated yeast cells were 74.76±1.04%, 76.42±1.13% and 90.99±1.42%, respectively, and the phagocytic index were 1.328±0.023, 2.386±0.041, 3.844±0.037, respectively. (2) Candida albicans-treated DCs showed an increases expression of the costimulatory molecules CD80 and major histocomatibility complex classesⅡ(MHCⅡ) surface antigens after 2h of incubation(P<0.05) and then decreased after 4h. The expression of CD86 did not significantly change(P>0.05). (3) A short-term treatment with Candida albicans provoked a strong appearance of Ik. Itraconazole on its own did not affect Ik when it was added to DC(P>0.05), but when DC were preincubated with itraconazole and were then stimulated by Candida albicans, Candida albicans provoked a lower peak K+ current amplitude(P<0.05). (4) In the presence of Candida albicans, the [Ca2+]i increased in a dose-dependent manner. Itraconazole on its own decreased [Ca2+]i when it was added to DC(P<0.05). When DC were preincubated with itraconazole and were then stimulated by Candida albicans, the amplitude of increasing of [Ca2+]i caused by Candida albicans was smaller(P<0.05). Fluconazol on its own did not affect DC [Ca2+]i and the effect of Candida albicans on DC [Ca2+]i(P>0.05).【Conclusions】(1) Such interaction between DC and Candida albicans may facilitate the induction of CD80 and MHCⅡ, elevate functional state of DC by increasing DC Ik and [Ca2+]i. Antimycotic agent itraconazole may suppress DC fuctions by decreasing DC [Ca2+]i and the effects of Candida albicans on DC Ik and [Ca2+]I and have immunosuppressive effects, acting on the host's immunity. By contrast, fluconazole may have no inhibitory effect on DC functions.