| Sporotrichosis is a commonly seen world-wide distributed deep mycosis which involves skin, sub-cutaneous tissue and deep organs. It is a serious hygiene problem in many places. Northeast China is one of the most endemic regions of sporotrichosis. The causative fungus is Sporothrix schenckii complex containing six species. The strain causing sporotrichosis in China is Sporothrix globosa. As a dematiaceous fungus, Sporothrix can produce a very important virulence factor, melanin, which is also confirmed to be related with many other kinds of pathogenic fungi. However, researches focusing on Sporothrix melanin is quite limited in number. It is only clear that Sporothrix melanin can inhibit cell immunity by disrupting phatocytosis and oxidative killing functions of macrophage.To further elucidate pathogenic mechanism of Sporothrix melanin, we induced an albino strain of Sporothrix globosa and extracted melanin ghosts in this research. In-vitro cell culture method and mice infection model were used to investigate the influence of Sporothrix melanin on antigen presentation and CD4+ T cell activation induced by macrophage.The wild type strain used in this study was confirmed to be Sporothrix globosa by CAL gene sequencing.The best inoculation density and energy density for UV inducement were 1×103/ml and 52×10-3J/cm2~104×10-3J/cm2, respectively. We have acquired a stable albino strain(MEL-) of Sporothrix globosa with this method. Comparing with its original melanin-producing wild-type strain(MEL+), MEL- mutant had similar character except for its white color. We studied the superstructures of both strains and found that there were large number of melanin particles inside and on the surface of cell wall of MEL+ strain, while melanin was absent in MEL- cell wall. We also extracted melanin ghosts from MEL+ spores by enzymes digestion and HCl boiling. A melanin ghost remains its original shape as when it was in the spore and becomes an empty shell without any cell structure. It represents almost all the melanin contained by one spore. Extracted melanin ghosts were indentified by positive dopa staining, negative cotton blue staining, negative PAS staining, specific absorbance curve and TEM manifestation. In the mice infection model, both MEL- and MEL+ strains were injected into abdominal cavity of BALB/c mice. After 21 days, Sporothrix could be isolated from all mice ’s lung, liver, spleen, kidney and testicles in MELgroup, while only from spleen and kidney of 20% mice in MEL- group. The organ fungal load per gramme of MEL+ group was also significantly higher than that of MEL- group. And the average weight of spleen of MEL+ group(0.328±0.162g) was significantly higher than that of MEL- group(0.138±0.028g). These results showed that Sporothrix globosa melanin is related with pathogenicity.Spores of MEL- and MEL+ strains were respectively co-cultured with mice peritoneal mactophages for 24 h, while macrophages were cultured alone for 24 h as Control. Realtime PCR and flow cytometry were applied in this research to detect the expression levels of Class II Transactivator(CIITA), its promotor PI, PIII, PIV, and MHC class II, costimulators CD80 and CD86 of macrophages in each group. We have found that the CIITA and PIV levels in MEL- group were significantly higher than those of MEL+ group. The MFI of MHC class II in Control, MEL+ and MEL- group were 6010.66±597.74, 7524.33±1047.01 and 10161.67±1205.23, respectively(MFI of MEL- was significantly higher than that of MEL+); The MFI of CD86 in Control, MEL+ and MEL- group were 4306.00±396.31, 9065.67±659.80 and 11570.00±1004.75, respectively(MFI of MEL-was significantly higher than that of MEL+); The MFI of CD80 in Control, MEL+ and MEL- group were 12869.00± 1746.73,10171.00±1195.26 and 7469.67±1513.01, respectively(No statistical difference was found between MEL- and MEL+ group). These results indicate that melanin of Sporothrix globosa can inhibit antigen presentation of macrophage. In this process, PIV and CD86 may be the most important promotor and co-stimulator, respectively. In the mice infection model, the peritoneal macrophages were isolated 21 d after infection and the MFI of MHC class II in Control, MEL+ and MEL- group were 2287.33±55.52, 11901.33±904.87 and 14785.33± 1506.12, respectively(MFI of MEL- was significantly higher than that of MEL+); the MFI of CD86 in Control, MEL+ and MEL- group were 2415.00±404.47, 2092.00±136.92 and 2025.67±99.12, respectively(No statistical difference was found); the MFI of CD80 in Control, MEL+ and MEL- group were 14683.33±555.75, 7380.67±313.95 and 5720±163.70, respectively(MFI of MEL- was significantly lower than that of MEL+). The results told us that the inhibition of antigen presentation could even last long to when the dissemination occurs. We transformed MEL- and MEL+ strains to yeast phase and both strains’ colonies turned to white. The melanin production of both strains was also low under TEM. After 24 h co-culture of yeast form of MEL-/MEL+ strain with mice macrophage, the MFI of MHC class II in Control, MEL+ and MEL- group were 6586.33±1435.67, 5957.33±904.87 and 7226.00±518.37, respectively(no statistical difference was found amoung groups). This finding had further proved melanin’s inhibition of antigen presentation. However, after 24 h co-culture of melanin ghosts with macrophages, the MFI of MHC class II in Control and MG group were 12766.67±2150.19 and 12866.67±1594.78, respectively. No significant difference were found, indicating that melanin itself can’t down regulate MHC class II directly. Melanin’s suppression on antigen presentation may be related with its masking effect on fungal atigens by forming a protective layer outside the cell wall of Sporothrix.Further research compared the level of NO secretion and the rate of TNF-α positive macrophages in each group after 24 h of co-culture.It was found that the NO level in the culture medium of MEL- group(121.07±6.60μmol/L) was significantly higher than that of MEL+ group(102.50±7.30μmol/L), which was also significantly higher than that of Control group(93.27±5.35μmol/L); The rate of TNF-αpositive macrophages in MEL- group(28.63±7.88%) was significantly higher than that of MEL+ group(15.57±3.44%), which was also significantly higher than that of Control group(15.50±2.01%). Considering the fact that NO and TNF-αis the mark of M1 type(killing) macrophages, this result suggests that melanin can inhibit macrophage’s antifungal ability by influencing its M1 type activation. To study if the CD4+ T cells activation is influenced after the inhibition of antigen presentation function of macrophage, mice CD4+ T cells were co-cultured with macrophages stimulated by MEL-, MEL+ and Control, respectively.The rate of different cytokine positive T cells of each group was detected by flow cytometry. The rates of IL4 positive T cells in MEL-,MEL+ and Control group were 4.77±0.32%, 6.67±0.75% and 1.86±0.07%, respectively(MEL- group was significantly lower than MEL+) and the rates of IFN-γ positive T cells in MEL-, MEL+ and Control group were 6.90±0.30%, 3.34±0.44% and 3.59±0.53%, respectively(MEL- group was significantly higher than MEL+). This showed that melanin tempt to induce th2 response rather than th1 response. The inhibition of th1 response could impair antifungal immunity by weakening the killing function of macrophages. The down-regulation of IFN-γ can further inhibit antigen presentation for a long time, which may be the reason why that the antigen presentation was still suppressed in the late stage of infection in the mice model. There was no statistical difference of the levels of IL17, Foxp3, IL10 and IL2 between MEL- and MEL+ group. In conclusion, Sporothrix globosa melanin can help the fungus to escape from the immune system by inhibiting antigen presentation and M1 activation of macrophage, and by inducing a th2 CD4+ T cell response.At last, we performed a proteomics research to compare the proteins of MEL- and MEL+ strains. One hundred and seventeen proteins were identified as they have significant differences in expression levels between two strains. No major virulence factor was among these proteins which proved that the difference in pathogenicity between strains were becauses of different levels of melanin, not of mutation of other virulence factors. What was interesting is, the major factors in known melanin synthesis pathways were also not among these proteins. This indicated the complexity of melanin production regulation which may involve many regulators.This research have revealed the mechanism of a conservative but important fungal virulence factor, melanin, in the battle between Sporothrix globosa and host immune system. Elaboration of melanin’s influence on macrophage activation and antigen presentation have provided new insights of Sporothrix-host interactions. |
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