Study Of T And B Cells In Sporotrichosis Patients Infected With Sporothrix Globosa

Sporotrichosis is a subcutaneous mycotic infection caused by Sporothrix schenckii complexs.Up to now,Sporothrix schenckii complexs has been found to contain seven genotypes,among which Sporothrix schenckii sensu stricto,Sporothrix brasiliensis and Sporothrix globosa are most clinically relevant.Sporothrix globosa has a global distribution and is especially epidemic in Northeast China,and till now Sporothrix globosa is the only reported strain isolated in China.Compared with Sporothrix brasiliensis which are prone to caused disseminated infection,the clinical manifestations caused by Sporothrix globosa mostly present with fixed form,owing to its relatively low virulence,which indicates it may trigger different immune responses in the host.Sporotrichosis caused by Sporothrix globosa may disseminated to other organs under immunosuppressive factors like organ transplantation、HIV infection,which is always fatal.Up to now,the defense mechanisms against Sporothrix complexs in human are unclear.Researches in the mechanisms are mostly carried out in mice or in vitro.Previous studies have acquired a consensus that Th1 and Th17 responses are considered critical to host defense against Sporothrix schenckii infection.Besides,specific antibodies against Sporothrix brasiliensis and Sporothrix schenckii have been found in human and mice,like anti-Gp70 antibody,which indicates humoral immunity also participate in the defense.However,B cells and Tfh cells playing important roles in the humoral immunity were rarely investigated in sporotrichosis.In order to explore the cell-mediated immunity and humoral immunity changes in patients,and the role of immune response in determining the clinical outcome of sporotrichosis,we collected84 patients diagnosed of sporotrichosis and then studied the immune changes in circulation,skin lesions and in vitro co-culture system.Firstly,we collected the peripheral blood of 50 adult patients and 25 healthy control.The patients and control are matched in sex and age.We detected the percentages of CD4~+T-bet~+Th1、CD4~+GATA-3~+Th2 and CD4~+ROR-γt~+Th17 in the circulation by flowcytometry.The results revealed a Th2 biased immunity with an elevated percentage of Th2 in the whole patients(P=0.0119)and patients with different duration(duration<6 months(P=0.0413);duration>6 months(P=0.0226))while Th1and Th17 responses did not change significantly compared with healthy control.The proportion of Th2 increased significantly in the group of fixed form compared with healthy control(P=0.0058).We further explored the percentages of Th1,Th17 and Th2 again through detecting the specific cytokines in CD4~+T cells as they have shown the changes as described above.We collected another 18 patients and 20 healthy control.Although the percentage of CD4~+IFN-γ~+Th1 was not changed in the whole patients,we detected a significant elevation in the patients with duration less than 6 months(P=0.0006)compared with healthy control.The proportions of CD4~+IL-4~+Th2 and CD4~+IL-17A~+Th17 were both elevated significantly in whole patients and patients with different duration(Th2:duration<6 months(P<0.0001);duration>6 months(P=0.0018);Th17:duration<6 months(P<0.0001);duration>6 months(P<0.0001)).Besides,the ratio of Th1/Th2 was significantly lower than healthy control in whole patients and patients with different duration.We also analysis frequency of CD4~+CD25~+CD127~-Treg cells by flowcytometry.In order to assess the tendency between pro-inflammatory and anti-inflammatory responses in sporotrichosis,we compared the CD4+effective T cell/Tregs(Teff/Tregs)ratio between patients and healthy control.We found a significant increased percentage of Th2/Tregs not only in whole patients but also in groups with different duration and clinical types(whole patients(P=0.0014);duration<6 months(P=0.0059);duration>6 months(P=0.0067));fixed type(P=0.0016);lymphocutaneous type(P=0.0383)).The ratio of Th17/Tregs was significantly elevated in whole patients(P=0.0425)and fixed type(P=0.016)compared with healthy control,while in patients of lymphocutaneous type and patients with different duration the ratio of Th17/Treg did not change remarkably.The ratio of Th1/Treg showed no significant changes.Collectively,these results suggested that a positive Th2 biased immunity,as well as relatively weak Th17 and Th1 responses in the circulation of patients infected by Sporothrix globosa,which did not change with disease duration.We also analyzed the frequencies of CD4~+CXCR5~+PD1~+Tfh cells,CD19~+B cells and its subtypes(Ig D~+CD27~-na(?)ve B cells(NB),Ig D~+CD27~+unswitched memory B cells(USM),Ig D~-CD27~+switched memory B cells(SM)and Ig D~-CD27~-double negative B cells(DN))to assess the humoral immunity in the circulation of patients infected with Sporothrix globosa.Although the frequencies of Tfh and CD19~+B didn’t change significantly,the frequency of USM B decreased(P=0.0354)while frequencies of SM B and DN B increased(P=0.0349;P=0002),which were also found in patients with duration less than 6 months(USM B(P=0.0335),SM B(P=0.0076),DN B(P=0.0086)).Only DN B changed significantly with an elevated proportion in patients of duration more than 6 months or patients with different clinical types compared with healthy control(duration>6mon(P=0.0018),fixed type(P=0.0071),lymphocutaneous type(P=0.0013))which indicates that USM B and SM B mainly participate the immune response during the early stage of infection.We also found that DN B was positive correlated with Th2 and Treg cell(P=0.01,P=0.04).These results suggest that B cell-mediated immunity are envolved in the early stage of Sporothrix globosa infection,moreover DN B probably interact with T cell.We detected the concentration of cytokines(IL-4、IL-17A、IFN-γand TGF-β1),Ig G and its subtypes(Ig G1、Ig G2、Ig G3、Ig G4)、Ig M and Ig E in serum of patients and healthy control.The serum level of IFN-γand IL-4increased(P=0.04,P=0.008)while the serum level of IL-17A and TGF-β1decreased(P=0.001,P=0.001)in whole patients.A significantly increased concentration of IL-4(P=0.0089)and decreased IL-17A(P=0.0012)were found in fixed form while a significantly increased IFN-γ(P=0.0355)was detected in lymphocutaneous form compared with healthy control.Our data suggested that a different cytokine profile exists between fixed form and lymphocutaneous form,which did not show strong association with disease duration.Ig G、Ig M and Ig E are comparable in patients with healthy control,but decreases in Ig G2 and Ig G4 were detected in patients of duration less than months.To verify the immune profile in situ,the local IFN-γ、IL-4 and IL-17A expression in skin lesions were evaluated by immunohistochemistry staining.We performed the staining in 16 sporotrichosis skin lesions,healthy skin(negative control)and lymph nodes(positive control).Sporotrichosis can induce these cytokines in situ,but the positive rates of IFN-γand IL-4(10/15 and 9/15)are higher than that of IL-17A(4/15).Immunohistochemistry staining was negative in healthy control.Obviously,Th2 and Th1 responses form in situ similar with that in serum of patients.Fixed form secreted high amounts of IFN-γ、IL-4 than lymphocutaneous form.Our data suggested that the clinical presentation could be influenced by different profile of the cytokines secretion in situ.In the third part,we performed in vitro co-culture of PBMCs from healthy donors with Sporothrix globosa.Freshly isolated PBMCs were co-cultured with live Sporothrix globosa yeasts at different ratios(Y/P ratio),and we next analyzed the frequencies of CD4~+IFN-γ~+Th1、CD4~+IL-4~+Th2 and CD4~+IL-17A~+Th17 by flowcytometry.Except the the highest Y/P ratio(10:1),frequencies of all these Teff were significantly elevated under the stimulation of yeast cells in a dose dependent manner compared with control.Our results suggested that Sporothrix globosa induced equal Teff subtype differentiation unlike immune changes in the circulation and local lesion of sporotrichosis patients.We for the first time demonstrated the Th2 inclined immunity and remarkably altered B cell subsets as well as the symphony of DN B with Th2 and Treg in patients with sporotrichosis caused by Sporothrix globosa.Based on these facts,Sporothrix globosa with relative weak virulence has triggered a different immune profile in human from those infected by Sporothrix schenckii and Sporothrix brasiliensis.Furthermore,the different profile of cytokines in systemic and in situ between fixed and lymphocutaneous form suggests that the clinical forms are influenced by cytokines induced.These findings may provide theory basis for therapeutic method enhancing Th1、Th17 as well as reducing Th2 responses.