Preliminary Study On Immune Response Against Sporothrix Globosa And The Effect Of Melanin On The Function Of THP-1 Macrophage

Sporotrichosis is a common deep mycosis involving skin,subcutaneous tissue and even viscera,bone and other organs.The course of the disease can is prolonged,and even life-threatening.Northeast China,especially Jilin Province,is one of the most serious epidemic areas.The pathogen of the disease is the Sporothrix schenckii complex,which has been found to contain seven genotypes,including three main pathogenic types: S schenckii sensu stricto,Sporothrix brasiliensis and Sporothrix globosa,while the epidemic strains in China are mainly Sporothrix globosa,and so far,it is the only genotype found in Northeast China.However,the pathogenesis of sporotrichosis is not completely clear,especially after the different genotypes is explored,there is little research on the Sporothrix globosa,which is prevalent in China.Sporothrix is a dark fungus,which has the ability to synthesize melanin.Melanin is one of important virulence factors,the researches on the melanin of Sporothrix are few.In order to preliminarily clarify the local immune response state of sporotrichosis patients,the mechanism of immune response of macrophage induced by sporotrichosis globosa and the role of melanin in the immune response,we studied the skin lesions of patients with sporotrichosis in Jilin Province by immunohistochemistry and protein research.In vitro,the wild strain of sporotrichosis globosa and the albino mutant strain induced by ultraviolet were used.In addition,the molecular mechanism of immune response of THP-1 macrophage to sporotrichosis globosa was explored.In the first part of this study,the skin lesions of 15 patients with sporotrichosis in Jilin Province were collected and compared with 3 normal skin samples The results of q PCR array detection showed that there were 5 genes with high expression in the skin lesions of sporotrichosis patients,included STAT1,TLR2,LYN,My D88 and PYCARD;17 genes with low expression,included IL-12 ?,FOS,TLR9,CHIA,MBL2,IL-2,COLEC12,NPTX1,C5AR1,IL-12?,CSF2,TIRAP,IKBKB,IL-10,CARD9,IL-23?,MAPK8.With the immunohistochemistry study,the expression of IFN-?,TNF-?,IL-18 in skin lesions was significantly higher than that in normal skin(p < 0.01),in which IFN-?was mainly expressed in epidermis;IL-1 ?,IL-4 and IL-10 were not significantly expressed in skin lesions,in which IL-1 ? expression was not significantly different from that in normal skin(p=0.250),but IL-4 and IL-10 were found in normal skin,there was significant difference between the normal skin and the lesion skin(p < 0.01).Western blot analysis of the proteins in the skin lesions showed that the expression of TLR2 in the skin lesions of patients with sporotrichosis was higher than that in the normal skin,while the expression of TLR4 and NLRP3 showed no significant change.TLR2 may be an important pattern recognition receptor that mediates the immune response in sporotrichosis patients.In the second part,we used the strain which was confirmed to be Sporothrix globosa by CAL gene sequencing,and the sequence of the strain was uploaded to Gen Bank.A stable albino mutant strain(MEL-)was obtained by UV light inducing.MEL-strain did not produce melanin,and other characters were highly similar to the wild strain(MEL+).MEL+ and MEL-were co-cultured with THP-1 macrophages respectively,immunofluorescence staining,ELISA,flow cytometry,q PCR and Western blot were used to detect the immune response and the expression of pattern recognition receptor of THP-1 macrophages stimulated by Sporothrix globosa.The results showed that the phagocytic index of THP-1 macrophages increased with the prolongation of co-culture time.Compared with MEL+,the phagocytic index of THP-1 macrophages to MEL-was higher,and the phagocytic index of THP-1 macrophages was significantly different(p < 0.05)at the time of 4 h of co-culturing.After lysing THP-1 macrophages that engulf conidia,inoculate the suspension released to SDA plate culture medium.After 7 days of culture at 28 ?,MEL+ strains showed colonies growing,but MEL-strains hardly grew.Moreover,with the prolongation of co-culture time,the number of MEL+ colonies gradually reduced,indicating that with the prolongation of co-culture time,THP-1 macrophages could enhance the ability of killing conidia of MEL+ strains;while the killing effect of THP-1 macrophage on MEL-strains was much stronger than that of MEL+ strain.MEL+,MEL-and melanin particles purified from wild strains were co-cultured with THP-1 macrophages to detect the level of NO and ROS released by THP-1 macrophages.The results showed that the level of NO and ROS released by THP-1 macrophages were significantly higher in MEL-group than that in MEL+ group at 4h of co-culturing.The level of cytokines secreted by THP-1 macrophages stimulated by conidia of sporotrichosis globosa was measured,the results showed that the levels of IL-6 and IL-18 were significantly increased when THP-1 macrophages were co-incubated with MEL+ conidia for 2 h,but there was no significant difference of IL-18 level at the time of 3 h and 4 h,TNF-? was significantly increased at 4 h,and IL-1 ? was not significantly different at any time point.When THP-1 macrophages were co-incubated with MEL-conidia for 2 h,the levels of IL-6,IL-1 ? and IL-18 increased significantly,and TNF-? increased significantly at 3 h.At 3h,TNF-? and IL-18 levels of THP-1 macrophages stimulated by MEL-conidia were higher than that of MEL+ conidia;at 4h,the levels of four cytokines stimulated by MEL-conidia were all significantly higher than that of MEL+ conidia.However,melanin alone had no significant effect on NO,ROS or cytokine secretion.The expression of TLR2,TLR4 and NLRP3 in THP-1 macrophages was detected by q PCR and Western blotting.The results showed that TLR2 and TLR4 expressed higher,and TLR2 expressed higher than TLR4,but the expression of NLRP3 showed no significant change.This suggest that TLR2 plays an important role in the recognition of Sporothrix globosa by the macrophages,while melanin can inhibit the immune efficacy of THP-1 macrophages against Sporothrix globosa.Finally,si RNA was used to knock down TLR2 in THP-1 cells,to verify the role of TLR2 in recognition of Sporothrix globosa by THP-1 macrophages and the subsequent inflammatory response activated by THP-1 macrophages.The results showed that the phagocytic index of normal THP-1 macrophage and TLR2 knockdown THP-1 macrophage was 30.01 ± 5.32 and 6.09 ± 1.62 respectively,which was significantly lower than that of normal group(p = 0.00174);TNF-? secreted by normal THP-1 macrophage was(2475.93 ± 245.12)pg / ml,IL-6 was(743.75 ± 55.34)pg / ml,which were significantly higher than those of the blank control group(p < 0.01),while TNF-? secreted by TLR2 si RNA THP-1 macrophages was(1476.18 ± 313.79)pg / ml,IL-6 was(452.65 ± 29.93)pg / ml,with no significant change compared with that of the blank control group(p = 0.951 and 0.068).It is suggested that TLR2 plays an important role in the process of recognizing and mediating the secretion of inflammatory factors in resisting the infection of sporotrichosis globosa.In this study,we preliminarily found the immune status and differential expressed genes in the skin lesions of patients with sporotrichosis.In vitro experiments further clarified the role of melanin as an important virulence factor of sporotrichosis globosa which could inhibit the host immune response,and TLR2 was confirmed to be an important receptor for the host to recognize Sporotrichosis globosa.This study provided a new theoretical basis for the pathogenesis of sporotrichosis.