Study On Influence And Mechanism Of THP-1 Macrophage Autophagy Induced By Sporothrix Globosa Melanin And TLRs

Sporotrichosis is a commonly chronic deep mycosis involving mainly the skin and subcutaneous tissue with neighbouring lymphatics caused by the dimorphic fungus Sporothrix schenckii complex.It often causes enormous harm to the patients.The main aetiological agents of Sporothrix schenckii complex are S schenckii sensu stricto,Sporothrix brasiliensis and Sporothrix globosa.S.globosa is distributed worldwide but is particularly endemic in northeast China.The pathogenesis of sporotrichosis remains largely obscure.Exacting data suggest the various virulence factors of Sporothrix spp.,including melanin production may participate in the progress of sporotrichosis.Melanin is the most widespread and important virulence factor of pathogenic fungi,and could suppress the host immune function.It has been demonstrated that melanin could not resist the phagocytosis and killing of the fungus by macrophages but also inhibit antigen presentation by down-regulating MHC II expression in macrophages.Recent research has indicated that the functions of antigen presentation and phagocytosis in macrophages were regulated by autophagy,which implied the anti-macrophage effect of melanin may be associated with its interference to cell autophagy.Moreover,TLR2/TLR4 as the important pattern recognition receptors in recognition of antifungal immunity,are also the key receptors in the inducement of macrophage autophagy.However,whether TLR2/TLR4 could mediate the macrophage autophagy induced by S.globosa is still unknow.Thus,in order to preliminarily elucidate the impact of melanin in autophagy of S.globosa by macrophages and clarify the influence of TLR2/TLR4 induced pathways in macrophage autophagy,the wild strain of S.globosa and the albino mutant strain induced by ultraviolet were used to compare the temporal and spatial regulation of THP-1 macrophage autophagy,and the influence of melanin on autophagy in vitro cell culture method.si RNA was used to knock down TLR2/TLR4 in THP-1 macrophages to study the molecular regulatory mechanism of TLR2/TLR4 in macrophage autophagy induced by S.globosa.In the first part of this study,the wild type strain confirmed to be Sporothrix globosa(Mel+,FHJU12082703)was used to infect THP-1 macrophages,Western blotting and q PCR were used to detect the expression of autophagy molecular markers of LC3,Beclin-1 and P62 after co-culture with S.globosa,and THP-1 macrophages infected with Ad-m RFP-GFP-LC3 adenovirus was to monitor autophagic flux.The results showed that autophagosomes and autolysosomes were significantly increased in THP-1 macrophages infected with S.globosa(Mel+)through laser scanning confocal microscopy observation,and obvious enhancements in the expression of autophagy molecular markers in THP-1 macrophages after stimulating with S.globosa(Mel+)in a time-dependent and MOI-dependent manner were observed.Furthermore,THP-1macrophages infected with S.globosa(Mel+)conidia triggered a large increase in ROS production and secreted increase levels of IL-6,TNF-?,IL-1? and IFN-?.ROS and the levels of cytokines presented corresponding trends during the process of suppression or activation of autophagy,indicating that the production of ROS and cytokines induced by S.globosa(Mel+)in THP-1 macrophages may be regulated by autophagy.In the second part of the study,a stable albino mutant strain of S.globosa(Mel-)obtained by UV light inducing was used to investigate the influence of melanin on autophagy induced by S.globosa in THP-1 macrophages.Mel+ and Mel-were cocultured with THP-1 macrophages respectively,Western blotting,q PCR and ELISA were used to detect the immune response and the expression of autophagy molecular markers of THP-1 macrophages stimulated by S.globosa.The results revealed that Melalso induced a time-and MOI-dependent autophagic response.Mel-conidia group showing remarkably higher expression levels of autophagy molecular markers and autophagic flux compared with the Mel+ conidia group,even in the presence of autophagy inhibitor(chloroquine,wortmannin)or activator(rapamycin),suggesting melanin could inhibit macrophage autophagy induced by S.globosa.Same as above,cells were found to produce increased levels of ROS and cytokines after stimulated by Mel+ and Mel-conidia,and melanin also reduced the production of ROS and cytokines.Autophagy process positively correlated to the expression levels of ROS and IL-6,TNF-? and IFN-?,while negative correlated with IL-1? in the presence of autophagy inhibitor(chloroquine)or activator(rapamycin).In the third part,elevated expression of TLR2 and TLR4 detected by Western blotting and q PCR was shown in THP-1 macrophages co-cultured with S.globosa,implying TLR2 and TLR4 may play crucial roles in recognition of S.globosa by THP-1 macrophages.To determine whether TLR2 and TLR4 mediated S.globosa-infected autophagy in THP-1 macrophages,cells infected with S.globosa after transfection with control si RNA,TLR2 si RNA or TLR4 si RNA were used to evaluate the autophagy molecular markers and autophagic flux.We have found that the S.globosa-stimulated expression levels of autophagy molecular markers and autophagic flux were significant difference in cells transfected TLR2 si RNA,whereas no statistical differences were found in TLR4 si RNA group,suggesting TLR2 as the upstream signal may play a major role in sporotrichosis immunity and involve in mediating autophagy.And,S.globosainduced autophagy on the ROS and cytokines levels significantly decreased when TLR2 was knockdown.This indicated that ROS and cytokines produced by THP-1macrophages induced by S.globosa may be mediated by TLR2.This study demonstrated that THP-1 macrophages could be induced by S.globosa,and we also found that melanin may inhibit autophagy in macrophages.Further studies have elucidated the role of TLRs as pattern recognition receptors in macrophage autophagy induced by S.globosa.