Role And Mechanism Of Heparan Sulfate In The Killing Of Candida Albicans Hyphae By Neutrophils

Background and purpose:Candida spp,a group of opportunistic fungi,frequently cause diverse infections of the skin,mucous membranes,internal organs,and blood,which are collectively known as"candidiasis".Presently,Candida albicans is still the most common pathogen detected in clinical candidiasis,which poses great threats to people’s health.Colonizing the surface of the mucous membrane and skin in yeast form,C.albicans is capable of transforming from yeast to hyphae to enhance its virulence and invasion.Once that occurs,host immunity is initiated to clear the infection.Neutrophils are a key member of the host innate immunity,and can kill C.albicans through various approaches,such as phagocytosis,degranulation,production of reactive oxygen species,and the formation of neutrophil extracellular traps(NETs).However,an unexpected result was observed in vaginal C.albicans infection that a high level of neutrophils only resulted in more severe local inflammation and failed to reduce fungal load.A series of experiments was then conducted in mouse models,suggesting that heparan sulfate(HS)existing in local environment inhibits the recognition and killing of C.albicans by neutrophils.Whether this conclusion still works in the interaction between human neutrophils and C.albicans remains to be examined.HS is a linear polysaccharide widely distributed on the cell surface and in the extracellular matrix in the form of heparan sulfate proteoglycan(HSPG).Furthermore,in order to successfully control C.albicans infection,it’s essential to ensure effective killing of hyphae that are regarded as the key form of C.albicans causing diseases.Consequently,our study focused on the interplay among C.albicans hyphae,human neutrophils and HS.In the past few years,studies have confirmed that hyphae are too large to be engulfed,and are mainly removed by neutrophils via NETs-mediated extracellular killing.It’s reasonable for us to hypothesize that the effect of HS on C.albicans killing by neutrophils could be related to NETs.To clarify the reason why neutrophils have trouble in clearing C.albicans infection in local environment,in-vitro experiments were designed to explore the role and mechanism of HS in the killing of C.albicans hyphae by neutrophils.In this study,we intended to extract neutrophils from human peripheral blood and assess their killing activity on C.albicans hyphae in vitro.In addition,the effect of HS intervention on fungal killing was evaluated.Neutrophils were triggered to release NETs,and their extracellular killing activity on C.albicans hyphae was also measured.The effects of HS intervention on the formation of NETs and their fungal killing activity were analyzed respectively,so as to shed light on the potential mechanism.The whole study includes the following two sections:Section Ⅰ:Role of heparan sulfate in in-vitro killing of C.albicans hyphae by neutrophilsMethods:1.Neutrophils were extracted from peripheral blood of healthy volunteers using density gradient centrifugation.Wright-Giemsa staining was performed to identify cell types,trypan blue staining was performed for viability analysis,and flow cytometry was conducted for purity analysis.2.C.albicans hyphae were induced and co-incubated with neutrophils in different proportions.The killing activity of neutrophils against C.albicans hyphae was measured using plate colony counting method.A suitable MOI was selected.3.HS was added into the co-incubation environment with suitable MOI to assess its role in in-vitro killing of C.albicans hyphae by human peripheral blood neutrophils.Results:1.Human peripheral blood neutrophils with good activity and 99%purity were successfully extracted.Their in-vitro killing activity against C.albicans hyphae was confirmed.2.With an MOI of 0.1,the killing effect was significant.Therefore,this value can serve as the suitable MOI in the subsequent experiments.3.HS inhibited the in-vitro killing of C.albicans hyphae by human peripheral blood neutrophils.Section Ⅱ:Heparan sulfate weakens the in-vitro killing of neutrophils against C.albicans hyphae by inhibiting the formation of NETsMethods:1.SYTOX Green dye was used to stain the co-incubated samples of neutrophils and C.albicans hyphae.NET structure was observed using confocal microscopy,which was further verified by scanning electron microscopy.2.PMA and C.albicans hyphae were used to stimulate neutrophils to release NETs.In each group,two subgroups with or without HS were set up.Comparisons between groups and in group were made to analyze the effect of HS on the release of NETs.3.PMA was used to induce NET release in advance,which was then co-incubated with C.albicans to assess the fungal killing activity of NETs using plate colony counting method.HS was added to evaluate its effect on the fungal killing activity of NETs that had already been released.Results:1.C.albicans hyphae are capable of activating human peripheral blood neutrophils to release NETs in vitro.Localization of NETs can be achieved by fluorescent staining with SYTOX Green dye alone.2.After incubating for 3 h under the same conditions,the NET-inducing ability of C.albicans hyphae was almost equivalent to that of PMA.HS inhibited the formation of NETs induced by hyphae,but had no influence on the NET-inducing ability of PMA.3.HS had no influence on the fungal killing activity of NETs that had already been released.Conclusions:HS weakens the in-vitro killing activity of human peripheral blood neutrophils against C.albicans hyphae by inhibiting the formation of NETs induced by hyphae.