Culture Medium Of Keratinocytes Challenged With Candida Albicans Modulate The Activity Of Macrophages

Introduction:Candida albicans is the leading conditional pathogen isolated from clinic samples,and the interesting thing about Candida albicans is that it is part of man's normal flora.Accumulating evidence has shown that Candida albicans could train some immune cells to enhance immune response against invading microorganisms.Previous studies have been mainly focused on the direct communications between Candida albicans and the aimed immune cell.Rare literatures have been published whether and how Candida albicans affect one cell by crosstalk with another cell.Without doubt,skin is the biggest organ of the human body.It plays an important role in the host defense against pathogenic attack.Structurally positioned at the outmost layer of the skin,keratinocytes are inevitably involved in skin immunity against infection and injury.Keratinocytes and keratinocyte-derived molecules transfer the danger signals to the nearby immune cells,which induce subsequent inflammatory responses and immune responses.Macrophages are major cellular immunological players in the skin,playing an important role in both innate immunity and adaptive immunity.We designed a series of experiments to investigate whether Candida albicans could regulate macrophage-induced immunity indirectly by cross-talk between keratinocyte and macrophage.Keratinocyte conditioned medium was obtained by co-culture with heat-killed yeast cells.After 24-hour conditioned culture,the chemotaxis,phagocytosis,NO production,PRR,MHC? molecules and co-stimulating molecules expression of macrophages was detected.At last,we performed a shot-gun proteomic study of the keratinocyte culture medium stimulated by Candida albicans to determine if there were any effective molecules that possibly mediated the cross-talk between keratinocytes and macrophages.Materials and methods:1.Cells and Candida albicansCandida albicans standard strain ACTT 11006;HaCaT human keratinocyte cell line;U-937 human monocyte/macrophage-like cell line.2.MaterialsCell culture medium for HaCaT human keratinocyte cell line and U-937 human monocyte/macrophage-like cell line;Griess NO detection kit;Real time RT-PCR,Western-Blot,Transwell assay,flow cytometry related regents.3.MethodsChemotaxis detection by Transwell assay;phagocytosis by flow cytometry;NO production by Griess program;PRR expression by Real time RT-PCR,Western-Blot;MHCII molecules and co-stimulating molecules expression by Real time RT-PCR and flow cytometry;comparative proteomic analysis by shot-gun.4.Statistical analysisAll the statistical analysis of data were performed using the statistical software SPSS(version 20.0)by ANOVA and LSD when compared between groups.All the data are presented as mean ± standard deviation.Significance was set at P<0.05.Results:1.Conditioned medium obtained from keratinocyte challenged by Candida albicans could enhance phagocytosis.migration of macrophage,and also elevate NO production when insulted by Candida albicans.2.Conditioned medium obtained from keratinocyte challenged by Candida albicans could increase MHC II molecules and co-stimulating molecules expression of macro-phage at mRNA level,but had no significant effect at protein level.3.Conditioned medium obtained from keratinocyte challenged by Candida albicans had no impact on the polarization of macrophage.4.Conditioned medium obtained from keratinocyte challenged by Candida albicans could improve macrophage expression of TLR-4,but not TLR-2 or dectin-1.5.A list of functional molecules associated with cross-talk between keratinocyte and macrophage was obtained from comparative proteomic study of the conditioned medium of keratinocyte with or without Candida albican stimulating.Conclusions:1.Candida albicans could elevate nonspecific activity of macrophage such as chemotaxis and phagocytosis by cross-talk between keratinocyte and macrophage.2.Candida albicans contributed to maintaining awakness of macrophage by cross-talk between keratinocyte and macrophage,which were not enough to achieve activation of macrophage.3.Macrophage might cross talk with keratinocyte by certain soluble factors derived from keratinocyte when challenged by Candida albicans.