Regulatory Role Of MiR-155 In Candida Albicans Induced Innate Immune Response In Dendritic Cells And Its Mechanisms

Candida albicans(C.albicans)is an opportunistic fungal pathogen,commonly colonizing on the surface of skin and mucosa(oral cavity,digestive tract and vagina).C.albicans rarely causes infectious diseases in immunocompetent individuals.However,under conditions that immune system is suppressed,the balance between C.albicans and the host immune system is broken,and C.albicans could tansform from commensal fungi to pathogens.C.albicans is the most prevalent fungal pathogen and causes various types of candidiasis,ranging from local mucosal infections to life-threatening disseminated candidiasis.In recent years,due to antibiotic abuse and increased number of cancer patients receiving radiotherapy and chemotherapy,as well as the number of HIV-infected patients,the morbility and motality of C.albicans infection has been dramatically increased.Therefore,investigating the pathogenesis of C.albicans,especially the mechanisms underlying anti-C.albicans immune response is crucial,as it might be helpful for the treatment of C.albicans infection.Innate immunity is the first defense line against C.albicans infection in the host.It consists of dendritic cells(DCs),neutrophils,and macrophages.The innate immune system could nonspecificly kill pathogens immediately,with short duration while with no enduring immune memory function.DCs,as a kind of specific antigen presenting cells(APCs),expresses high levels of pattern recognition receptors(PRRs)on its surface.The major biological function of these PRRs lies on their ability to sense the conserved structures presented on the surface of pathogens,termed as pathogen-associated molecular patterns(PAMPs).The interaction between PAMPs and PRRs activates DCs and triggers the immune response against C.albicans.Activated DCs could secrete a variety of inflammatory factors and chemokines,thereby promoting nonspecific immune response against C.albicans.The mechanisms of DCs activation are very complicated.The core event is the nuclear translocation of transcription factors such as NF-?B and nuclear transcription factor activation protein-1(AP-1).After nuclear translocation,these factors could regulate the expression of various inflammatory cytokines.Furthermore,DCs could phagocytize pathogens through PRRs,process and present the pathogenic antigens to Th cells,thereby inducing the polarization of T cells and framing the type and duration of adaptive immune responses.Innate immunity is the start and driving force of adaptive immunity,and the adaptive immunity presents a more permanent and memory-based immune effect.It is noteworthy that the immune response against C.albicans should be tightly controlled.If the immune response is too weak,pathogens could not be effectively cleared.On the contrary,if the immune response is too strong or the duration is too long,it might result in immune pathogenical damage.MicroRNAs(miRNAs)is a type of single-stranded non-coding RNAs with approximately 20 bases in length.As a post-transcriptional regulator,miRNA could combine with the 3'untranslated regions(UTRs)of the target genes,thereby impairing the stability of target genes mRNA or inhibiting their translation.A miRNA could regulate the expression of multiple target genes,meanwhile a single gene could be regulated by multiple miRNAs.Several studies have shown that miRNAs play important regulatory roles in immune cells development and immune response.In our previous study,miRNAs expression profile of DCs stimulated by C.albicans was detected using microRNA microarray and the expression of miR-155 was dramatically increased.Furthermore,previous studies have shown that miR-155 had complex regulatory roles in anti-bacterial and viral immune responses.However,rare researches on the role of miR-155 in immune response to fungal infections were reported.Therefore,the study was performed to investigate the regulatory role of miR-155 in the innate immune response against C.albicans infection and its underlying mechanisms.Part one The effect of heat-killed C.albicans on the activation of dendritic cells and the polariation of CD4~+Th cells.Objective:To investigate the effects of heat-killed C.albicans on the maturation and activation of dendritic cells,as well as DCs'function on the polariation of CD4~+Th cells.Methods:CD14~+monocytes and CD4~+Th cells were positively selected using CD14 or CD4 MACS microbeads from freshly peripheral blood,according to the instructions of the manufacturer.Immature DCs were induced from monocytes using GM-CSF and IL-4.DCs were stimulated with heat-killed C.albicans,then its maturation and activation were detected by the expression of mature markers and the medium level of IL-1?,IL-6,IL-23,TNF-?and IFN-?.Furthermore,to detect the ability of DCs on Th cells polarization,C.albicans-loaded DCs were co-cultured with CD4~+Th cells,and the intracellular IFN-?and IL-17 were measured using flow cytometry.Results:The expression of mature markers CD83 and CD86,and the release of cytokines IL-1?,IL-6,IL-23,TNF-?and IFN-?were significantly upregulated in DCs stimulated by inactivated C.albicans.C.albicans-loaded DCs significantly upregulated the expression of CD69,an activation marker of T cells.The secretion of IFN-?and IL-17was also dramatically increased.Conclusions:Heat-killed C.albicans could promote the maturation of DCs and induce the differentiation of CD4~+Th cells.Part two The expression of miR-155 in DCs could be upregulated by heat-killed C.albicans.Objective:To investigate whether heat-inactivated C.albicans could upregulate the expression of miR-155.Methods:DCs were stimulated with heat-killed C.albicans,and the expression of miR-155 was detected by qRT-PCR.Exosomes in medium of DCs with C.albicans treatment were isolated and the expression of miR-155 in supernatant and exosomes was detected.DCs,monocytes,THP-1 cells and RAW264.7 cells were stimulated with C.albicans to detect the time-dependent expression of miR-155.Results:Significant upregulation of miR-155 was observed in DCs exposed to heat-killed C.albicans.The expression of miR-155 in supernatant and exosomes was also increased.The expression of inflammatory cytokines elevated rapidly after C.albicans stimulation and gradually decreased in the late phase,contrary to the sustained upregulation of miR-155.A similar significant increased miR-155 in response to C.albicans was also found in monocytes,THP-1 and RAW264.7 cells.Conclusions:Heat-killed C.albicans could sustainably upregulate the expression of miR-155 in DCs,and the over-expressed miR-155 could be secreted to the extracellular matrix through exosomes.The trend of miR-155 and inflammatory factors in the late phase of immune response is opposite.Part three Regulation of miR-155 on the secretion of cytokines in DCs exposed to C.albicans and its mechanismsObjective:To investigate the regulatory role of miR-155 on the production of cytokines in DCs stimulated with C.albicans and its possible mechanisms.Methods:MiR-155 mimics or inhibitors were transfected into DCs prior to heat-killed C.albicans stimulation.The expression and concentration of IL-6,IL-23,TNF-?and IFN-?were measured by qRT-PCR and ELISA.The bioinformatic softwares including TargetScan,miRDB and microRNA.org were used to predict the target genes of miR-155.The regulatory role of miR-155 on the target genes was tested by western blotting and luciferase reporter assay.SiRNAs of target genes were tranfected to DCs and its effects on the secretion of cytokines were analyzed by qRT-PCR and ELISA.Results:Overexpression of miR-155 markedly suppressed the expression of IL-6,IL-23,TNF-?and IFN-?induced by C.albicans.Inhibition of endogenous miR-155further increased C.albicans-induced expression of these cytokines.In DCs exposed to C.albicans,miR-155 repressed NF-?B p65 or BCL10 expression through binding to their3'UTRs.In addition,knocking down BCL10 significantly inhibited the activation of NF-?B signaling pathway,thus attenuating the capacity of C.albicans to upregulate the expression of inflammatory cytokines.Conclusions:The sustained upregulation of miR-155 in DCs could inhibit the secretion of inflammatory cytokines induced by C.albicans.This might be related to the inhibition of BCL10 and NF-?B p65,and further influencing the activation of NF-?B pathway.Part four Mechanisms of up-regulated expression of miR-155 in DCs activated by C.albicans.Objective:To investigate the mechanisms of miR-155 induced by heat-killed C.albicans.Methods:DCs were treated with Dectin-1 receptor blockers or siRNAs before stimulation with C.albican,then the expression of miR-155 was measured using qRT-PCR.Immunofluorescence and western blotting were used to detect the nuclear translocation of NF-?B p65 and the phosphorylation of MAPK signaling pathway molecules.To study the role of NF-?B and MAPK on the upregulation of miR-155 by C.albicans,specific inhibitors of NF-?B or MAPK signaling were added before C.albicans stimulation and the expression of miR-155 was measured using qRT-PCR.Results:Exposure to Dectin-1 receptor agonists significantly upregulated miR-155 in DCs,while Both Dectin-1 receptor blockers and siRNA attenuated the increased expression of miR-155 by C.albicans.In C.albicans stimulated DCs,obvious nuclear translocation of NF-?B p65 were observed,and inhibitor of NF-?B p65 significantly suppressed the induction of miR-155.In addition,heat-killed C.albicans could activate ERK,JNK pathway and the phosphorylation of downstream transcription factor c-Jun.Inhibitor of ERK or JNK pathway impaired the upregulation of miR-155 by C.albicans stimulation.Conclusions:Dectin-1 on DCs and their intracellular NF-?B,MAPK signaling pathways are involved in the upregulation of miR-155 induced by C.albicans.