The Study Of The Modulation Of Candida Albicans On Platelet Activation And Its Influence On Immunomodulatory Effect Of Monocytes

Background and objectivesCandida albicans,the most commonly encountered fungal pathogen,causes diseases varying from superficial mucosal complaints to life-threatening systemic disorders.Platelets are traditionally viewed as crucial mediators of haemostasis.Recent studies suggests that they have an essential role in infection and immunity,especially when they are activated.They contribute to host defence against infection(bacteria,viruses and fungi)by secreting microbicidal proteins,promoting phagocytosis of phagocytes and facilitating antigen presentation.Platelets can recognize microorganisms by pattern recognition receptors(PRRs),adhesion receptor GP Ⅱb/Ⅲa,ect.,leading to intracellular signaling pathway transduction and platelet activation.However,little is known about how platelets recognize C.albicans and whether platelets can be activated by C.albicans.Monocytes have an important role in defense against systemic C.albican infection.In some bacterial and fungal infections,platelets are capable of enhancing the phagocytosis and the secretion of pro-inflammatory cytokines of monocytes,and therefore play an indirect role in the efficient clearance of pathogens.However,little is known about whether platelets promote monocytes to defend against C.albicans.Under the above research background,this experiment aims to clarify whether C.albicans can induce platelet activation and secretion of antimicrobial effectors;to explore the receptor mediating platelet activation induced by C.albicans;and to explore whether platelets affect the immune response of monocytes against C.albicans.Materials and methods1.After live C.albicans or heat-killed(HK)C.albicans incubating with platelets,flow cytometry(FACS)was used to detect platelet activation by P-selectin staining;enzyme-linked immunosorbent assay(ELISA)was used to detect platelet secretions of PF-4,CCL5,TNF-α,IL-8,IL-1β and β-defensin-2;and western-blot(WB)was used to detect the expression of AKT,Stat-3,P38 MAPK and ERK1/2 MAPK phosphorylation in C.albicans-treated platelets.2.To explore whether C.albicans could activate platelets after inhibiting PI3K-AKT pathway,platelets were pretreated with the PI3K inhibitor LY294002 and Akt inhibitor MK2206 and then incubated with C.albicans.WB was used to detect the inhibitory effect of AKT phosphorylation,and FACS was used to detect platelet activation by P-selectin staining.3.FACS,WB and real-time PCR were used to detect the expression of TLR2 and TLR4 in C.albicans-treated platelets.4.To explore whether C.albicans could activate platelets after blocking TLR2 and TLR4 on platelets,platelets were pretreated with anti-TLR2 and anti-TLR4 neutralizing antibodies and then incubated with C.albicans.FACS was used to detect platelet activation by P-selectin staining.5.To explore whether TLR2 and TLR4 activation could induce platelet activation,platelets were treated with the TLR2 agonists Pam3CSK4 and FSL-1 and the TLR4 agonist LPS.FACS was used to detect platelet activation by P-selectin staining;ELISA was used to detect platelet secretions of PF-4.6.To explore whether C.albicans could activate platelets after inbibiting GP Ⅱb/Ⅲa on platelets,platelets were pretreated with GP Ⅱb/Ⅲa inhibitor tirofiban and then incubated with C.albicans.FACS was used to detect the inhibitory effect of GP Ⅱb/Ⅲa and platelet activation;and WB was used to detect the expression of AKT phosphorylation.7.To explore whether C.albicans could activate platelets after blocking GP Ⅱb/Ⅲa on platelets,platelets were pretreated with anti-GP Ⅱb/Ⅲa neutralizing antibodies and then incubated with C.albicans,FACS was used to detect platelet activation by P-selectin staining.8.THP-1 monocytes or primary human monocytes were incubated with the fluorescent dye FUN-1-labeled C.albicans in the presence of platelets or not.Phagocytosis of FUN-1-labeled C.albicans by THP-1 cells or primary human monocytes was observed with microscope and analyzed by FACS.Real-time PCR was used to detect the expression of inflammatory cytokines IL-1β,IL-6,TNF-α and CXCL8 in THP-1 cells or primary human monocytes.9.To explore whether platelet-regulated immune response of monocytes is dependent on GP Ⅱb/Ⅲa-mediated platelet activation,platelets were pretreated with anti-platelet agent cilostazol or GP Ⅱb/Ⅲa inhibitor tirofiban and then incubated with THP-1 monocytes in the presence of FUN 1-labeled C.albicans.Phagocytosis of FUN-1-labeled C.albicans by THP-1 cells was analyzed by FACS.Real-time PCR was used to detect the expression of inflammatory cytokines IL-1β.Results1.C.albicans increased the expression of P-selectin(the marker of platelet activation)on platelets,and elevated the secretion of PF-4,CCL5,TNF-α,CXCL8 and β-defensin-2.2.C.albicans elevated the expression of AKT phosphorylation,but did not affect the expression of Stat3,ERK1/2 MAPK and p38 MAPK phosphorylation.Inhibition of the PI3K-AKT signaling pathway reversed C.albicans-induced platelet activation.3.C.albicans increased the expression of TLR2 and TLR4 on the surface of platelets,but did not affect the expression of TLR2 and TLR4 on total protein and mRNA levels.Blocking TLR2 or TLR4 did not suppress the expression of P-selectin on C.albicans-treated platelets.Platelets stimulated with the TLR2 agonists Pam3CSK4 and FSL-1 and the TLR4 agonist LPS did not increase the expression of P-selectin or the secretion of PF-4.These data suggest that C.albicans-mediated platelet activation is independent of TLR2 and TLR4.4.Inbibiting and blocking GP Ⅱb/Ⅲa reversed C.albicans-induced platelet activation.5.Platelets enhanced THP-1 monocyte and primary human monocyte phagocytosis of C.albicans and expression of inflammatory cytokines.6.Treatment of platelets with anti-platelet agent cilostazol before coculture with THP-1 cells and C.albicans removed the elevated phagocytosis effect and the expression of IL-1β in platelet-treated THP-1 cells.And treatment of platelets with GP Ⅱb/Ⅲa inhibitor tirofiban inhibited platelet activation and therefore removed the elevated phagocytosis effect and the expression of IL-1β in platelet-treated THP-1 cells.These data suggested that platelet-regulated immune response of monocytes was dependent on GPⅡb/Ⅲa-mediated platelet activation.Conclusions1.C.albicans induces platelet activation and secretions of antimicrobial effectors.2.C.albicans induces platelet activation through the PI3K-AKT signaling pathway.3.C.albicans induces platelet activation in a GP Ⅱb/Ⅲa-dependent manner,independent of TLR2 and TLR4.4.Platelets enhance human monocyte innate immune responses against C.albicans infection,which are dependent on platelet activation mediated by GP Ⅱb/Ⅲa.