Ting the production of TNF in response to C. albicans was investigated by comparing RPM from cPLA2+/+ and cPLA2-/- mice, and by treating the macrophages using a cyclooxygenase inhibitor NS398 (Figure 3A). The production ofResultsThe production of eicosanoids by RPM is initiated by the activation of cPLA2, which happens rapidly in response to C. albicans or zymosan as a consequence of post-translational processes [9?2]. The big arachidonic acid metabolites produced by RPM in response to C. albicans and zymosan are PGI2, PGE2, and LTC4, and their production is dependent on cPLA2 activation to provide arachidonic acid substrate [12?4]. As shown in Figure 1A, eicosanoids were produced most swiftly through the initial 30 min soon after C. albicans addition. Prostaglandin production occurred before the boost in COX2 expression stimulated by C. albicans, which was detected three h right after addition of C. albicans but not at 1 h (Figure 1B). In contrast, COX1 was constitutively expressed in RPM and expression was not impacted by C. albicans infection. Microarray information also confirmed that COX2 expression was really low compared to COX1 in unstimulated cPLA2+/+ RPM, but there was a substantial improve in expression of COX2 (Ptgs2) but not COX1 (Ptgs1) in cPLA2+/+ RPM treated with C. albicans for three h (Table 1). The results recommend that cPLA2-mediated release of arachidonic acid couples to COX1 for early production of prostaglandins.Ethyl 6-hydroxybenzofuran-3-carboxylate uses PLOS One particular | plosone.(t-Bu)PhCPhos Pd G3 site orgcPLA2 Regulates Gene Expression in MacrophagesFigure 1.PMID:24103058 Time course of C. albicans-stimulated eicosanoid production. (A) RPM have been incubated with C. albicans for the indicated times. The culture medium from unstimulated (open squares) or C. albicans-stimulated (closed squares) RPM was analyzed for eicosanoids by mass spectrometry. The data are the typical of triplicate samples ( .D.) from a representative experiment. (B) Cell lysates from unstimulated RPM (US) or RPM stimulated with C. albicans (CA) for 1, 3 and six h had been analyzed for COX1 and COX2 expression by Western blotting. Sample loading was evaluated by probing with antibodies to -actin.doi: 10.1371/journal.pone.0069002.gFigure 2. Part of PRRs in regulating C. albicans-stimulated TNF production. Wild variety (open bars) and Dectin-1-/- (A), MyD88-/- (B) and TLR4-/- (C) RPM (shaded bars) have been incubated with C. albicans for 6 h. In panel C, RPM had been stimulated together with the parental wild variety C. albicans (WT), the Capmr1 null mutant and the re-integrant strain (Capmr1+CaPMR1). The data are the typical of three experiments .E. (*p0.05). Levels of TNF inside the culture medium had been determined by ELISA.doi: 10.1371/journal.pone.0069002.gPLOS 1 | plosone.orgcPLA2 Regulates Gene Expression in MacrophagesFigure three. Part of prostaglandins in regulating C. albicans-stimulated TNF production. cPLA2+/+ and cPLA2-/- RPM were incubated with (A) NS-398 (10 ), or (B) iloprost (1 ) or butaprost (10 ) for 30 min followed by incubation with C. albicans for 6 h. Levels of TNF in the culture medium have been determined by ELISA. The information are the average of three experiments .E. (*p0.05).doi: 10.1371/journal.pone.0069002.gTable 1. Relative expression values of cyclooxygenases and prostaglandin receptors in RPM.Official Symbol Entrez_Gene_ID Unstimulated Imply Expression Ptgs2* Ptgs1 Ptger2 Ptger4 Ptgir* 19225 19224 19217 19219 19222 67 ?14 4694 ?2731 204 ?18 257 ?24 589 ?C. albicans-treated Mean Expression 11243 ?2938 2027 ?655 177 ?65 248 ?50 1168 ?cPLA2+/+ RPM have been stimulated with C. albicans for 3 h and gen.
