E expression determined by microarray analysis. The * denotes a substantial (p0.05) boost in expression by C. albicans remedy.TNF was decrease in cPLA2+/+ RPM in comparison to cPLA2-/- RPM measured 6 h right after C. albicans addition. NS398 therapy enhanced TNF production in cPLA2+/+ but not in C. albicansstimulated cPLA2-/- RPM suggesting that prostanoids suppress TNF expression. NS398 completely blocked production of PGE2 and PGI2 in RPM stimulated with C. albicans for six h (information not shown), and at the concentration applied (ten ) inhibits both murine COX1 and COX2 [36]. To further investigate the role of prostanoids inside the autocrine regulation of TNF production, RPM were treated with agonists for the PGE2 receptor EP2 (butaprost) and also the PGI2 receptor IP (iloprost) (Figure 3B). Microarray data showed that RPM express the IP receptor (Ptgir), the EP2 (Ptger2) and EP4 (Ptger4) receptors (Table 1). The agonists had no effect on the levels of TNF made by cPLA2+/+ RPM that generate endogenous prostaglandins in response to C. albicans (Figure 3B). Even so, the greater levelof TNF made by C. albicans-stimulated cPLA2-/- RPM, which usually do not create endogenous prostaglandins, was decreased by the receptor agonists towards the level made by cPLA2+/+ RPM. The data suggest that prostaglandins acting via the EP2 and IP receptors suppress TNF production due to the fact it can be enhanced by inhibiting prostaglandin production in C. albicans-stimulated cPLA2+/+ RPM and suppressed by prostaglandin receptor agonists in cPLA2-/- RPM. The EP2 and IP receptors mediate increases in cAMP, which is implicated in regulating Tnf gene expression [37,38]. As shown in Figure 4A, the steady cAMP analogue 8-Br-cAMP suppressed C. albicans-stimulated TNF production in cPLA2-/- RPM, as observed for the prostanoid receptor agonists, but had no effect on the reduce level of TNF created by cPLA2+/+ RPM. The results suggest that prostaglandins produced by C. albicans-stimulated cPLA2+/+ RPM act in an autocrine manner via prostaglandin receptors that improve cAMP to suppress TNF production. That is supported by final results displaying that levels of cAMP have been larger in cPLA2+/+ RPM than cPLA2-/- RPM within 5-30 min following C. albicans addition (Figure 4B).Impact of C. albicans on gene expression in RPMWe subsequent determined the impact of C. albicans on worldwide gene expression in RPM by microarray and then evaluated how cPLA2 activation modulates the transcriptional response.Ethyl 2-diazo-3-oxobutanoate Chemical name C. albicans stimulated a rise in expression of 427 genes (four.0-fold, p0.05, n=3) in cPLA2+/+ Balb/c RPM at 3 h. Relative expression levels for these genes and the fold transform in response to C.BuyN2-Isobutyryl-2′-O-methylguanosine albicans are shown in Table S1A.PMID:24118276 Many of the genes that raise in response to C. albicans representPLOS 1 | plosone.orgcPLA2 Regulates Gene Expression in MacrophagesFigure 4. cAMP production is enhanced by cPLA2 activation and suppresses TNF production. (A) cPLA2+/+ and cPLA2-/RPM had been incubated with 8-Br-cAMP (1 mM) for 30 min followed by incubation with C. albicans for 6 h. Levels of TNF within the culture medium were determined by ELISA. (B) cPLA2+/+ (WT, squares) and cPLA2-/- RPM (KO, circles) have been incubated with (closed symbols) or with no (open symbols) C. albicans (CA) for the indicated instances. Cell lysates were processed for cAMP determinations as described in Experimental Design. The data will be the average of 3 experiments .E. (*p0.05). In panel B, CA treated WT vs. CA treated KO at 5 and 15 min are compared for significance.do.