Critical role of TLR2 and MyD88 for functional response of macrophages to a group IIA-secreted phospholipase [A.sub.2] from snake venom

The snake venom MT-III is a group IIA secreted phospholipase [A.sub.2] (sPL[A.sub.2]) enzyme with functional and structural similarities with mammalian pro-inflammatory sPL[A.sub.2]s of the same group. Previously, we demonstrated that MT-III directly activates the innate inflammatory response of macrophages, including release of inflammatory mediators and formation of lipid droplets (LDs). However, the mechanisms coordinating these processes remain unclear. In the present study, by using TLR[2.sup.- /-] or [MyD88.sup.-/-] or C57BL/6 (WT) male mice, we report that TLR2 and MyD88 signaling have a critical role in MT-III- induced inflammatory response in macrophages. MT-III caused a marked release of PG[E.sub.2], PG[D.sub.2], PG[J.sub.2], IL- 1β and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR[2.sup.-/-] macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In [MyD88.sup.-/-] macrophages, MT-III-induced release of PG[E.sub.2], IL- 1β and IL-10 was abrogated, but release of PG[D.sub.2] and PG[J.sub.2] was maintained. In addition, COX-2 protein expression seen in MT- III-stimulated WT macrophages was abolished in both TLR[2.sup.-/-] and [MyD88.sup.-/-] cells, while perilipin 2 expression was abolished only in [MyD88.sup.-/-] cells. We further demonstrated a reduction of saturated, monounsaturated and polyunsaturated fatty acids and a release of the TLR2 agonists palmitic and oleic acid from MT-III-stimulated WT macrophages compared with WT control cells, thus suggesting these fatty acids as major messengers for MT-III-induced engagement of TLR2/MyD88 signaling. Collectively, our findings identify for the first time a TLR2 and MyD88-dependent mechanism that underlies group IIA sPL[A.sub.2]-induced inflammatory response in macrophages.