Abstract
The snake venom MT-III is a group IIA secreted phospholipase A2 (sPLA2) enzyme with functional and structural similarities with mammalian pro-inflammatory sPLA2s 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 TLR2−/− or MyD88−/− 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 PGE2, PGD2, PGJ2, IL-1β and IL-10 and increased the number of LDs in WT macrophages. In MT-III-stimulated TLR2−/− macrophages, formation of LDs and release of eicosanoids and cytokines were abrogated. In MyD88−/− macrophages, MT-III-induced release of PGE2, IL-1β and IL-10 was abrogated, but release of PGD2 and PGJ2 was maintained. In addition, COX-2 protein expression seen in MT-III-stimulated WT macrophages was abolished in both TLR2−/− and MyD88−/− cells, while perilipin 2 expression was abolished only in MyD88−/− 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 sPLA2-induced inflammatory response in macrophages.
Highlights
Secreted phospholipases A2 are enzymes that hydrolyse glycerophospholipids at the sn-2 position of the glycerol backbone in a calcium-dependent manner, releasing fatty acids and lysophospholipids
We initially evaluated the involvement of TLR2 and MyD88 signaling in MT-III-induced PGE2, PGD2 and prostaglandin J2 (PGJ2) production in TLR2- and MyD88- deficient macrophages
As Toll-like receptors (TLRs) are recognized to trigger a pro-inflammatory program in macrophages and respond to exogenous molecules and host-derived endogenous ligands [20,61], in the present study we investigated the participation of TLR2 and MyD88 adaptor molecule in the stimulatory effects of the venom secreted phospholipase A2 (sPLA2) MT-III in macrophages
Summary
Secreted phospholipases A2 (sPLA2s) are enzymes that hydrolyse glycerophospholipids at the sn-2 position of the glycerol backbone in a calcium-dependent manner, releasing fatty acids and lysophospholipids. An Asp sPLA2, named myotoxin-III (MT- III), has been purified from Bothrops asper snake venom [9] This group IIA sPLA2 has been reported to induce inflammatory events in in vivo experimental models [10,11]. This venom sPLA2 exhibits the ability to directly activate inflammatory functions in isolated macrophages, such as increased phagocytic capacity, release of cytokines [11,12], upregulation of lipid mediator cascade, with release of prostaglandins [13], and increased formation of lipid droplets (LDs) [14], which are organelles implicated in the differentiation of macrophages into foam cells. The initial steps leading to this sPLA2induced activation of macrophages are still unknown
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