TLR2, TLR4 and CD14 Recognize Venom-Associated Molecular Patterns from <i>Tityus serrulatus</i> to Induce Macrophage-Derived Inflammatory Mediators

<div><p>Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense <i>Tityus serrulatus</i> venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.</p></div

TsV and Ts1 induce IL-6, TNF-α, PGE₂ and LTB₄ production in peritoneal macrophages.

<p>Adherent macrophages from C57Bl/6 (WT) mice were stimulated with TsV or Ts1 (50 µg/ml), and the supernatants were collected after 24 h, following incubation in a 5% CO₂ atmosphere at 37°C. The levels of IL-6 (a), TNF-α (b), PGE₂ (c) and LTB₄ (d) in the supernatants were measured by ELISA. Medium alone was used as the negative control. *<i>p</i><0.05 (one-way ANOVA) compared to medium alone. The data from 3 independent experiments are shown (<i>n</i> = 12).</p

TLR4, TLR2 and CD14 mediate the recognition of TsV and Ts1 and modulate IL-6, TNF-α, PGE₂ and LTB₄ production.

<p>Peritoneal macrophages from C57Bl/6 (WT) mice, TLR2^−/−, TLR4^−/− or CD14^−/− mice were stimulated with TsV (50 µg/ml) (a, b) for 30 min or 24 h or with TsV (e, f) or Ts1 (50 µg/ml) (c, d, g, h) for 24 h in a 5% CO₂ atmosphere at 37°C. The concentrations of IL-6 (a, c), TNF-α (b, d), PGE₂ (e, g) and LTB₄ (f, h) in the culture supernatants were determined by ELISA. *<i>p</i><0.05 (one-way ANOVA) compared to the WT mice. The values represent the means ± SD (<i>n</i> = 8), and the data are from 2 independent experiments.</p

TsV and Ts1 stimulation increases the mRNA expression of <i>Tlr2, Cd14, Myd88</i> and <i>Ptgs2</i> in peritoneal macrophages.

<p>Adherent macrophages from C57Bl/6 (WT) mice were treated with TsV or Ts1 (50 µg/ml) for 4 h. Unstimulated macrophages were used as the negative control. The cells were lysed, and total RNA was extracted. qRT-PCR was performed to determine the relative expression levels of transcripts encoding lipid metabolism enzymes, TLRs and adaptor proteins. The results were normalized to the expression levels of the endogenous internal controls <i>Actb</i>, <i>Gapdh</i> and <i>Tbp</i>. The 2^–ΔΔCt method was used for the analysis of the qRT-PCR data. *<i>p</i><0.05 (one-way ANOVA followed by Dunnett’s post-test) compared to medium alone. Statistically significant changes were considered when <i>p</i><0.05 and any gene presented a fold-change >2.0. The results are presented as the fold-change measured from 2 independent experiments.</p

A schematic diagram showing the increased pro-inflammatory cytokine production in peritoneal macrophages stimulated with TsV and Ts1.

<p>Pro-inflammatory cytokine production occurs via the following two routes: (1) MyD88-dependent signaling, where TsV and Ts1 are recognized by TLR4/CD14/TLR2, resulting in NF-kB nuclear translocation; and (2) MyD88-independent signaling, where TsV is recognized by TLR4/CD14 and activates ERK1/2 and p38 phosphorylation and c-Fos/Jun expression.</p

Activation of NF-κB/AP-1 in RAW-Blue™ cells.

<p>These cells were derived from RAW 264.7 macrophages and contain a secreted embryonic alkaline phosphatase (SEAP) reporter construct that is integrated into the cellular DNA and that can be induced by NF-κB. The cells were incubated with either (A) anti-mTLR2-IgG (100 ng/ml) or (B) LPS-RS (10 ng/ml) for 30 min, with or without LPS (0.5 µg/ml), and TsV or Ts1 (50 µg/ml) for 24 h. The QUANTI-Blue™ substrate was used to measure the SEAP at 650 nm with an ELISA reader. The measurements were performed in triplicate, and a representative experiment is shown. *<i>p</i><0.05 (one-way ANOVA) compared to medium alone (dashed line). The values represent the means ± SD (<i>n</i> = 8), and the data are from 2 independent experiments.</p

Ts1 induces TLR2- or TLR4/MyD88-dependent activation of NF-κB and TLR2- or TLR4/MyD88-independent activation of AP-1 in stimulated macrophages.

<p>Adherent peritoneal macrophages from WT (C57Bl/6), TLR2^−/−, TLR4^−/− or MYD88^−/− mice were stimulated with Ts1 (50 µg/ml) for 10 or 120 min in a 5% CO₂ atmosphere at 37°C. The p-NF-κB (a, d), p-IκBα (b, e) and p-c-Jun (c, f) protein levels were determined using the PathScan Inflammation Multi-Target Sandwich ELISA kit, as described in the Materials and Methods section. The results are presented as a percentage of the phosphoprotein level in non-stimulated control cell lysate (dashed line). *<i>p</i><0.05 (one-way ANOVA) compared to WT. The values represent the means ± SD (<i>n</i> = 4), and the data are from 2 independent experiments.</p