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 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

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

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

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

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

MMP2 and MMP9 are Associated with Apical Periodontitis Progression and Might be Modulated by TLR2 and MyD88

<div><p>Abstract The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.</p></div

LXA₄-MS increased collagen deposition and angiogenesis and affected VEGF production.

<p>(A) Collagen deposition was measured using the ImageJ software with the Color Deconvolution plug-in, which measured densitometry in at least 12 random 400× fields in all groups, at days 2, 7 and 14. Percentages of collagen deposition are represented as means ± SEM (n = 6 ulcers/group). One-way ANOVA was used to determine statistical significance (<i>p</i> < 0.05) and is indicated as follows: *, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^{, LXA₄-MS vs. soluble LXA₄. (B) Photomicrographs of wounds stained with Picro Sirius Red (200×) show collagen deposition at days 2, 7, and 14. (C) Histogram showing quantitative analysis of vascular density using the ImageJ software with the Cell Counter plug-in on 200× images. One-way ANOVA was performed to determine statistical significance (p < 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS vs. Vehicle (PBS/glue);}, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄. (D) VEGF was quantified in all groups at days 2, 7 and 14 (represented as bars) in wounds via ELISAs as proxy to blood vessel density. Values are means ± SEM (n = 6 wounds/group). One-way ANOVA was performed to determine statistical significance (<i>p</i>< 0.05), which is indicated as follows: *, significant VEGF increase as compared to normal tissue (dashed line); ^#, soluble LXA₄ or LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^$, LXA₄-MS or soluble LXA₄ <i>vs</i>. Un-MS; and ^&, LXA₄-MS <i>vs</i>. soluble LXA₄.</p

WRW4, a selective LXA₄ receptor antagonist, reversed wound healing properties of LXA₄-MS.

<p>(A) qRT-PCR analysis was performed to assess the LXA₄ receptor <i>ALX</i> mRNA’s abundance in skin ulcers collected on days 2, 7, and 14 in the control (vehicle—PBS/glue), Un-MS, soluble LXA₄, and LXA₄-MS groups. Data represent means ± SEM (n = 5 ulcers/group). One-way ANOVA was done to determine statistical significance (<i>p</i> < 0.05), which is indicated as follows: *, LXA₄-MS <i>vs</i>. Vehicle (PBS/glue); ^#, LXA₄-MS <i>vs</i>. Un-MS; and ^$, LXA₄-MS <i>vs</i>. soluble LXA₄. (B) Representative images of 1.5 cm dorsal wounds at days 2 and 7, with day 0 images serving as pre-injury images, are presented for the following groups: WRW4 (25 μl per animal—from a main peptide solution of 1 mg/ml), WRW4 + LXA₄-MS (WRW4 applied 10 minutes before MS application– 10 mg of LXA₄-MS), and LXA₄-MS (10 mg). (C) Wound healing index values for the groups outlined in (B). Index values range from 0 to 1, where a value of 0 indicates the original wound, and a value of 1 represents a completely closed wound. Data represent means ± SEM (n = 9 ulcers/group); One-way ANOVA was done to determine statistical significance (*<i>p</i> < 0.05).</p