5-LO deficiency increases the inflammatory response in the lung.

<p>Representative lung sections from WT and 5-LO^−/− mice infected with <i>H. capsulatum</i> (A). Hematoxylin-eosin staining for leukocytes (magnifications ×100) and GMS staining for yeast cells (black arrow) (magnifications ×400). (B) Neutrophils recruitment from lung parenchyma (C) TNF-α production from homogenized lungs. Cells and cytokines were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section from mice after i.t. injection of PBS or i.t. infection with <i>H. capsulatum</i>. Cells were enumerated and identified after Rosenfeld staining, and TNF-α levels were determined by ELISA. Data are expressed as the mean ± SEM from one experiment representative of a total of two experiments (n = 6). *, p<0.05 vs. PBS; #, p<0.05 vs. WT.</p

LTB₄ and CysLTs production in lung tissue.

<p>Enzyme immunoassay quantification of LTB₄ and CysLTs concentrations in lungs from mice that had received either an i.t. PBS injection (uninfected) or an i.t. infection with <i>H. capsulatum</i>. Data are presented as the mean ± SEM and are representative of one of two independent experiments (n = 6). *, p<0.05 vs. PBS.</p

Deficiency of 5-LO impairs T cell recruitment to the lung.

<p>Cells were obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section from mice after i.t. injection of PBS or i.t. infection with <i>H. capsulatum</i>. The lymphocyte population was gated for forward/side scatters and analyzed the percentage of T cells expressing a phenotype effector (CD44^high/CD62^low). (A) CD4⁺ T cells, CD8⁺ T cells (B) and T cell proliferation(C). Data are presented as the mean ± the SEM from three experiments. *, WT and 5-LO^−/− vs. PBS; #, WT vs. 5-LO^−/−. p<0.05.</p

Effect of 5-LO deficiency on survival, fungal burden and NO₂⁻ production.

<p>(A) 5-LO^−/− and WT mice were infected i.t. with 3×10⁶ yeast <i>H. capsulatum</i> and survival was monitored for 30 days (n = 6). CFU numbers in lungs (B) and spleen (C) were evaluated at 7 and 14 days post <i>H. capsulatum</i> infection. (D) NO₂⁻ levels were quantified in the supernatant of lung homogenates at different time points using a Griess reaction. Data are expressed as the mean ± SEM from one experiment representative of a total of two experiments (n = 6). *, p<0.05 vs. PBS; #, p<0.05 vs. WT.</p

5-LO deficiency affects the ability of macrophages to phagocytose <i>H. capsulatum</i>.

<p>(A) PMs from WT and 5-LO^−/− mice were incubated for 1 h with a yeast:macrophage ratio of 1∶5 in the absence or presence of IgG. PMs were pretreated with LTB₄ (B) and LTC₄ (C) for 5 min before the addition of opsonized <i>H. capsulatum</i>. Phagocytosis was calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031701#s3" target="_blank"><i>Material and Methods</i></a> section and was expressed as a percentage of the control. Data are expressed as the mean ± SEM from one experiment representative of a total of three experiments (n = 6). *, p<0.05 vs. control; #, p<0.05 vs. WT cells; & p<0.05 vs. 5-LO^−/− cells.</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

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

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

BLT₁ expression on C57BL/6 (A) and 129/Sv (B) macrophages.

<p>The resident cells were obtained as described in the Material and Methods, and the expression of the higher affinity receptor for LTB₄ was evaluated by flow cytometry. The mononuclear population was gated using the forward/side scatters and analyzed to determine the fluorescence intensity on the cells. The numbers in the histograms indicate the percentage of cells expressing the BLT₁ receptor. The results shown are from one experiment and are representative of two independent experiments.</p