Additional file 2: of Hematobin is a novel immunomodulatory protein from the saliva of the horn fly Haematobia irritans that inhibits the inflammatory response in murine macrophages

Figure S2. Aedes aegypti SGE induces lymphocyte death in vitro. Spleen cells were cultured in the presence of A. aegypti SGE and stimulated by Con A (0.5 μg/ml, final concentration) according to [23] as a positive control for the assay presented in Fig. 5. Flow cytometry evaluated annexin V+ events in CD3+-gated cells after 2 h and 18 h. The data are expressed as the percentage of annexin V+ events for each condition. (TIF 115 kb

Additional file 1: of Hematobin is a novel immunomodulatory protein from the saliva of the horn fly Haematobia irritans that inhibits the inflammatory response in murine macrophages

Figure S1. Evaluation of the cross-reactivity between native and recombinant HTB. Whole salivary gland extract of H. irritans was separated on a 12% gradient poly-acrylaminde gel and transferred onto a PVDF membrane. The membranes were blocked with PBS containing 5% of soy milk and were probed with either pre-immune rabbit serum or with serum from rabbits immunized with the recombinant HTB. Then, the membranes were incubated with HRP-conjugated anti-rabbit IgG and bands were detected with 3,3’-Diaminobenzidine. Lane A: pre-immune rabbit serum; Lane B: serum from rabbit immunized with recombinant HTB (immune serum). The immune serum recognized a single band with approximately 15 kDa from the salivary gland extract corresponding to the native protein. MW: molecular weight. (TIF 127 kb

B and T cells are increased in the BAL of mice sensitized by mosquito bites followed by SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Differential analyses of lymphocytes were performed by flow cytometry in cells stained with florescence-conjugated antibodies for cell surface markers. <b>(A)</b> Sensitization protocol; <b>(B)</b> total number of CD19⁺ cells, <b>(C)</b> total number of CD4⁺ cells; <b>(D)</b> total number of CD8⁺ cells. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Mice sensitized by mosquito bites develop eosinophilic airway inflammation after intranasal SGE challenge.

<p>BAL collection from control and sensitized groups was performed 24 h after the last challenge with PBS or SGE. Total cell number and differential cell counts were analyzed by microscopic evaluation of cytocentrifuged slides. <b>(A)</b> Sensitization protocol; <b>(B)</b> total cells; <b>(C)</b> eosinophils; <b>(D)</b> neutrophils; <b>(E)</b> macrophages; <b>(F)</b> lymphocytes. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 when compared with PBS group; ^<i>#</i><i>p</i> < 0.05 when compared with the 1x group.</p

Th2 cytokine levels are upregulated in BAL of sensitized mice in response to SGE challenge.

<p>BAL of control and sensitized mice was collected 24 h after the last challenge with PBS or SGE, and the cytokine levels in the free-cell supernatant were determined by ELISA. <b>(A)</b> Sensitization protocol; <b>(B)</b> IL-4; <b>(C)</b> IL-5; <b>(D)</b> IL-13; <b>(E)</b> IFN-γ; <b>(F)</b> IL-17. Results were expressed as mean ± SEM (n = 6). *<i>p</i> < 0.05 compared with PBS group; ^<i>#</i><i>p</i> < 0.05 compared with SGE group.</p

Respiratory pattern and tracheal responsiveness were not affected in SGE-challenged sensitized mice.

<p>Twenty-four hours after the last challenge, the respiratory pattern was assessed in control and mosquito bite-sensitized mice using whole-body plethysmography, as described in Material and Methods. Airway sensitivity was tested in the presence of increasing concentration of methacholine and maximal resistance values was recorded after 5 minutes. <b>(A)</b> Sensitization protocol; <b>(B)</b> results expressed as Penh. For the tracheal reactivity assay, mice were sensitized or not by mosquito bites and challenged twice with PBS or SGE. Basal values were obtained from the control group and concentration-effect curves for methacholine were constructed using an organ bath system. <b>(C)</b> Sensitization protocol; <b>(D)</b> trachea responsiveness. Results are expressed as mean ± SEM (n = 6–8). *<i>p</i> < 0.05 when compared with PBS group; ^#<i>p</i> < 0.05 when compared with SGE group.</p

Tempol inhibits transcription and functional expression of tissue factor in endothelial cells, and cytokine production. A, Inhibition of tissue factor (TF) activity.

<p>MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing the wells and addition of lipopolysaccharide (LPS) (200 ng/mL). A mixture containing FX and FVIIa was then added to the cells, and FXa generation was estimated in the supernatant using chromogenic substrate (S2222), as described in Materials and Methods. B, Inhibition of TF generation. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL) for six hours. Wells were washed and cells were lysed with 0.1% Triton X-100. The supernatant was used to detect TF antigen by ELISA. C, Inhibition of TF transcription. Cells were incubated overnight with Tempol (0, 1, and 3 mM) followed by addition of LPS (200 ng/mL) for 2 h. Cells were washed and trypsinized for extraction of mRNA. Real-time PCR of the samples was evaluated as described in Materials and Methods. The figure shows a representative result from two independent experiments. D–F, Inhibition of cytokine generation. MVEC were incubated overnight with Tempol (0, 1, and 3 mM) followed by washing of the wells and addition of LPS (200 ng/mL). After six hours, the supernatant of the cells was collected and used for detection of D, IL-6, E, IL-8, and F, MCP-1 by ELISA (n = 8). *, P≤0.05 (analysis of variance, Bonferroni post-test).</p

Tempol inhibits dendritic cell (DC) functions.

<p>Bone marrow-derived DCs (10⁶ cells/mL) were preincubated overnight with medium or Tempol at indicated concentrations and stimulated with lipopolysaccharide (LPS) (200 ng/mL) for 24 hours. Detection of <b>A,</b> TNF-α was evaluated in cell-free culture supernatants after six hours, and the levels of <b>B,</b> IL-6 and <b>C,</b> IL-12p70 were evaluated after 24 hours. <b>D,</b> Cells were stained with fluorochrome-labeled monoclonal antibodies to CD11c, CD40, CD80, CD86, and MHC class II and analyzed by flow cytometry. <b>E,</b> DCs incubated with medium or Tempol were pulsed with OVA (100 µg/mL) plus LPS (200 ng/mL) for four hours. After repeated washings, DCs were co-incubated with purified CD4⁺ cells from DO11.10 mice (DC:CD4⁺ ratio  = 1:4) for 72 hours, and proliferation was measured as described in Materials and Methods. <b>F,</b> IL-2 and <b>G</b>, IFN-γ were evaluated in the culture supernatants from the proliferation assay. *, P≤0.05 versus control group (^–/–); ^#, P≤0.05 versus LPS group (^+/−) (analysis of variance).</p

Pathologic events in malaria lead to radical oxygen species (ROS) generation and inflammation.

<p><i>Plasmodium falciparum</i> infection is associated with coagulation and complement activation, cytokine release, host response to infection, and activation of different cells types, including endothelial cells, platelets, dendritic cells and monocytes, and neutrophils. It is also associated with release of <i>Pf</i>-GPI, iron overload, heme release and Fenton reaction, and diminished levels of antioxidants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Miller1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Ruf1" target="_blank">[9]</a>. Superoxide contributes to platelet aggregation, tissue factor expression, cytokine release, NF-κB activation, DNA damage, NET formation, recruitment of inflammatory cells, conversion of nitric oxide (NO) to peroxynitrate (ONOO⁻) which in turn leads to DNA damage, PARP activation, lipid peroxidation, and protein nitration. These events result in sustained inflammation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Finkel1" target="_blank">[52]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087140#pone.0087140-Smith1" target="_blank">[56]</a>. NO, nitric oxide; O₂⁻, superoxide; ONOO⁻, peroxynitrate.</p

Tempol increases survival of mice in a cerebral malaria model.

<p>Comparison with other antioxidants. <i>Plasmodium berghei</i> Anka strain parasitized red blood cells (10⁶) were injected in mice intraperitoneally (i.p.). Then, <b>A,</b> Tempol (20 mg/kg, n = 40), <b>B,</b> PBN (50 mg/kg, n = 10), <b>C,</b> MnTE-2-PyP (5 mg/kg, n = 10), <b>D</b>, MnTBAP (10 mg/Kg, n = 10), <b>E</b>, Mitotempo (3 mg/Kg, n = 10), <b>F</b>, Mitoquinone (10 mg/Kg, n = 10), <b>G,</b> M30 (7.5 mg/kg, n = 10), and <b>H,</b> Epigallocatechin gallate (25 mg/kg, n = 10), were injected i.p. starting from day 1 (all reagents) or day 4 p.i. (for Tempol only) as indicated. One hundred µL of each antioxidant was injected i.p. daily/mice. The percent of non-treated and treated mice that survived over time is given in Kaplan–Meier curves. *, P≤0.05 (Log-rank test). NS, non-significant.</p