AvCystatin treatment in primary RSV infection.

<p>A) Schematic of the primary RSV model with AvCystatin treatment regimen: i.p. intraperitoneal; i.n. intranasal application. Neutrophil influx in the BAL was shown (B). RSV L gene copy numbers in the lungs (C) and viral load (D) measured 4 days post RSV/mock challenge. RSV-specific IgG2a detected in serum 8 days post infection (E). IFN-γ, TNFα, IL-6, CCL3, and CCL5 cytokine and chemokine production (F). Naïve (dark grey bars): mock infected and mock treated with PBS, RSV challenged day 0 (black bars); AvCystatin/RSV; AvCystatin treatment i.n. or i.p. on day -1 (white bars or light grey bars, respectively). Representative data of at least 2 independent experiments, 5 mice per group. Error bars indicate SEM. <i>P</i> values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.</p

Altered immune responses in mice infected with Tm-sAChE.

<p>(A) Specific antibody responses (end-point titres) to <i>T</i>. <i>musculi</i> at d14 post-infection. (B) Cytokine responses from splenocytes at d14 post-infection. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. **p<0.01, ***p<0.001. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

Expression of <i>N</i>. <i>brasiliensis</i> AChE B in <i>T</i>. <i>musculi</i>.

<p>(A) Detection by western blot. NbSP: Secreted products from <i>N</i>. <i>brasiliensis</i>; TmE: <i>T</i>. <i>musculi</i> extracts; TmSP: <i>T</i>. <i>musculi</i> secreted products. WT: Wild type trypanosomes; AChE; <i>T</i>. <i>musculi</i> expressing cytosolic AChE; sAChE: <i>T</i>. <i>musculi</i> expressing secreted AChE. (B) Tm-sAChE is glycosylated. Extracts and secreted products as in A), either with (+) or without (-) PNGase F treatment. Molecular mass markers are shown in kDa. (C) Tm-sAChE stained with antibody to <i>N</i>. <i>brasiliensis</i> AChE B and DAPI and viewed by indirect immunofluorescence. (D) Visualisation of AChE activity after non-denaturing gel electrophoresis, abbreviations as in panel A. (E) AChE activity measured by Ellman assay, abbreviations as in panel A. TmE: <i>T</i>. <i>musculi</i> extracts from 5 x 10⁵ trypanosomes; TmSP: <i>T</i>. <i>musculi</i> secreted products from 5 x 10⁴ trypanosomes cultured for 24 hrs. Data are shown as the mean ±SEM, assayed in triplicate.</p

Cellularity of spleens from mice infected with transgenic <i>T</i>. <i>musculi</i>.

<p>Phenotyping was performed by flow cytometry as described in Materials and Methods. Data are shown as the mean ± 1SEM (n = 5) and are representative of two independent experiments with 5 mice in each group. *p<0.05, **p<0.01. White bar = uninfected mice; Light grey bar = mice infected with Tm-luc; Dark grey bar = mice infected with Tm-sAChE.</p

Generation of transgenic <i>T</i>. <i>musculi</i> and expression of GFP.

<p>(A) Schematic depiction of expression vector pSSUGFP, utilising 5´ and 3´ regions to integrate into the 18S SSU rRNA gene locus, and both PFR and β-α tubulin intergenic regions to effect RNA processing of eGFP and blasticidin resistance genes. (B) Detection of eGFP expression by western blot. Lane 1: Wild type <i>T</i>. <i>musculi</i>; Lane 2: Parasites transformed with expression cassette in which eGFP was incorporated into the tubulin gene array (pTubGFP); Lane 3: Expression cassette with eGFP incorporated into the SSU rRNA locus (pSSUT7GFP) in a cell line with T7 RNA polymerase incorporated into the tubulin array (pT7polyNeo); Lane 4: Expression cassette with eGFP incorporated into the SSU rRNA locus alone (pSSUGFP). Molecular mass markers are shown in kDa. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.s002" target="_blank">S2 Fig</a> for different constructs. (C) Detection of eGFP expression by fluorescence microscopy: eGFP incorporated into the SSU rRNA locus.</p

AvCystatin induced IL-10 production by CD4⁺ T cells in airways and lungs.

<p>Total CD4⁺ T cell counts in lungs (A) and BAL (B). CD4⁺ T cells were stimulated with PMA/ionomycin for 3h, IL-10⁺ and IFN-γ⁺ cells were gated in lung (B+C) and BAL (E+F). Ratios of IL-10⁺ versus IFN-γ⁺ CD4⁺ T cells in lung and BAL (G). Naïve (light grey): mock infected and mock treated with PBS; vvG/RSV (black bars): scarified with vvG (d-14) and challenged with RSV (d0); vvG/Av17/RSV (white bars): model plus injection of AvCystatin (d-14 and -7 i.p., d-2 and -1 i.n.). (A) Combined data from 2 independent experiments, (B-G) Representative data from 2 independent experiments, 5 mice per group. Error bars indicate SEM. <i>P</i> values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.</p

AvCystatin reduced chemokine and cytokine release in the airways.

<p>A) IL-4, B) IL-13, C) CCL11/Eotaxin, D) CCL5 / RANTES, E) TNF- α, F) IFN-γ, G) CCL3 / MIP-1α, Detection limit: 0.3pg/ml; 7.8pg/ml; 2.2pg/ml; 5.2pg/ml; 3.4pg/ml; 1.8pg/ml; 12.5pg/ml, respectively). Day 4 post RSV/mock challenge. Naïve (light grey bars): mock infected and mock treated with PBS; vvG/RSV (black bars): scarified with vvG and challenged with RSV; FI-RSV/AvCystatin/RSV (white bars). Representative data from 2 experiments, 5 mice per group. Error bars indicate SEM. <i>P</i> values reflect Mann-Whitney t-test: * p<0.05, **p<0.01, ***p<0.001.</p

Growth and survival of transgenic <i>T</i>. <i>musculi</i> in vitro and in vivo.

<p>(A) Growth <i>in vitro</i> of wild type <i>T</i>. <i>musculi</i> (WT), those engineered to express secreted AChE (Tm-sAChE) and expressing cytosolic luciferase (Tm-luc). (B) Survival of Tm-sAChE compared to Tm-luc <i>in vivo</i>. Parasitaemia in peripheral blood monitored over the course of infection of female BALB/c mice. Data are shown as the mean ±SEM (n = 5) and are representative of three independent experiments. *p<0.05, **p<0.01, ***p<0.001. (C). Detection of AChE in serum of mice 8 days post-infection with Tm-luc and Tm-sAChE by activity-based gel assay [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005998#ppat.1005998.ref023" target="_blank">23</a>]. Samples of serum (5 μl) from individual mice were loaded in each lane.</p

IL-10⁺ CD4⁺ T cell induction by AvCystatin conditioned macrophages.

<p>Lung and BAL CD4⁺ T cells detected by flow cytometry (A and D). Total number of IL-10 producing T cells determined by intracellular staining for IL-10 after restimulation with PMA/ionomycin for 3h in Lung and BAL (B and E). Number of CD4⁺ T cells positive for FoxP3 in lung (C) and BAL (F). Percentage of F4/80⁺CD11b⁺ cells infiltrating the lungs 24h post RSV challenge (G) and total cell number (H). Total cell number of CD4⁺ IL-10⁺ T cells infiltrating the BAL, 8 days post PEC transfer (I). Naïve (dark grey bars): mock infected and mock treated with PBS, RSV challenged day 0 (black bars); AvCystatin/RSV; AvCystatin treatment i.n. or i.p. on day -1 (white bars or light grey bars, respectively). Representative data of 2 experiments, 5 mice per group. Error bars indicate SEM. <i>P</i> values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.</p

AvCystatin treatment reduced RSV-induced immunopathology in Th2-based models of viral lung eosinophilia.

<p>A) Schematic of the vvG RSV model: e.d. epidermabrasive; i.p. intraperitoneal; i.n. intranasal application. B) Weight loss in vvG RSV over an eight day period C) Eosinophil, neutrophil, and macrophage influx into BAL: total numbers in vvG RSV. D) RSV-specific serum immunoglobulin levels. E) L gene copy numbers in lungs, d4 after RSV/mock challenge. Naïve (light grey bars): mock infected and mock treated with PBS; vvG/RSV (black bars): scarified with vvG and challenged with RSV; vvG/AvCystatin/RSV (white bars): vvG/RSV plus intraperitoneal (d -14 and -7) and intranasal (d -2 and -1) injection of AvCystatin. Representative data from 2 independent experiments, 5 mice per group. Error bars indicate SEM. <i>P</i> values reflect Mann-Whitney t-test: * p<0.05, **p<0.01.</p