Leukocyte recruitment and cfDNA levels in BALF after IAV infection upon OmCI and Zileuton treatment.

<p>C57BL/6J mice were infected intranasally with 10⁴ PFU of Influenza A/WSN/33 H1N1, or received PBS intranasally (Mock group). The 5-LO inhibitor Zileuton (30 mg/kg) was given alone or in combination with OmCI. Mice received the treatment prior to the infection and daily until day 5 after infection, while vehicle group received PBS both, via i.p, Zileuton was given by oral gavage. At the sixth day after infection, mice were euthanized, BAL performed and lungs were harvested. A) Total number of leukocytes; B) number of neutrophils; C) cfDNA levels measured in BALF. Data are presented as Mean ± SEM. * and *** for p<0.05 and p<0.001 respectively, when compared to Mock group; # and ## for p<0.05, p<0.01 respectively, when compared to Vehicle group (One-way ANOVA, Newman-Keuls Multiple Comparison test).</p

Neutrophil accumulation in lung parenchyma of vehicle and OmCI treated mice.

<p>C57BL/6J mice were infected and assigned to treatment groups as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064443#pone-0064443-g002" target="_blank">Figure 2</a>. At 1, 3 and 6 days after infection, mice were euthanized, lungs harvested and MPO assayed, to measure neutrophil accumulation in tissue. A) Relative numbers of neutrophils in lungs. At day 6, lungs were harvested for assessment of neutrophil infiltration by analysis of H&E stained lung slides. B) Pathologic score (0–5) of neutrophil accumulation in lungs performed by a pathologist. Representative slides of H&E stained lungs of a C) mock mouse; D) vehicle mouse; E) OmCI treated mouse. Asterisks indicate areas with neutrophils infiltration and arrowheads indicate bronchial epithelial damage. Data are presented as Mean ± SEM. * and *** for p<0.05 and p<0.001, respectively, when compared to Mock group (One-way ANOVA, Newman-Keuls Multiple Comparison test). Bars represent 100 µm.</p

Number of CD8+ T cells and pulmonary viral load after IAV infection and OmCI treatment.

<p>C57BL/6J mice were infected and assigned to treatment groups as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064443#pone-0064443-g002" target="_blank">Figure 2</a>. A) Numbers of CD3+ CD8+ cells recovered by BAL and analyzed by FACS are reduced in OmCI treated group 6 days after infection. ** and *** for p<0.01 and p<0.001 respectively, when compared to Mock group; # for p<0.05, when compared to Vehicle group (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test). B) Viral titers 6 days after infection in lungs homogenates shown are not changed between vehicle and OmCI treated groups, as assessed by MDCK plaque formation (Unpaired t test).</p

Effects of C5 activation inhibitor OmCI during IAV infection on leukocyte transmigration and protein leakage.

<p>C57BL/6J mice were infected intranasally with influenza or Mock infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064443#pone-0064443-g001" target="_blank">Figure 1</a>. OmCI treated mice received the protein prior to IAV infection and daily thereafter, while vehicle group received PBS, until one day before the indicated time point. At the 1, 3 and 6 days after infection, mice were euthanized and BAL performed. A) Total number of leukocytes, B) number of neutrophils and C) number of macrophages, D) number of lymphocytes recovered from the airways; E) total protein quantification in BALF. Data are presented as Mean ± SEM. *, ** and *** for p<0.05, p<0.01 and p<0.001 respectively, when compared to Mock group; # and ## for p<0.05, p<0.01 respectively, when compared to Vehicle group sampled on the same day (One-way ANOVA, Newman-Keuls Multiple Comparison test).</p

Effects of OmCI on cfDNA levels and number of dead cells in BALF after IAV infection.

<p>C57BL/6J mice were infected and assigned to treatment groups as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064443#pone-0064443-g002" target="_blank">Figure 2</a>. At1, 3 and 6 days after infection, mice were euthanized and BAL was performed. A) Cell free DNA (cfDNA) levels were measured in BALF by the Quant-iT PicoGreen dsDNA quantification kit. At six days after infection, leukocytes recovered from airways of Mock, Vehicle and OmCI treated mice were analyzed for cellular death by Annexin V and PI incorporation. B) Total apoptotic leukocytes, Annexin V+ PI-, in BALF; C) Total necrotic or late apoptotic leukocytes, Annexin V+ PI+, in BALF. Data are presented as Mean ± SEM. ** and *** for p<0.01 and p<0.001 respectively, when compared to Mock group; # and ## for p<0.05, p<0.01 respectively, when compared to Vehicle group (Kruskal-Wallis test, Dunn’s Multiple Comparison post-test).</p

Inflammatory mediator levels after IAV infection.

<p>C57BL/6J mice were infected and assigned to treatment groups as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064443#pone-0064443-g002" target="_blank">Figure 2</a>. At 1, 3 and 6 days after infection, mice were euthanized, BAL performed and lungs harvested. BALF concentrations of C5a (A), CXCL1 (B) and pulmonary concentrations of CXCL1 (C), and IFN-γ (D) were measured by ELISA. Data are presented as Mean ± SEM. ** and *** for p<0.01 and p<0.001 respectively, when compared to Mock group; # and ### for p<0.05 and p<0.001 respectively, when compared to vehicle group sampled on same day (One-way ANOVA, Newman-Keuls Multiple Comparison test).</p

SsE abolishes PAF-induced activation and chemotaxis of neutrophils.

<p>(A) SsE treatment of PAF abolishes PAF-induced Ca²⁺ mobilization in human neutrophils. Neutrophils (2×10⁵ cells/well) loaded with Fluo-4 acetoxymethyl ester dye were mixed with 0.05 ng/ml PAF, 0.05 ng/ml SsE^S178A-treated PAF, 50 ng/ml SsE-treated PAF, or SsE alone control, and Ca²⁺ flux was recorded. Presented are time courses of the fluorescence intensity after addition of PAF, SsE-treated PAF, or SsE or buffer controls. (B) Number of neutrophils migrated to PAF, SsE- and SsE^S178A-treated PAF, SsE, SsE^S178A, and buffer in a transwell cell migration assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002624#ppat.1002624-Kirpotina1" target="_blank">[58]</a> (See the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002624#s4" target="_blank">Methods</a> section for experimental details).</p

Evidence for the role of SsE in evasion of bactericidal neutrophil responses.

<p>(A) Deletion of <i>sse</i> enhances neutrophil recruitment. BALB/c mice were subcutaneously inoculated on the back with 1.0×10⁸ cfu MGAS5005 or 1.1×10⁸ Δ<i>sse</i>^MGAS5005. Neutrophil numbers at 24 h post-inoculation were determined by the myeloperoxidase assay. *** (One-way ANOVA analysis): The data is significant for Δ<i>sse</i> site versus buffer control (PBS), MGAS5005 inoculation site (5005inj), MGAS5005 spread area (5005sp), and reverse-complement strain of Δ<i>sse</i> (Δ<i>sse</i>-<i>sse</i>). (B) Significant reduction in neutrophil ingress to Δ<i>sse</i> site, but not to MGAS5005 site, in PAFR^−/− mice compared with BALB/c control mice. Five PAFR^−/− (KO) or BALB/c (wt control) mice per group were subcutaneously inoculated with 0.2 ml PBS, MGAS5005, or Δ<i>sse</i>^MGAS5005 suspension at OD₆₀₀ of 0.8. The statistic analysis data (***, significant; ns, not significant) were obtained from one-way ANOVA analysis of the combined data of two experiments. (C) Efficient clearance of Δ<i>sse</i>^MGAS5005. Numbers of viable GAS at sites of MGAS5005 and Δ<i>sse</i>^MGAS5005 infection at 1, 24, and 48 h after inoculation are shown. Six mice were used for each time point and each strain. Inoculum: MGAS5005, 1.1×10⁸ cfu; Δ<i>sse</i>, 1.2×10⁸ cfu; Δ<i>sse-sse</i>, 1.0×10⁸ cfu.</p

Antigen-induced arthritis in mice is mainly associated with the presence of neutrophils.

<p>(A) Intensity of nociception. (B) Number of neutrophils, (C) myeloperoxidase activity, and (D) CXCL1 chemokine levels in the peri-articular tissue. (E) Representative photos of the morphologic alterations in the knee (x100 and x400) and (F) arthritis index. Arrows indicate the synovial and asterisks represent the inflammatory infiltrate. Adipose tissue and liver (G) myeloperoxidase activity, and (H) CXCL1 levels at 1, 3, 24 and 48 hours after the challenge with the antigen-induced arthritis (AIA). Bars represent the mean values±SEM (n = 6–8). Neutrophils number in (I) epididymal adipose tissue and liver accumulated in Lysm-eGFP mice and (J) representative confocal microscopy image, 3 and 24 hours after the challenge with the AIA (x100). Bars represent the mean values±SEM (n = 3–5), *<i>P</i><0.05 vs. PBS.</p

Histological analyses showing the difference in levels and patterns of inflammatory cell infiltration between the MGAS5005 and Δ<i>sse</i>^MGAS5005 infections.

<p>BALB/c mice were subcutaneously inoculated on the back with 1.0×10⁸ cfu MGAS5005 or 1.1×10⁸ cfu Δ<i>sse</i>^MGAS5005, and the skin samples were collected at 24 h after inoculation. The microscopic pictures of the Gram and H&E-stained samples were each combined from three snapshots that were taken at a 40× magnification. The five zones in panel A represent different morphologies. Panels: (A), MGAS5005/Gram stain; (B), MGAS5005/H&E Stain; (C), Δ<i>sse</i>^MGAS5005/Gram stain; and (D), Δ<i>sse</i>^MGAS5005/H&E stain.</p