Figure 1.
Effects of IAV infection on C5a levels in BALF (left) and serum (right).
C57BL/6J mice were infected intranasally with 104 PFU of Influenza A/WSN/33 H1N1, or received PBS intranasally (Mock group). At the indicated time points after infection 5 mice per group were euthanized, blood was collected and BAL performed. Serum and BALF concentrations of C5a were measured by ELISA. Data are presented as Mean ± SEM. *, ** and *** for p<0.05, p<0.01 and p<0.001 respectively, when compared to Mock group or indicated group (One-way ANOVA, Newman-Keuls Multiple Comparison test).
Figure 2.
Effects of C5 activation inhibitor OmCI during IAV infection on leukocyte transmigration and protein leakage.
C57BL/6J mice were infected intranasally with influenza or Mock infected as described in Figure 1. 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).
Figure 3.
Neutrophil accumulation in lung parenchyma of vehicle and OmCI treated mice.
C57BL/6J mice were infected and assigned to treatment groups as in Figure 2. 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.
Figure 4.
Inflammatory mediator levels after IAV infection.
C57BL/6J mice were infected and assigned to treatment groups as in Figure 2. 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).
Figure 5.
Histopathologic changes caused by IAV are reduced in OmCI treated mice.
C57BL/6J mice were infected and assigned to treatment groups as in Figure 2. At the sixth day after infection, mice were euthanized and lungs harvested. Representative slides of H&E stained lungs of mock (A, B, C); vehicle (D, E, F); and OmCI treated mice (G, H, I). “p” represents parenchyma, “v” represents vessels and “a”, airways. Asterisks indicate foci of inflammatory infiltrates and arrowheads indicate areas of epithelial injury. (J) Histopathological score (maximal of 23) evaluated airway, vascular, parenchymal inflammation, neutrophilic infiltration and epithelial injury. 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 (One-way ANOVA, Newman-Keuls Multiple Comparison test). Bars represent 100 µm.
Figure 6.
Effects of OmCI on cfDNA levels and number of dead cells in BALF after IAV infection.
C57BL/6J mice were infected and assigned to treatment groups as in Figure 2. 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).
Figure 7.
Number of CD8+ T cells and pulmonary viral load after IAV infection and OmCI treatment.
C57BL/6J mice were infected and assigned to treatment groups as in Figure 2. 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).
Figure 8.
Leukocyte recruitment and cfDNA levels in BALF after IAV infection upon OmCI and Zileuton treatment.
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).