Innate Immune Induction and Influenza Protection Elicited by a Response-Selective Agonist of Human C5a

<div><p>The anaphylatoxin C5a is an especially potent mediator of both local and systemic inflammation. However, C5a also plays an essential role in mucosal host defense against bacterial, viral, and fungal infection. We have developed a response-selective agonist of human C5a, termed EP67, which retains the immunoenhancing activity of C5a at the expense of its inflammatory, anaphylagenic properties. EP67 insufflation results in the rapid induction of pulmonary cytokines and chemokines. This is followed by an influx of innate immune effector cells, including neutrophils, NK cells, and dendritic cells. EP67 exhibits both prophylactic and therapeutic protection when tested in a murine model of influenza A infection. Mice treated with EP67 within a twenty-four hour window of non-lethal infection were significantly protected from influenza-induced weight loss. Furthermore, EP67 delivered twenty-four hours after lethal infection completely blocked influenza-induced mortality (0% vs. 100% survival). Since protection based on innate immune induction is not restricted to any specific pathogen, EP67 may well prove equally efficacious against a wide variety of possible viral, bacterial, and fungal pathogens. Such a strategy could be used to stop the worldwide spread of emergent respiratory diseases, including but not limited to novel strains of influenza.</p> </div

EP67 induced cytokines and chemokines.

<p>Mice were insufflated with 30 micrograms EP67 in a total volume of 30 microliters, and then sacrificed at various time points between two hours and fourteen days later. Cytokine analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040303#s2" target="_blank">Materials and Methods</a>. Three individual samples (not pooled) were tested for each time point. For each cytokine graph, the X-axis is time in days (except for the initial two hour time point) after EP67 insufflation. The Y-axis is specific cytokine concentration in pg/ml. Data is displayed as group average ± SEM for each time point.</p

EP67 prevents morbidity and mortality following lethal influenza infection.

<p>Mice (five mice/group) were infected with a lethal dose of influenza (3.3×MLD₅₀) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040303#s2" target="_blank">Materials and Methods</a>. One day later, groups were insufflated with either EP67 (240 µg/mouse) or saline. Mice were sacrificed when weight loss reached 30% of starting body weight, and the infection counted as lethal. A. Survival curve. B. Group average weight loss.</p

Kinetics of innate effector cells in the airways following EP67 insufflation.

<p>X-axis is time in days following EP67 insufflation. A. Absolute cell number following EP67 insufflation. B. Subpopulations of BAL cells as % of BAL cell population. C. Total cell number of individual BAL cell subpopulations. AM: open triangles; Neutrophils: <b>x</b>; exMacs: closed squares; mDC: closed triangles. NK cells are not shown, as they never represented more than 1% of the total BAL population. Results are shown as mean ± SD.</p

Innate effector cells in the BAL following EP67 insufflation.

<p>Mice were insufflated with EP67 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040303#pone-0040303-g001" target="_blank">Figure 1</a>. BAL cells were isolated either two hours or one day later, then stained with fluorescent antibodies and analysed by FACS. FACS plots shown are from individual mice representative of at least 10 mice per time point. For surface marker analysis, empty histograms represent negative control stains and filled histogram overlay represents the stain listed on the X-axis. FSC: forward scatter (size); SSC: side scatter. A. Gating strategy for BAL cell analysis, using a non-EP67 stimulated control mouse. All samples were initially gated by size and side scatter and stained with CD45.2 to exclude any non-hematopoetic cells or red blood cells from analysis. All CD45 positive cells were stained with CD11c and CD11b to identify discrete cell populations. The identity of individual cell populations was confirmed using additional surface stains, as shown. B. BAL cells isolated two hours after EP67 insufflation. Boxed cells indicate cell populations not present as a large percentage of BAL prior to EP67 insufflation. C. BAL cell analysis one day after EP67 insufflation. Representative staining data is shown (10–30 mice per time point).</p

EP67 displays protection against influenza-induced morbidity.

<p>Four groups of mice were infected with a non-lethal dose of influenza (0.33×MLD₅₀) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040303#s2" target="_blank">Materials and Methods</a>. Three groups were treated one time with EP67, either the day before infection (EP67 day −1), at the time of infection (EP67 day 0), or the day after infection (EP67 day 1). Mice were weighed daily. A. Weight change following influenza infection. Group average weight change is shown as mean ± SEM. Day 8 weight loss results are statistically significant (using a two-tailed Student’s t-test) as reported in the text. One experiment is shown; experiment was repeated twice. B. Viral load in lungs was measured using quantitative RT-PCR of the influenza matrix gene. Lung RNA was isolated three days after infection. Results are shown as matrix gene copies per 100 ng RNA. (^∧p<0. 0005, two-tailed Student’s t-test). C. ELISA analysis of anti-influenza antibodies. Individual serum samples were analyzed for total IgG, IgG1, IgG2b, and IgG2c distribution. Results are displayed as mean ± SEM. The total IgG response shown is from the 1∶10,000 dilution of serum. The subclass responses shown are from the 1∶1000 serum dilution. *p<0.05, two-tailed Student’s t-test.</p