Social network visualisation a) male and female <i>M. nattereri</i>, b) male and female <i>M. daubentonii</i>, c) female <i>M. daubentonii</i>, and d) male <i>M. daubentonii.</i>

<p>a) <i>M. nattereri</i> male (n = 85) and female (n = 214), modularity  = 0.74, b) <i>M. daubentonii</i> (n = 344), modularity  = 0.66, c) female <i>M. daubentonii</i> (n = 145), modularity  = 0.67, d) male <i>M. daubentonii</i> (n = 199), modularity  = 0.64. Nodes represent individual bats (males, circles; females, triangles) and associations are represented by the lines that join them. Colours indicate the assignment of individuals to social groups using the Girvan-Newman algorithm. Colours do not correspond between panels. Colours in a) and c) are comparable to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112225#pone-0112225-g003" target="_blank">Figure 3</a>. The position of individuals within these networks indicates their position in social space and is not an indication of an individual's geographical location.</p

Lagged association rates within and between sexes of <i>M. daubentonii</i> (left) and <i>M. nattereri</i> (right).

<p>Standard error is calculated by jackknifing over a 30-day period.</p

Distribution of a) <i>M. nattereri</i> both sexes and b) female <i>M. daubentonii</i> social groups in Wytham Woods.

<p>Roosts used by bats, and home range estimates are coloured according to social group - colours are comparable to Figure 2, panels a) and c) – symbols indicate colony size and roosts identified by radio-tracking. Roost home ranges are estimated using 100% minimum convex polygons (MCPs). MCPs exclude roosts occupied by a single individual (<i>M. nattereri</i>, n = 42; <i>M. daubentonii</i>, n = 44) or separated by over 1 km from a roost of the same social group (n = 1 for each species). Four adult female <i>M. daubentonii</i> were radio-tracked; two from each of two social groups. The daytime roosts (including trees) used by these individuals are indicated by asterisks and are coloured according to the social group to which they belonged.</p

Japanin homologues modulate DC maturation.

<p>(A) Three Japanin homologues were successfully expressed in Sf9 cells, as shown by Western blotting with an anti-His tag antibody. ∼10 ng protein was loaded per lane. (B) & (C) Dendritic cells were cultured in the presence or absence of Japanin or Japanin homologues (500 ng/ml) and LPS (100 ng/ml) for 18–20 hours. CD86 (B) and CD274 (C) were then assessed by flow cytometry. Modelled means ±95% confidence intervals using data from three (cells with LPS) or four (cells without LPS) experiments are shown. * p<0.05, as compared to cells without Japanin or a Japanin homologue.</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

Japanin blocks differentiation of DC from monocytes.

<p>Monocytes were cultured with GM-CSF (1000 U/ml) and IL4 (500 U/ml) with or without Japanin (500 ng/ml). Before the culture, and again after 3 and 5 days of culture, CD1a and CD14 expression were assessed by flow cytometry, in order to monitor differentiation into CD1a⁺CD14^low dendritic cells. Data shown is from one experiment, representative of three independent experiments using cells from different donors.</p

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

Japanin modulates dendritic cell maturation, rather than simply inhibiting it.

<p>Dendritic cells were cultured in the presence or absence of Japanin (500 ng/ml) and LPS (100 ng/ml) for 18–20 hours. (A) CD40, CD83, CD86, CD274 and HLA-DR expression were then assessed by flow cytometry, and (B)the concentration of pro-inflammatory cytokines in the culture supernatant was measured by Luminex1. Modelled means ±95% confidence intervals using data from at least four experiments are shown, except where marked ‡ where above-scale readings in the LPS-only made it impossible to calculate meaningful confidence intervals; the graphs show the lowest possible mean value (taking an above-scale value to be equal to the maximum possible on-scale value). ** p<0.01, * p<0.05, p<0.1, NS p>0.05.</p

Pretreatment of dendritic cells with Japanin inhibits their upregulation of CD86 in response to LPS.

<p>Dendritic cells were incubated with japanin for 24 hours prior to the addition of LPS (100 ng/ml) for a further 18–20 hours. CD86 expression was then analysed by flow cytometry. (A) The results from a representative experiment using 500 ng/ml japanin. (B) Titration of Japanin concentration, showing a dose-dependent inhibition of CD86 upregulation. The range and mean of duplicate measurements from one representative experiment are shown. This experiment was performed four times, with dose-dependency demonstrated each time, but with EC₅₀ varying between donors.</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