Immunization inhibits embryogenesis in female worms.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Seventy days after infection female worms were analyzed for their embryonic stages. Representative pictures of oocyte (A; micron bar 10 µm), divided egg (B; 10 µm), pretzel stage (C; 15 µm) and stretched Mf (D; 30 µm) are shown. (E) Embryogram illustrating the composition of embryonic stages in female worms. If present, three female worms of each mouse were investigated (27 females in the control group, 28 females from the immunized group, additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s004" target="_blank">Figure S4</a>). Statistical analysis was performed with Mann-Whitney U-test (mean ± SEM, ** <i>P</i><0.01, *** <i>P</i><0.001).</p

Immunization reduces adult worm burden, but does not affect their development.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. Numbers of worms on days 15 (A), 56 (B), 70 (C) and 90 (D) p.i. (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5A</a>–C), gender balance (E) (individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5D</a>, E), as well as length of males (F) and females (G) at day 90 p.i. (10/90 percentile, outliers are indicated, individual experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s005" target="_blank">Figure S5F</a>–I) were analyzed with Student's t-test (** <i>P</i><0.01, *** <i>P</i><0.001).</p

Mice immunized with Mf in alum have reduced numbers of Mf.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum. Control mice received alum alone. <i>L. sigmodontis</i> infection was performed one week after the last immunization. Microfilaraemia was monitored twice a week throughout patency. (A) Kinetics of Mf load of sham-treated (dashed line) and immunized (black line) mice in the peripheral blood. One representative of three independent experiments with ten mice per group is shown (2-way ANOVA, mean ± SEM), including both Mf⁻ and Mf⁺ mice. For additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">figure S3A</a>, B. (B) Percentage of Mf⁺ mice of three independent experiments was analyzed using Student's t-test. Each mouse with peripheral Mf at any given time point was defined as Mf⁺. (C, D) Mf burden in the pleural space days 70 (C) and 90 (D) p.i.. Graphs show one representative of three (C) and two (D) independent experiments (at least seven mice each group, see also <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s003" target="_blank">Figure S3C</a>–E) and were analyzed with Welch-corrected t-test. Numbers below the symbols indicate the number of Mf⁺ mice (median, * <i>P</i><0.05, ** <i>P</i><0.005).</p

Immunization induces Mf-specific IgG1 and IgG2.

<p>Mice were immunized three times s.c. with 100,000 Mf in alum (Al-Mf/naïve, Al-Mf/Inf). Control mice received alum alone (Al/naïve, Al/Inf). <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization (Al/Inf, Al-Mf/Inf) or left uninfected (Al/naïve, Al-Mf/naïve). Plasma levels of Mf-specific IgG1 (A) and IgG2a/b (B) were measured. Two-way ANOVA was used for statistical analysis, day 0 indicates day of challenge infection. Asterisks indicate significant differences between the immunized and infected, and the corresponding control group (*** <i>P</i><0.001) and pound signs between the immunized but uninfected, and the corresponding control group (^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001). (C–F) Pleural space lavage was analyzed for specific IgG1 and IgG2a/b on days 22 (C, D) and 70 p.i. (E, F). Data analyzed with Welch-corrected t-test (mean, *** <i>P</i><0.001). Graphs show representatives of three independent experiments with eight to ten mice each group (additional experiments see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001558#pntd.0001558.s006" target="_blank">Figure S6A, B, E–J</a>).</p

Immunization strategies that failed to protect mice from peripheral microfilaraemia.

<p>Mice were immunized with 100,000 Mf either three times i.v. (A, B) or first s.c. followed by an i.p. and i.v. immunization (C, D). All control mice received PBS. <i>L. sigmodontis</i> challenge infection was performed one week after the last immunization. (B) After immunization mice were treated i.v. with IVM. (D) Mice were immunized with irradiated (400 Gy) Mf. Microfilaraemia was monitored throughout patency. Data obtained from single experiments with at least six mice per group are shown. Two-way ANOVA (mean ± SEM) was used for statistical analysis including both Mf⁻ and Mf⁺ mice.</p

Immunization enhances IFN-γ responses.

<p>(A) At day 22 p.i. the pleural lavage was analyzed for IL-5 and IFN-γ. Combined data of three independent experiments with five mice each group are shown. (B, C) At day 22 p.i. cells from the site of infection were restimulated for 72 h with 5 µg/ml Concanavalin A (ConA), 100 µg/ml complete adult (Ls) or microfilarial (Mf) crude extract of <i>L. sigmodontis</i> and IFN-γ (B) and IL-5 (C) secretion were measured (mean ± SEM). Representative data of two independent experiments with five mice each group. Analysis was done using the 2-way ANOVA, for significances see text.</p

SREC-I on nasal epithelila cells.

<p>Surface exposure of SREC-I on HNECs and CRNECs was monitored by FACS with the same anti-human SREC-I antibody and isotype control. Statistical evaluation on HNECs (A) and on CRNECs (B). Data represent means ± SD of 3 or 4 independent experiments. Statistical analysis was performed by Student's t-test. Significant differences are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01), or three (<i>P</i><0.001) asterisks (<b>*</b>).</p

Deficiency in ST2 leads to pronounced microfilaremia and increased adult worm length.

<p>A, microfilariae count per 50 μl of peripheral blood of ST2-ko mice and wild type (WT) controls throughout <i>L. sigmodontis</i> infection. B, microfilarial burden in the thoracic cavity 60 days post <i>L. sigmodontis</i> infection. C, percentage of ST2-ko mice and WT controls that develop patent infections. D, embryogram of female worms 60dpi (6 mice per group and two female worms per mouse). E, adult worm burden in ST2-ko mice and WT controls 35, 60 and 100 dpi, (F and G) length of male and female <i>L. sigmodontis</i> worms in WT and ST2-ko mice during infection. A and C show pooled data from three independent experiments with a minimum of 8 mice per group. B shows pooled data from two independent experiments and E-G show representative data of two independent experiments with a minimum of 6 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05.</p

Impact of SREC-I on adhesion of <i>S. aureus</i> to CHO epithelial cells and impact of SREC-I on the early phases of nasal colonization in the cotton rat model.

<p><i>S. aureus</i> adhesion to the cell line CHO expressing or not expressing SREC-I assayed in chamber slides under shear stress (A) and under static conditions (B). Attachment of <i>S. aureus</i> wt, <i>tagO</i>, and <i>tagO</i> compl. evaluated microscopically by counting at least 8 visible fields per well or chamber slide containing CHO cells without SREC-I or expressing full length SREC-I (A) or consecutive truncations of the extracellular EGF-like domains (B). Means and SD of 4 independent experiments (static) or 3 independent experiments (flow) are shown. Statistical analysis was performed by one-way ANOVA with Bonferroni's multiple comparison test (A and B). Significant differences are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01), or three (<i>P</i><0.001) asterisks (<b>*</b>). Nasal colonization was assayed in a cotton rat model (C). Bacterial numbers were determined 8 h and 6 days after inoculation. 15 min prior to inoculation cotton rats were pretreated with 2 µg anti-SREC-I Fab₂-fragment per nose. After 8 h and 6 days the noses were dissected and the bacterial CFU was evaluated on <i>S. aureus</i> selective highchrome agar. Statistical analysis was performed by D'Agostino & Pearson omnibus normality test and a subsequent Kruskal-Wallis non-parametric ANOVA with Dunn's Multiple Comparison Test (C). Significant differences between groups are indicated by one (<i>P</i><0.05), two (<i>P</i><0.01), or three (<i>P</i><0.001) asterisks (<b>*</b>).</p

Model of SREC-I role in nasal colonization.

<p>SREC-I can be detected on growing nasal epithelial cells in the inner nasal cavity. <i>S. aureus</i> WTA binds to SREC-I which mediates <i>S. aureus</i> adhesion to cells from the inner nasal cavity. This WTA/SREC-I interaction has a considerable impact on nasal colonization. Therefore, we postulate a reservoir of <i>S. aureus</i> cells in the inner nasal cavity as a source for continuous colonization, which is at least partially maintained by WTA dependent adhesion to epithelial cells.</p