Inhibition of IgE-IC-mediated toxoplasmacidal activity of human macrophages by endogenous and exogenous IL-10.

<p>(<b>A</b>) Simultaneous addition of recombinant IL-10 (10 ng/ml) to CD23-engaged macrophages significantly decreased their ability to eliminate <i>T. gondii</i>. Addition of neutralizing anti-IL-10 (20 µg/ml) McAb increased cell resistance to infection in the absence of CD23 engagement. Results show mean±SD from 3 distinct MDM preparations following 72 h incubation, each done in duplicates. (<b>B</b>) Infection with <i>T. gondii</i> induces IL-10 generation by human macrophages. Results show mean±SD from two distinct MDM preparations, each done in duplicates. (<b>C</b>) Infected or uninfected cells were incubated with anti-CD23 or chemical NO (SNAP) and were collected 24 h later for iNOS- and IL-10-mRNA quantification and cell supernatants were harvested (72 h) to assess the levels of nitrites and IL-10. CD23 engagement reduced <i>T. gondii</i>-mediated IL-10 increase at both mRNA and protein levels and was inversely correlated with iNOS expression by infected human macrophages. Chemical NO (SNAP) also reduced the IL-10 expression from infected macrophages. Results show mRNA quantification from one representative macrophage preparation, out of two and mean±SD from 2 distinct MDM preparations following 72 h incubation for the quantification of mediators, each done in duplicates.</p

The presence of IL-10, IgE and nitrites in sera from patients infected with <i>T. gondii</i>.

<p>Freshly isolated <b>s</b>era from 18 <i>T. gondii</i> infected patients or 11–18 uninfected controls were tested for the levels of IL-10, IgE and nitrites. (<b>A</b>) Infected patients have significantly high serum levels of IL-10 compared to controls. (<b>B</b>) Elevated IgE levels were detected in infected patients compared to controls, while concentrations of serum nitrites were close to normal values except to 6/8 patients with high IgE (>200 IU) levels (p<0.018).</p

Involvement of iNOS pathway during IgE-IC-mediated activation of <i>T. gondii</i> killing by human macrophages.

<p>Infected cells were incubated in the presence of IL-4, IgE-IC or anti-CD23 McAb. (<b>A</b>) Cells were then collected (24 h) for iNOS-mRNA quantification and cell supernatants were harvested (72 h) to assess the levels of nitrites. Both conditions induced iNOS mRNA expression and consequent NO generation. (<b>B</b>) Cells were also incubated with an iNOS inhibitor (L-NMMA), a negative control (D-NMMA), or chemical NO-donor (SNAP). Following 72 h incubation, the percentage of infected macrophages was assessed. Addition of L-NMMA inhibited IgE-IC- or CD23-mediated parasiticidal activity (p<0.0008) and was reversed by L-arginine supplementation. Addition of chemical NO completely destroyed parasites without cell toxicity (>75% viable cells found in control cultures). Results show mean±SD for data from 3 distinct macrophage preparations, each done in duplicates.</p

IgE-IC induces toxoplasmacidal activity of human macrophages through FcεRII/CD23 ligation.

<p><i>T. gondii</i>-infected macrophages were incubated in the presence of IgE-IC, cross-linking anti-FcεRI (20 µg/ml), or anti-FcεRII (10, 20 µg/ml) McAb and the percentage of infected cells was assessed following 72 h incubation. Only IgE-IC and anti-FcεRII induced parasite elimination (upper panel). Infected cell incubation with recombinant IL-4 or IFN-γ induced parasite killing, reversed by the simultaneous blockade of CD23 cross-linking by Fab fragments of anti-CD23 McAb (20 µg/ml) (median panel). Cells were also treated 24 h with IL-4 and IgE-IC or anti-CD23 prior to cell infection. The lower panel shows that pretreatment of macrophages during 24 h with CD23 ligands enabled them to resist to <i>T. gondii</i> infection. Results show mean±SD from 3 distinct macrophage preparations, each done in duplicates. Asterisks show significance compared to infected cells cultured in medium alone.</p

Adhesion rate of <i>C. glabrata</i> strains to Caco-2 cells.

<p>Adhesion level rate is expressed in percentage after normalization to ATCC2001 reference strain adherence level used in each experiment as a control. Results are presented based upon the clonal complex belonging as previously determined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069664#pone.0069664-EnacheAngoulvant1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069664#pone.0069664-Brisse1" target="_blank">[14]</a>. Strains EF0806Blo, EF1859Blo, EF2918Blo, EF1210Blo, EF0904Blo, EF2501Blo are of alpha mating type. The mating type of strains EF2502Blo, EF0612Blo, EF2903Blo, IHEM15749 have not been determined. Other strains are of A mating type.</p

A Mouse Model for <i>Candida glabrata</i> Hematogenous Disseminated Infection Starting from the Gut: Evaluation of Strains with Different Adhesion Properties

<div><p>Adhesion to digestive mucosa is considered a crucial first step in the pathogenicity of invasive <i>Candida</i> infections. <i>Candida glabrata</i> disseminated infections predominantly start from the gut. A mouse model of disseminated infection starting from the gut was set up. Hematogenous dissemination was obtained after a low-protein diet followed by a regimen of cyclophosphamide-methotrexate and an oral inoculation of the yeasts via the drinking water. The liver was the first organ infected (day 7 post-infection), and lethality was 100% at day 21 post-infection. This new mouse model was used to compare the mortality rate and fungal burden in deep organs induced by 5 strains exhibiting different levels of adhesion to enterocyte Caco-2 cells, as determined in a test on 36 <i>C. glabrata</i> strains. In this model, no statistical difference of lethality was demonstrated between the strains, and fungal burden varied in kidneys and lungs but without correlation with the level of adhesion to enterocytes. Further studies using the model developed here allow analysis of the crossing of the digestive mucosa by yeasts, and help relate this to yet-poorly understood adhesion phenotypes.</p></div

Survival curves for mice infected with six different <i>C. glabrata</i> strains.

<p>Adhesion levels of the strains used for the infection are as follows: 100% for ATCC2001, 98.43% for IHEM15749, 27% for EF1121Blo, 151% for EF1237Blo, 142% for EF2502Blo and 23% for EF0901Blo. No statistically significant differences in survival were determined using the Mantel-Cox log rank test (Prism 5.0).</p

Pairs of <i>C. glabrata</i> strains with significant difference in adhesion to Caco-2 cells as tested with the Dunn’s post-test.

<p>Pairs of <i>C. glabrata</i> strains with significant difference in adhesion to Caco-2 cells as tested with the Dunn’s post-test.</p

Kinetics of <i>C. glabrata</i> dissemination in organs of infected mice. Mice were housed in three groups of 6 animals.

<p>After sacrifice of an animal from each group at different time points after infection, organs were aseptically removed and homogenized for CFU count (n = 3/day).</p

Experimental protocol of <i>C. glabrata</i> colonization and dissemination in animal model.

<p>Five weeks old female DBA2/J mice are infected with 5×10⁷ CFU/ml in drinking water for five days after 14 days of low-protein diet and 24 h starvation. A chemotherapy protocol including a single MTX ip injection and an injection of CPA/day for five consecutive days follows the infection. Mice are provided with sterile water supplied with enrofloxacine from the beginning of chemotherapy until the end of experiment. MTX: methotrexate, CPA: cyclophosphamide.</p