B-1CDPs are one of the components of the peritoneal macrophage population after LPS stimulation.

<p>A) Absolute number of peritoneal B-1 cells (CD19⁺CD23⁻CD11b⁺) detected in BALB/<i>Xid</i> mice submitted to the specified treatment. ** p<0.01 and ***p<0.001 when the indicated groups are compared with the non-marked groups. p<0.001 when the two last groups are compared. B) Absolute number of peritoneal macrophages (CD19⁻CD11b⁺F4/80⁺) detected in BALB/<i>Xid</i> mice submitted to the specified treatment. ** p<0.01 and ***p<0.001 when the indicated groups are compared with the non-marked groups. C) Absolute number of macrophages (CD19⁻CD11b⁺F4/80⁺) and the number of B-1CDPs (CFSE⁺ cells) detected in this population from BALB/<i>Xid</i> mice submitted to the specified treatment. **p<0.01 and ****p<0.001 when the CFSE⁺ cell number of the indicated groups are compared with all other groups. The results are representative of two independent experiments using 4 or 5 mice per group per experiment.</p

LPS induces B-1CDP differentiation and proliferation.

<p>A) Analysis of B-1 cell purification and CFSE⁺ staining. B-1 cells were obtained from the total peritoneal cells based on the selection of CD19⁺CD23⁻ cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034570#pone.0034570.s001" target="_blank">Figure S1</a>. The first dot plot represents the B-1 cell gate selected to perform cell sorting. The second dot plot illustrates B-1 cells after cell sorting. Subsequently, these cells were subjected to CFSE staining as demonstrated in the last dot plot. B) Analysis of expression of CD19 and CD23 by peritoneal lymphocytes confirms the transfer of B-1 cells to BALB/<i>Xid</i> mice. B-1 cells are almost absent in BALB/<i>Xid</i> and BALB/<i>Xid</i>+LPS mice but are present in significant numbers in BALB/<i>Xid</i>+B-1 and BALB/<i>Xid</i>+B-1+LPS mice, indicating the efficiency of the B-1 cell transfer. C) Absolute number of B-1 cells (CD19⁺CD23⁻CD11b⁺) in the peritoneal cavity of BALB/<i>Xid</i> (□) and BALB/<i>Xid</i>+B-1 (▪) after LPS or saline (−) injection. p<0.001 when the LPS-stimulated group is compared to the non-stimulated group. D) Analysis of the presence of B-1CDP (CFSE⁺ cells) in the peritoneal macrophage population (CD19⁻CD11b⁺F4/80⁺) from BALB/<i>Xid</i>, BALB/<i>Xid</i>+B-1, BALB/<i>Xid</i>+LPS and BALB/<i>Xid</i>+B-1+LPS mice. E) Absolute number of peritoneal macrophages (CD19⁻CD11b⁺F4/80⁺) from BALB/<i>Xid</i> mice (□) and BALB/<i>Xid</i>+B-1 mice (▪) after LPS or saline (−) injection. * p<0.01 when the indicated group is compared to all groups. The results are representative of two independent experiments using 5 mice per group per experiment.</p

Characterization of peritoneal cell subpopulations in the peritoneal cavity of op/op^(−/−) mice.

<p>(A) The number of cells in the peritoneal cavity of op/op^(−/−) mice is significantly reduced when compared to the littermates. Peritoneal cells were collected from op/op^(−/−) mice and their normal littermates (op/op^(+/?)), and the cells were counted (p<0.01). (B) Percentage of B-1 cells, B-2 cells and macrophages in the peritoneal cavity of op/op^(−/−) mice and littermates (op/op^(+/?)). (C) Absolute number of B-1 cells (ns), B-2 cells (ns) and macrophages (Mφ - p<0.0001). Cell populations were determined by flow analysis based on the following surface marker expression: B-1 cells (CD23⁻CD19⁺CD11b⁺), Mφ (CD11b⁺F4/80⁺CD19⁻), and B-2 (CD23⁺CD19⁺CD11b⁻). Graphs are representative of two independent experiments using 6 mice per group per experiment.</p

LPS increases the number of B-1 cells and macrophages in the peritoneal cavity of op/op^(−/−) mice.

<p>(A) Number of peritoneal cells collected and counted individually from 6 mice (op/op^(−/−)) and their littermates (op/op^(+/?)) in control or LPS-treated groups. (B) Absolute number of B-1 cells and macrophages in untreated control (□) or LPS stimulated (▪) littermates op/op^(+/?) and op/op^(−/−) mice. Cell populations were determined by flow analysis based on surface marker expression: B-1 cells (CD23⁻CD19⁺CD11b⁺) and Mφ (CD11b⁺F4/80⁺CD19⁻). Flow cytometric analysis showed significantly increased percentages of B-1 cells in both mice lineages after LPS stimulation when compared to untreated mice. LPS also increased the number of macrophage cells in both groups of mice. Significant differences between the sample means are indicated as follows: **p<0.05, ***p<0.01, and ****p<0.001. The results are representative of two independent experiments.</p

Interaction of the Rattlesnake Toxin Crotamine with Model Membranes

Crotamine is one of the main constituents of the venom of the South American rattlesnake <i>Crotalus durissus terrificus</i>. A common gene ancestry and structural similarity with the antimicrobial β-defensins (identical disulfide bond pattern and highly positive net charge) suggested potential antimicrobial activities for this snake toxin. Although crotamine demonstrated low activity against both Gram-positive and Gram-negative bacteria, a pronounced antifungal activity was observed against <i>Candida</i> spp., <i>Trichosporon</i> spp., and <i>Cryptococcus neoformans</i>. Crotamine’s selective antimicrobial properties, with no observable hemolytic activity, stimulated us to evaluate the potential applications of this polypeptide as an antiyeast or candicidal agent for medical and industrial application. Aiming to understand the mechanism(s) of action underlying crotamine antimicrobial activity and its selectivity for fungi, we present herein studies using membrane model systems (i.e., large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs), with different phospholipid compositions. We show here that crotamine presents a higher lytic activity on negatively charged membranes compared with neutral membranes, with or without cholesterol or ergosterol content. The vesicle burst was not preceded by membrane permeabilization as is generally observed for pore forming peptides. Although such a property of disrupting lipid membranes is very important to combat multiresistant fungi, no inhibitory activity was observed for crotamine against biofilms formed by several <i>Candida</i> spp. strains, except for a limited effect against <i>C. krusei</i> biofilm

<i>In vivo</i> and <i>ex vivo</i> analysis of iRBC uptake by splenic DCs during crisis.

<p><b>(A-D)</b> B6.CD11-YFP mice were i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed by CIVM after eight days, at a time of day when mature parasite stages predominated. <b>(A)</b> A snapshot shows the subcapsular RP. <b>(B)</b> CIVM 3D animation shows the presence of few mCherry-<i>Pc</i> iRBCs (red) attached to YFP⁺ cells (green). <b>(C)</b> Percentage of mCherry⁺ cells in the YFP⁺ cell population is shown (mean ± SEM). <b>(D)</b> YFP⁺ cell volume and sphericity are shown. Black, red and blue dots are from three different experiments. Horizontal lines represent mean values and SEM. <b>(E</b> and <b>F)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs. At eight days p.i., half of the B6 mice were re-infected i.v. with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min. <b>(E)</b> Representative contour plots show CTV staining in the CD11c⁺ cells of <i>Pc</i>-infected mice that were re-infected or not with CTV-<i>Pc</i> iRBCs. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of CTV⁺ cells in the CD11c⁺ cell population. <b>(F)</b> The numbers of total and CTV⁺CD11c⁺ cells per spleen were calculated from the data obtained in <b>E</b> (means ± SD). <b>(G</b> and <b>H)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBCs. Spleens were analyzed after eight days, at a time of day when mature parasite stages predominated. <b>(G)</b> Representative contour plots obtained by flow cytometry show GFP staining in CD11c⁺ cells of mice that were infected with <i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBC. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of GFP⁺ cells in the CD11c⁺ cell population. <b>(H)</b> Numbers of total and GFP⁺CD11c⁺ cells per spleen were calculated from the data obtained in <b>G</b>. In <b>A</b> and <b>B</b>, the scale bars correspond to 50 µm and 30 µm, respectively. One representative experiment out of three (n = 2) is shown. In <b>C</b> and <b>D</b>, data were obtained using Imaris software. Data from three experiments (n = 2) are shown. In <b>E-H</b>, one representative experiment out of three (n = 5) is shown.</p

<i>In vivo</i> analysis of iRBC uptake by subcapsular RP DCs during pre-crisis.

<p><b>(A-D)</b> B6.CD11c-YFP mice were i.p. infected with 1 × 10⁶ mCherry-<i>Pc</i> iRBCs. Spleens were analyzed by CIVM after five days, at a time of day when mature parasite stages predominated. <b>(A)</b> Serial snapshots show the subcapsular RP and, in the right panel, a detailed image of a YFP⁺ cell (green) upon phagocytosis of an mCherry-<i>Pc</i> iRBC (red). <b>(B)</b> CIVM 3D animation shows the presence of mCherry-<i>Pc</i> iRBC remnants (yellow spots of merged mCherry/YFP-3D signal) inside the YFP⁺ cells. <b>(C)</b> Percentage of mCherry⁺ cells in the YFP⁺ cell population is shown (mean ± SEM). <b>(D)</b> YFP⁺ cell volume and sphericity are shown. Black, red and blue dots are from three different experiments. Horizontal lines represent mean values and SEM. <b>(E</b>-<b>H)</b> B6.CD11c-YFP mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs. At five days p.i., mice were i.v. infected with 1 × 10⁸ mature CMTPX-<i>Pc</i> iRBCs. After 15 min, mice were injected i.v. with a fluorescent anti-F4/80 mAb and the spleens were analyzed by CIVM. <b>(E)</b> Snapshots taken 30 min later show YFP⁺ cells (green), iRBCs (red), F4/80⁺ cells (blue) and merged F4/80⁺YFP⁺ cells (white) in the subcapsular RP. <b>(F)</b> Percentage of F4/80⁺ cells in the YFP⁺ cell population is shown (mean ± SEM). <b>(G)</b> Percentages of CMTPX⁺ cells in the F4/80⁺YFP⁺ and F4/80^-YFP⁺ cell subsets are shown (means ± SEM). <b>(H)</b> The relative proportions of F4/80⁺ and F4/80^- cells in the CMTPX⁺YFP⁺ cell population were calculated from the data obtained in <b>F</b> and <b>G</b> (means ± SEM). In <b>A, B</b> and <b>E</b>, the scale bars correspond to 50 µm. One representative experiment out of three (n = 2) is shown. Data were calculated using Imaris software. In <b>C, D, F, G</b> and <b>H</b>, data were calculated using Imaris software. Data from three experiments (n = 2) are shown. In <b>G</b>, significant differences (p < 0.05) between the indicated groups are designated by *.</p

<i>Ex vivo</i> analysis of iRBC uptake by splenic DCs soon after <i>Pc</i> infection.

<p><b>(A-C)</b> B6 mice were i.v. infected with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs (flow cytometry) or GFP-<i>Pc</i> iRBCs (immunofluorescence). Spleens were analyzed after 15 min and in non-infected controls. <b>(A)</b> A representative immunofluorescence image (10x magnification) shows the spleen of an infected B6 mouse. The staining of MOMA-1⁺ metallophilic macrophages and CD19⁺ B cells delineates the RP/MZ from the white pulp (WP). The lower panel details a merged GFP⁺CD11c⁺ cell in the RP. <b>(B)</b> Percentage of CD11c pixels colocalized with GFP pixels and percentages of GFP pixel distribution in the splenic RP/MZ and WP were obtained from immunofluorescence images (means ± SD). <b>(C)</b> Representative contour plots obtained by flow cytometry show CTV staining in the CD11c⁺ cells of naïve mice (-) and recently infected mice. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed, while excluding T cells, B cells, NK cells and RBCs. Data in contour plots show the percentages of CTV⁺ cells in the CD11c⁺ cell population. The numbers of total and CTV⁺CD11c⁺ cells per spleen in recently infected mice were calculated from the data obtained in contour plots (means ± SD). <b>(D-F)</b> B6.CD11c-YFP mice were i.v. infected with 1 × 10⁸ purified mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min and in non-infected controls. <b>(D)</b> Representative contour plots show CTV staining in the CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells. Data show the percentages of CTV⁺ cells in each population. <b>(E)</b> The numbers of total and CTV⁺ CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells per spleen were calculated from the data obtained in <b>D</b> (means ± SD). <b>(F)</b> The relative proportions of CD11c⁺ and F4/80⁺ cells in the CTV⁺YFP⁺ cell population were calculated from the data obtained in <b>E</b> (means ± SD). In <b>A</b>, the scale bars correspond to 50 µm. In <b>B</b>, data were calculated using FIJI software. In <b>B</b> and <b>E</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>A-F</b>, one representative experiment out of three (n = 3-4) is shown.</p

Effects of DC depletion on acute <i>Pc</i> malaria.

<p><b>(A-F)</b> B6 and B6.CD11c-DTR mice were treated with either DTx to deplete CD11c⁺ cells or PBS as a control. The mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs 24 h later. <b>(A)</b> Representative contour plots obtained 24 h after treatment by flow cytometry confirm the efficiency of DTx-induced depletion of splenic CD11c⁺I-A⁺ cells in B6.CD11c-DTR mice. Data show the percentages of CD11c⁺I-A⁺ cells in the splenocyte population. <b>(B)</b> Parasitemia curves are shown (means ± SD). <b>(C)</b> Variations in body weight relative to day 0 are shown (means ± SD). <b>(D)</b> Survival curves are shown. <b>(E)</b> Data show the numbers of CD3⁺CD4⁺ T cells per spleen at days 0 and 4 p.i. (means ± SD). <b>(F)</b> Data show the percentages of proliferating CFSE^lowCD4⁺ T cells and IFN-γ concentrations in the supernatants of spleen cell cultures stimulated for 72 h with iRBCs (means ± SD). In <b>B-D</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>E</b> and <b>F</b>, significant differences (p < 0.05) between all other groups are designated by *. In <b>A-F</b>, one representative experiment out of three (n = 5) is shown.</p

<i>Ex vivo</i> analysis of iRBC uptake by splenic DCs during pre-crisis.

<p><b>(A</b> and <b>B)</b> B6 mice were i.p. infected with 1 × 10⁶ GFP-<i>Pc</i> iRBCs. Spleens were analyzed after five days, at a time of day when mature parasite stages predominated. <b>(A)</b> A representative immunofluorescence image (10x magnification) represents the spleens of GFP-<i>Pc</i>-infected mice. The lower panel details a merged GFP⁺CD11c⁺ cell. <b>(B)</b> Percentage of CD11c pixels colocalized with GFP pixels in the immunofluorescence images is shown (mean ± SD). <b>(C</b> and <b>D)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs. At five days p.i., half of the B6 mice were i.v. re-infected with 1 × 10⁸ mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min. <b>(C)</b> Representative contour plots show CTV staining in the CD11c⁺ cells of <i>Pc</i>-infected mice that were re-infected or not with CTV-<i>Pc</i> iRBCs. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of CTV⁺ cells in the CD11c⁺ cell population. <b>(D)</b> Numbers of total and CTV⁺CD11c⁺ cells per spleen in re-infected mice were calculated from the data obtained in <b>C</b> (means ± SD). <b>(E</b> and <b>F)</b> B6 mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBCs. Spleens were analyzed after five days, at a time of day when mature parasite stages predominated. <b>(E)</b> Representative contour plots obtained by flow cytometry show GFP staining in the CD11c⁺ cells of mice that were infected with <i>Pc</i> iRBCs or GFP-<i>Pc</i> iRBCs. CD11c⁺ cells in the CD3^-CD19^-DX5^-Ter119^- cell population were analyzed. Data show the percentages of GFP⁺ cells in the CD11c⁺ cell population. <b>(F)</b> Numbers of total and GFP⁺CD11c⁺ cells per spleen in GFP-<i>Pc</i>-infected mice were calculated from the data obtained in <b>E</b>. <b>(G</b>-<b>I)</b> B6.CD11c-YFP mice were i.p. infected with 1 × 10⁶<i>Pc</i> iRBCs. At five days p.i., half of the B6.CD11c-YFP mice were i.v. re-infected with 1 × 10⁸ mature CTV-<i>Pc</i> iRBCs. Spleens were analyzed by flow cytometry after 15 min. <b>(G)</b> Representative contours plots show CTV staining in the CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells. Data show the percentages of CTV⁺ cells in each population. <b>(H)</b> The numbers of total and CTV⁺ CD11c⁺YFP⁺ and F4/80⁺YFP⁺ cells per spleen were calculated from the data obtained in <b>G</b> (means ± SD). <b>(I)</b> The relative proportions of CD11c⁺ and F4/80⁺ cells in the CTV⁺YFP⁺ cell population were calculated from the data obtained in <b>H</b> (means ± SD). In <b>A</b>, the scale bars correspond to 50 µm. In <b>B</b>, data were calculated using FIJI software. In<b>H</b>, significant differences (p < 0.05) between the indicated groups are designated by *. In <b>A-I</b>, one representative experiment out of three (n = 3-4) is shown.</p