Inhibition of NADPH oxidase decreases the oxidative burst.

<p>Monocytes from CL patients (n = 15) and HS individuals (n = 7) were preincubated with either DPI (10mM), an inhibitor of the NADPH oxidase, or L-NMMA (1mM), an iNOS inhibitor, for 10 minutes. The monocytes were pre-incubated with DHR (10 minutes) and infected with <i>L</i>.<i>braziliensis</i> promastigotes (5:1cells) or stimulated with PMA (1 ug/mL) for 25 minutes. Cells were stained with anti-CD14. Data were collected using flow cytometry and analyzed using FLOWJO software. (A) Representative contour plots. (B) The data represent the median of mean intensity of fluorescence (MIF) of DHR expression by monocytes from CL patients and HS individuals (C). Statistical analysis was performing using ANOVA with Bonferoni´s pos-test and Manny Whitney test. The results were considered significant with a p< 0.05 (** p<0.01; ***p<0.001).</p

<i>L</i>.<i>braziliensis</i> induces high levels of oxidative burst in monocytes from CL patients.

Monocytes from CL patients (n = 13) and HS individuals (n = 5) were treated with DHR (10ng/mL-10 min) and infected with L.braziliensis promastigotes for 25 minutes at a ratio of 5:1cells. PMA (1ug/ml) was used as positive control. Cells were stained with anti-CD14. Data were collected using flow cytometry and analyzed using FLOWJO software. (A) Representative gating strategy on CD14+ and DHR expression in monocytes from one CL patient (B) The data represent the median of mean intensity of fluorescence (MIF) of oxidative burst production by CL and HS monocytes. (C) The ex vivo expression of TLR2 and TLR4 was evaluated on CD14+ cells. Data were collected using flow cytometry and analyzed using FLOWJO software. Statistical analysis was performed using the Manny Whitney test and results were considered significant with a **p<0.01, ***p<0.001.</p

The role of NO and NO in the control of <i>L</i>.<i>braziliensis</i> infection by monocytes from CL patients.

<p>Monocytes from CL patients (n = 9) and HS individuals (n = 6) were treated with inhibitor of the NADPH oxidase (DPI-10mM) or with an iNOS inhibitor (L-NMMA-1mM) for 10 minutes and infected with <i>L</i>.<i>braziliensis</i> at a 5:1. After 72 hours, the medium of monocytes culture was replaced by Schneider’s medium and after 5 days the number of viable promastigotes was estimated. Statistical analysis was performed using the Manny Whitney test. (*** p < 0.001).</p

Phagocytosis and the killing of <i>L</i>. <i>braziliensis</i> by monocytes from CL patients.

<p>Monocytes from CL patients (n = 9) and HS individuals (n = 6) were infected with <i>L</i>. <i>braziliensis</i> promastigotes at a 5:1 ratio for 2, 24, 48 and 72 hours. The number of infected cells (A) and the number of intracellular parasites (B) were determined by microscopic evaluation after May-Grunwald-Giemsa staining from cytocentrifuge preparations. Monocytes were preincubated with either DPI (10mM) or L-NMMA (1mM), for 10 minutes and were infected with <i>L</i>. <i>braziliensis</i> promastigotes at a 5:1 ratio for 72 hours. (C) The number of infected cells. (D) The number of intracellular parasites. Statistical analysis was performed using the Kruskal-Wallis test (* p < 0.05, ** p < 0.01).</p

Oxidative burst production before and after therapy and cure of CL patients.

<p>Production of burst oxidative, NO and ROS by monocytes from CL patients (n = 6) after infection with <i>L</i>.<i>braziliensis</i> promastigotes or upon PMA stimulus, were determined before and after therapy (i.v. pentavalent antimonial, 20mg/kg body weight daily for 20 days) and cure of cutaneous leishmaniasis. The data represent the median of mean intensity of fluorescence (MIF) of oxidative burst production (A), frequency of NO production (B) and frequency of ROS production (C). Statistical analysis was performed using Wilcoxon test and results were considered significant (p<0.05).</p

Clinical and epidemiological characteristics of cutaneous leishmaniasis patients (CL) and Healthy Subjects (HS).

<p>Clinical and epidemiological characteristics of cutaneous leishmaniasis patients (CL) and Healthy Subjects (HS).</p

Monocytes from CL patients produced high levels of reactive oxygen species after infection with <i>L</i>.<i>braziliensis</i>.

<p>Monocytes from CL patients (n = 13) and HS individuals (n = 7) were stained with DAF-FM diacetate (NO probe, 10mM) and CMH-2DCFDA (ROS probe, 1 μM) for 10 minutes, infected with <i>L</i>.<i>braziliensis</i> promastigotes for 25 minutes at a ratio of 5:1cells, and stained with anti-CD14. PMA was used as positive control. Data were collected using flow cytometry and analyzed using FLOWJO software (A). Representative histograms of ROS production, (B) Frequency of <i>L</i>.<i>braziliensis</i>-infected monocytes expressing ROS, (C) Representative histograms of NO production, (D) Frequency of <i>L</i>.<i>braziliensis</i>-infected monocytes expressing ROS. Statistical analysis was performing using ANOVA with Bonferoni´s pos-test and Manny Whitney test. The results were considered significant with a p< 0.05 (*p<0.05).</p

Correlation between NO and ROS production by monocytes and lesion size of CL patients.

<p>Monocytes from CL patients (n = 8) were treated with DAF-FM diacetate (NO probe, 10mM) or CMH-2DCFDA (ROS probe, 1 μM) for 10 minutes and infected with <i>L</i>.<i>braziliensis</i> promastigotes for 25 minutes at a ratio of 5:1cells as described in materials and methods. Production of NO and ROS was evaluated by flow cytometry. (A) Correlation between NO production (%) and lesion size (mm). (B) Correlation between ROS production (%) and lesion size (mm). Statistical analysis was performed using the Pearson correlation.</p

IL-17 neutralization reverses the pathology induced after blockade of IL-10R signaling.

<p>C57BL/6, or <i>Il-10^−/−</i> mice were infected intradermally with <i>L. major</i> metacyclic promastigotes. Either isotype, anti-IL-10R or anti-IL-17 or both mAbs were administrated at day-1 and twice weekly during 4 weeks. Lesion development was assessed by measuring ear thickness (A, F). Values represent mean induration in mm (mean ± SEM) of 5 mice per group. Numbers of parasites in ear lesions were quantified using limiting dilution assays at 6 and 5 wk after infection (B, F). Mean ± SEM parasite numbers are shown as individual parasite counts per ear. Bar graph showing number of Ly6G⁺ cells per ear (C). Level of cytokines was measured in supernatants from <i>in vitro</i> stimulated draining lymph node cells with <i>L. major</i> FTAg (D, E) (ND: not detectable). (G) H&E staining of histological sections of paraffin-embedded ears at 6 wks of infection showing epithelial hyperplasia (head arrow), leukocyte infiltration in epidermis (black arrows) and localized neutrophils infiltration in deep dermis marked as * in the anti-IL-10R treated sections (Bars, 100 µm; EP: epidermis, D: dermis). The data shown are from one experiment and are representative of at least three experiments (*, <i>p</i><0.05).</p

IL-10R signaling blockade increases IFN-γ and IL-17A production.

<p>Cells from naïve or 5 week infected draining lymph nodes or from ears were analyzed for IFN-γ, IL-17A and IL-4 production. Antigen-specific cytokine release by draining lymph node cells from 5 week infected mice was determined by ELISA after restimulation with <i>L. major</i> freeze-thawed antigen (FTAg). Values represent mean ± SEM of 5 mice per group (ND: not detectable) (A). Cells from the ears of 5 week infected mice were stimulated with PMA and ionomycin for 4 h and analyzed for intracellular cytokine production. Contour plots and bar graphs show the frequency and number of IFN-γ (B) and IL-17A (C) in gated CD4 T cells at 1 (top) and 5 (bottom) weeks after infection. Frequency of double positive IL-17A/IL-10 (D) or IL-17A/IFN-γ (E) producer CD4 T cells at 1 or 5 weeks after infection, respectively. Quadrant values are the percentages from total gated population. Numbers between parentheses indicate the mean fluorescence intensity (MFI) for the IL-17 within the CD4 T cells. The data shown are from one experiment and are representative of at least three experiments (*, <i>p</i><0.05).</p