Nucleosides modulate <i>in vivo</i> expression of suppressive genes and mediate immunoregulatory effects by IL-10 during <i>Leishmania amazonensis</i> infection.

<p>Expression levels of IDO (<i>A</i>), arginase-1 (<i>B</i>), COX₂ (<i>C</i>), or IL-10 (<i>D</i>) mRNA into ears from C57BL/6 mice that were infected or not (naïve) with 10⁶<i>L</i>. <i>amazonensis</i> promastigotes in the presence of ADO+AMP or PBS at 11 weeks post infection. <i>E</i>, IL-10 production in cultured draining lymph node cells from ADO+AMP or PBS-treated mice was determined by ELISA. Data are shown as the means ± SEM of two separate experiments; each experiment was performed with four (naïve) or six (infected) mice per group (<i>n</i> = 4–6). *, <i>p</i><0.05 relative to naïve mice; ^#, <i>p</i><0.05 compared with the PBS-treated group. Ear lesion (<i>F</i>, <i>G</i>) and parasite burden (<i>H</i>)—by a limiting-dilution assay—in WT and IL-10^-/-mice at 11 weeks p.i. were determined. Data are shown as the means ± SEM of two separate experiments; each experiment was performed with five mice per group (<i>n</i> = 5). <i>G</i>, representative photographs of ears at 11 weeks post infection.</p

Salivary nucleosides induce iTreg in an A2_AR-dependent manner.

<p>In <i>A</i>, ears from mice infected with <i>Leishmania amazonensis</i> (10⁶ parasites/ ear) in the presence of ADO+AMP (■) or PBS (□) were harvested at 11 weeks post infection for quantification of A2_AR and A2_BR. <i>A</i>, mRNA expression. Data are shown as the means ± SEM from one of two independent experiments that were each performed with four mice per group (<i>n</i> = 4 per group). *, <i>p</i><0.05 relative to naïve mice. ^#, <i>p</i><0.05 relative to the PBS group. Ear lesion (<i>B</i>) and parasite burden in ear and draining lymph nodes (<i>C</i>) by a limiting-dilution assay in WT and A2_AR^-/-mice at 11 weeks post infection were determined. Expression levels of CD39, CD73, and CD103 (<i>E</i> and <i>G</i>) analyzed in CD4⁺CD25⁺ (<i>D</i>) and CD4⁺CD25^- (<i>E</i>) population. Data are shown as the means ± SEM of two separate experiments; each experiment was performed with five (burden parasite and flow cytometry) to ten (ear lesion) mice per group (<i>n</i> = 5–10).</p

Salivary nucleosides induce expression of a regulatory T cell profile in effector T cells.

<p>Ear cells from mice infected with <i>Leishmania amazonensis</i> in the presence of ADO+AMP (■) or PBS (□) were harvested at 11 weeks post infection. <i>A</i>, Lymphocytes from the ears were phenotyped by flow cytometry with FITC-conjugated anti-CD3, PerCP-conjugated anti-CD4, and PE-Cy7-conjugated anti-CD25 Lymphocytes were selected with CD4⁺CD25⁺ (<i>B</i>) or CD4⁺CD25^- (<i>C</i>) gates, and these populations were subsequently analyzed for expression of FoxP3, CD39, CD73, and CD103. CD4⁺CD25^-lymphocytes (2 × 10⁶ cells/ml) were isolated from draining lymph nodes of PBS- (□) or ADO+AMP- (■) infected mice at 8 weeks post infection. The lymphocytes were re-stimulated in vitro with αCD3 (2 μg/ml) plus αCD28 (1 μg/ml) for 96 h. CD4⁺CD25⁺ cells were added to some of the CD4⁺CD25^-culture wells. <i>D</i>, Suppressive activity was determined by ELISA to measure IL-10 secretion in the culture supernatants. Data are expressed as the means ± SEM and are representative of two independent experiments that were each performed with four mice per group (<i>n</i> = 4). ^#, <i>p</i><0.05 relative to the PBS-treated group.</p

In vitro and in vivo effects of ADO+AMP on dendritic cells (DC) during <i>Leishmania amazonensis</i> infection.

<p>Bone marrow-derived cells (BMDC) from wild type or IL-10^-/-mice were incubated without (□) or with (■) ADO+AMP for 1 h prior to infection with GFP-expressing <i>L</i>. <i>amazonensis</i>. Parasite growth in BMDCs was determined by GFP positivity and measured by flow cytometry. <i>A</i>/<i>C</i>, Bars display the relative GFP positivity and are representative of three independent experiments that were each performed in quadruplicate (<i>n</i> = 4). ^#, <i>p</i><0.05 relative to uninfected cells; *, <i>p</i><0.05 relative to the PBS group. <i>B/D</i>, Levels of cytokines TNF-α and IL-10 in the culture supernatants were measured by ELISA. BMDCs (1x 10⁶ cells/ml) were incubated ± ADO+AMP for 1 h. <i>E</i>, Cells were harvested 24 h after <i>L</i>. <i>amazonensis</i> infection for quantification of IL-10, COX₂, TGF-β and IDO mRNA expression. Data are shown as the means ± SEM from one of three independent experiments that were performed in quadruplicate (n = 4 per group). ^#, <i>p</i><0.05 relative to the control group; *, <i>p</i><0.05 relative to the parasite-infected group. PBS- or ADO+AMP-infected mice were euthanized at 8 wk post infection, the draining lymph nodes were harvested, and the cells were labeled with FITC-conjugated anti-CD11c or PE-conjugated anti-MHC class II mAbs to detect DC surface markers. <i>F</i>, Representative histograms of DCs from PBS- and ADO+AMP-infected mice are shown in each box and bars display the relative mean fluorescence intensity, and data are shown as the means ± SEM; <i>n</i> = 5. ^#, <i>p</i><0.05 relative to naïve mice; *, <i>p</i><0.05 relative to the ADO+AMP group.</p

Deamination of salivary nucleosides reversed the immunosuppressive effect of salivary gland extract (SGE).

<p>C57BL/6 mice were infected with <i>Leishmania amazonensis</i> (1 × 10⁶ parasites/ear) in the presence of PBS (Ο) or SGE (1 gland/ear) (●). In some groups, SGE (■) or PBS (□) had been previously incubated with ADA (4.3 U) for 3 h before infection. <i>A</i>, Ear lesion growth course was determined by difference in thickness between the infected and opposite uninfected ear for each mouse. Measurements are reported in millimeters (mm), and <i>n</i> = 4 mice per group. Parasite burdens in the ears and draining lymph nodes (LN) (<i>B</i>) at 12 weeks post infection were determined by a limiting-dilution assay. Data are shown as the means ± SEM of three separate experiments; each experiment was performed with five mice per group (<i>n</i> = 5). *, <i>p</i><0.05 relative to the PBS-treated group; ^#, <i>p</i><0.05 compared with the ADA or ADA-SGE-treated groups.</p

ADO+AMP exacerbates <i>Leishmania amazonensis</i> infection in vivo.

<p>C57BL/6 or BALB/c mice were infected with 10⁶<i>L</i>. <i>amazonensis</i> promastigotes in the presence of ADO+AMP (●) or PBS (Ο). The infection course was monitored weekly by measuring sizes of ear lesions with metric calipers. In <i>A</i> and <i>D</i>, lesion size was determined by the difference in thickness between the infected ear and the opposite uninfected ear on each mouse. Measurements are reported in millimeters (mm); <i>n</i> = 4 mice per group. Data are shown as the mean ± SEM and are representative of three different experiments. *, <i>p</i><0.05 relative to the PBS-treated group. Parasite burdens in ears (<i>B</i>) and draining lymph nodes (<i>E</i>) at 11 weeks post infection were determined by a limiting-dilution assay. Data are shown as the means ± SEM of three separate experiments; each experiment was performed with five mice per group <i>n</i> = 5. <i>C</i> and <i>F</i> are representative photographs of mouse ears at 11 weeks post infection. <i>In</i> G and H, BALB/c mice were infected with 10³ metacyclic promastigotes forms of <i>L</i>. <i>amazonensis</i> in the presence of ADO+AMP (●) or PBS (Ο). In I-J, as in D–E, but using infection with 10⁶ stationary-phase <i>L</i>. <i>major</i> promastigotes. Data are shown as the means ± EPM performed with five to six mice per group <i>n</i> = 5–6.</p

tDC generated by nucleosides present ability to induce regulatory profile on CD4⁺CD25^- cells.

<p>Bone marrow-derived cells (BMDC) treated with ADO or PBS and stimulated with LPS (50 ng/ml) were co-cultured with CD4⁺CD25^-under Treg polarizing conditions at the ratio of 1:10 (BMDC:T cells). Natural Treg (nTreg, CD4⁺CD25⁺) or Th0 (CD4⁺CD25^-) were used as positive and negative differentiation control. Representative histograms of CD39, CD73, CD103, and FoxP3 are shown in each box. Bars display the relative mean fluorescence intensity, and the results are expressed as the mean ± SEM obtained from one of three independent experiments made in triplicate (<i>n</i> = 3 per group).*, <i>p</i><0.05 relative to the BMDC/PBS group.</p

Anti-IL-4 mAb treatment reduces parasite loads in LTCP393(R)-inoculated BALB/c mice lesions.

<p>The animals were inoculated at right ear dermis with 1×10⁶ stationary phase promastigotes of <i>L. braziliensis</i>, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars), treated with anti-IL-4 monoclonal antibody (hatched bars) or rat IgG (full bars), and the parasite load estimated at 7 weeks post infection in the ears (A) and draining lymph nodes (B). Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. Statistical analysis: ANOVA and Tukey multiple comparision test. *p<0,05.</p

Flow cytometry of infected BALB/c mice ears inflammatory infiltrate.

<p>The animals were inoculated with 1×10⁶ stationary phase <i>L. braziliensis</i> promastigotes, isolates LTCP393(R) (black bars) or LTCP15171(S) (white bars). Analyses were conducted at 3, 5, 7 and 12 weeks post infection, FoxP3, CD103, CTLA-4 and GITR expression on CD4⁺CD25⁺ cells were evaluated at 7 and 12 weeks post infection. Results are expressed as total number of cells in the lesion (A, B, C and D), and as percentage of cells inside CD4⁺CD25⁺ gate (E and F). Results are expressed as mean ± standard error of mean, and are representative of 3 independent experiments. *p<0,05.</p

Anti-IL-4 mAb treatment reduces lesion thickness in LTCP393(R)-inoculated BALB/c mice.

<p>The animals were inoculated with 1×10⁶<i>L. braziliensis</i> stationary phase promastigotes, isolates (A) LTCP393(R) or (B) LTCP15171(S), in the right ear dermis, treated with anti-IL-4 monoclonal antibody (black signs) or rat IgG (white signs), and the course of lesion development was monitored for 7 weeks. Lesion thickness was determined as the difference between the infected ear and the contralateral one, noninfected. Results are expressed as mean ± standard error of mean, and are representative of 2 independent experiments. *p<0,05.</p