Image_2_NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.tif

<p>Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4⁺Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4⁺ T cells secreting IL-2⁺, TNF-α⁺, or IFN-γ⁺, or a combination of two or the three cytokines (IL-2⁺TNF-α⁺IFN-γ⁺). The CD8⁺ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.</p

Image_1_NH36 and F3 Antigen-Primed Dendritic Cells Show Preserved Migrating Capabilities and CCR7 Expression and F3 Is Effective in Immunotherapy of Visceral Leishmaniasis.tif

<p>Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4⁺Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4⁺ T cells secreting IL-2⁺, TNF-α⁺, or IFN-γ⁺, or a combination of two or the three cytokines (IL-2⁺TNF-α⁺IFN-γ⁺). The CD8⁺ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.</p

Analysis of cell morphology and motility.

<p>Cell morphology (<b>A</b>); cell motility (<b>B</b>) and cell circularity (<b>C</b>) of A549 cells treated in NG, HG or OG conditions with (right panel) or without TGF-β (left panel). Representative photos are presented. Tracks of 50 random individual cells on gold solution (<b>D</b>) were measured using the Scion Image program represented as squared pixels, and are shown as mean ± SD. NG (white bar), HG (black bar) or OG (gray bar). *P≤0,005.</p

Effect of hyperglycemia onfFN biosynthesis.

<p>Western blot of A549 cell lysates cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar) medium with (+) or without (−) TGF-β, showing expression of total FN (first lane) and onfFN (second lane) (<b>A</b>). Signal intensities were normalized, with GAPDH as loading control, and relative intensities of total FN (<b>B</b>) and onfFN (<b>C</b>) are shown. (<b>D</b>) Western blot of A549 total FN (first lane) and onfFN (second lane) immunoprecipitated from cell lysates by FDC-6 mAbs, submitted (+) or not (−) to the remotion of <i>O</i>-glycosylation. Human plasma FN (pFN, 0.5 µg) was used as control. qRT-PCR analysis of gene that codifies IIICS domain of onfFN (<b>E</b>) and GalNacT6 (<b>F</b>) respectively. Graph shows one of three independent experiments as mean ± SD. * <i>P≤0.005</i>. Effect of anti-TGF-β blocking antibody in the expression of total FN (first line) and onfFN (second line) (<b>G</b>).</p

TGF-β production and expression of mesenchymal markers in A549 cells.

<p>(<b>A</b>) Quantification of TGF-β in supernatants from cells cultured for 48 h in NG (white bar), HG (black bar) or OG (gray bar). (<b>B</b>) Western blot of cell lysates loads analyzing expression levels of N-cad, (first lane) and Vimentin (second lane) in cells cultured in NG (white bar), HG (black bar) or OG (gray bar) conditions with or without 2 ng/mL TGF-β. Signal intensities were normalized, with GAPDH as loading control, and relative intensities of N-cad (<b>C</b>) and Vimentin (<b>D</b>) are shown. The results are representative of 3 independent experiments. Quantitative analyses are shown as mean ± standard deviation. P values were calculated using the Student's t test. *<i>P≤0.01</i>; **P<i>≤0.005</i>.</p

Inhibition of CD4⁺ T cell proliferation is partially recovered upon neuraminidase-treatment of <i>T. cruzi</i> mucin.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 for 72 hr, in the presence or absence of increasing concentrations of native or desialylated Tc Muc (10 and 20 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between native or desialylated Tc Muc treatment <i>versus</i> anti-CD3 stimulated positive controls are significant (<i>P≤</i>0.05). #The inhibition of proliferation by Tc Muc was partially recovered when <i>T. cruzi</i> mucin was desialylated by previous treatment with neuraminidase (<i>P</i> = 0.0023). Results are the means ±SE of triplicate cultures. This experiment was repeated three times, with similar results each time.</p

Tc Muc induced G1 cell cycle arrest of CD4⁺ T cells correlates with upregulation of Cyclin D3 and downregulation of p27^kip1.

<p>Purified CD4⁺ T cells from naïve spleens were stimulated with plate bound anti-CD3 in the presence or absence of native or desialylated <i>T. cruzi</i> mucins (20 µg/ml). After 3 days, cell cycle analysis (A) was evaluated by flow cytometry based on propidium iodide (PI) intercalation into the cellular chromatin (for details see Materials and methods). The histograms represent the fluorescence intensity of PI for the indicated groups. (B) Flow cytometry cell cycle analysis revealed that the population of cells in the S and G2/M phase was remarkably decreased by Tc muc (<i>P</i>≤0.05). For determination of the cell cycle checkpoint regulators, cells were harvested after 3 days and whole-cell lysates were prepared for immunoblotting with specific atibodies against cyclin D3, p27^kip1, and actin antibodies used to assure uniform loading (bottom row). Optical densitometry of the western blots used NIH Image software, where cyclin D3 and p27^kip1 expression was normalized with the actin expression. (C) Expression of cyclin D3 was increased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this increase was not observed when stimulation was done in the presence of Tc Muc (<i>P</i> = 0.0383); *the sialylation abolished the property of Tc Muc to induce downmodulation of cyclin D3 expression (<i>P = </i>0.0067). Expression of p27^kip1 was decreased in anti-CD3 activated CD4⁺ T cells as compared to controls (<i>P≤</i>0.05); this decrease was not observed when stimulation was done in the presence of Tc Muc (<i>P</i>≤0.05); # the sialylation abolished the property of Tc Muc to induce upregulation of p27^kip1 expression (<i>P≤</i>0.05). These data are representative of two independent experiments.</p

Splenocytes from <i>T. cruzi</i> infected mice treated with Tc mucin produce low levels of IFN-γ.

<p>BALB/c mice were infected <i>i.p.</i> with 2×10⁵ chemically induced metacyclic forms of <i>Trypanosoma cruzi</i> Dm28c clone. The mice received <i>i.p.</i> injections of Tc Muc (20 µg/mouse) or PBS on alternate days starting at day of infection. Non-infected mice were used as control group. Twenty four days after infection, purified splenocytes were stimulated with PMA and ionomycin, as described in the Methods section, and supernatants were harvested at 24 h for determination of (<b>A</b>) IFN-γ and (<b>B</b>) TNFα by ELISA. The <i>y</i>-axis represents the levels of cytokines, detected by a specific ELISA assay, expressed in ng/ml. Asterisks represent statistical significance (p<0.05) as determined by the Student t test. To access the T cell activation profile CD69 expression on both CD4⁺ (<b>D</b>) and CD8⁺ (<b>E</b>) T cells were analysed by FACS analysis; the frequency of IFN-γ producing T cells from splenocytes polyclonally stimulated with PMA/ionomycin, and the percentages of both IFN-γ producing CD4⁺ T cells (<b>F</b>) and CD8⁺ T cells (<b>G</b>)<b>,</b> were determined by intracellular cytokine FACS-staining. #Infected group are statistically different from non-infected control mice (<i>P</i>≤0.05). Asterisks represent statistical differences between Tc mucin treated <i>versus</i> non-treated mice from infected groups as determined by the Student t test (<i>P</i>≤0.05). All experiments were repeated at least 3 times.</p

Tc Muc inhibits anti-CD3 restimulation of activated CD4⁺ T cells.

<p>Naϊve splenic CD4⁺ T cells were stimulated in 48-well culture plates coated with anti-CD3 (5 µg/mL) in the presence or absence of Tc Muc (20 µg/mL) or the same amount of the bovine mucin as control. After 72 hr of stimulation, activated CD4⁺ T cells were harvested and restimulated for an additional 3 days with plate-bound anti-CD3 in the presence or absence of the TcMuc (20 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. *Differences between Tc mucin treatment <i>versus</i> anti-CD3 stimulated positive control are significant (<i>P≤</i>0.05). The data represented above are representative of one of three experiments with similar results.</p

Tc Muc inhibits CD4⁺ T cell proliferation.

<p>(<b>A</b>) Purified CD4⁺ T cells from naïve spleens were stimulated with pre-coated anti-CD3 for 72 hr, in the presence or absence of increasing concentrations of Tc Muc (10, 20 and 50 µg/mL). Proliferation was measured 72 h after stimulation by [³H]thymidine incorporation. (<b>B</b>) The inhibition of proliferation by Tc mucin was not observed when control mucin derived from bovine submaxillary glands was used, nor was it reverted by addition of exogenous IL-2 when naïve splenic purified CD4⁺ T cells were stimulated with pre-coated anti-CD3 for 72 hr. Results are the means ±SE of triplicate cultures of three different experiments. *Differences between Tc mucin treatment <i>versus</i> anti-CD3 stimulated positive control are significant (<i>P≤</i>0.05).</p