ArtinM offers new perspectives in the development of antifungal therapy

The thermally dimorphic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM), the most frequent systemic mycosis that affects the rural populations in Latin America. Despite significant developments in antifungal chemotherapy, its efficacy remains limited since drug therapy is prolonged and associated with toxic side effects and relapses. In response to these challenges, it is now recognized that several aspects of antifungal immunity can be modulated to better deal with fungal infections. A common idea for halting fungal infections has been the need to activate a cell-based, pro-inflammatory Th1 immune response to improve the fungal elimination. ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has the property of modulating immunity against several intracellular pathogens. Here, we review the immunomodulatory activity of ArtinM during experimental PCM in mice. Both prophylactic and therapeutic protocols of ArtinM administration promotes a Th1 immune response balanced by IL-10, which outstandingly reduces the fungal load in organs of the treated mice while maintaining a controlled inflammation at the site of infection. A carbohydrate recognition-based interaction of ArtinM with Toll-like receptor 2 (TLR2) accounts for initiating the immunomodulatory effect of the lectin. The precise identification of the TLR2 N-glycan(s) targeted by ArtinM may support novel basis for the development of antifungal therapy

Vaccination of Mice with Salmonella Expressing VapA: Mucosal and Systemic Th1 Responses Provide Protection against Rhodococcus equi Infection

Conventional vaccines to prevent the pneumonia caused by Rhodococcus equi have not been successful. We have recently demonstrated that immunization with Salmonella enterica Typhimurium expressing the VapA antigen protects mice against R. equi infection. We now report that oral vaccination of mice with this recombinant strain results in high and persistent fecal levels of antigen-specific IgA, and specific proliferation of the spleen cells of immunized mice in response to the in vitro stimulation with R. equi antigen. After in vitro stimulation, spleen cells of immunized mice produce high levels of Th1 cytokines and show a prominent mRNA expression of the Th1 transcription factor T-bet, in detriment of the Th2 transcription factor GATA-3. Following R. equi challenge, a high H2O2, NO, IL-12, and IFN-γ content is detected in the organs of immunized mice. On the other hand, TNF-α and IL-4 levels are markedly lower in the organs of vaccinated mice, compared with the non-vaccinated ones. The IL-10 content and the mRNA transcription level of TGF-β are also higher in the organs of immunized mice. A greater incidence of CD4+ and CD8+ T cells and B lymphocytes is verified in vaccinated mice. However, there is no difference between vaccinated and non-vaccinated mice in terms of the frequency of CD4+CD25+Foxp3+ T cells. Finally, we show that the vaccination confers a long-term protection against R. equi infection. Altogether, these data indicate that the oral vaccination of mice with S. enterica Typhimurium expressing VapA induces specific and long-lasting humoral and cellular responses against the pathogen, which are appropriately regulated and allow tissue integrity after challenge

Immunological Basis for the Gender Differences in Murine Paracoccidioides brasiliensis Infection

This study aimed to investigate the immunological mechanisms involved in the gender distinct incidence of paracoccidioidomycosis (pcm), an endemic systemic mycosis in Latin America, which is at least 10 times more frequent in men than in women. Then, we compared the immune response of male and female mice to Paracoccidioides brasiliensis infection, as well as the influence in the gender differences exerted by paracoccin, a P. brasiliensis component with carbohydrate recognition property. High production of Th1 cytokines and T-bet expression have been detected in the paracoccin stimulated cultures of spleen cells from infected female mice. In contrast, in similar experimental conditions, cells from infected males produced higher levels of the Th2 cytokines and expressed GATA-3. Macrophages from male and female mice when stimulated with paracoccin displayed similar phagocytic capability, while fungicidal activity was two times more efficiently performed by macrophages from female mice, a fact that was associated with 50% higher levels of nitric oxide production. In order to evaluate the role of sexual hormones in the observed gender distinction, we have utilized mice that have been submitted to gonadectomy followed by inverse hormonal reconstitution. Spleen cells derived from castrated males reconstituted with estradiol have produced higher levels of IFN-γ (1291±15 pg/mL) and lower levels of IL-10 (494±38 pg/mL), than normal male in response to paracoccin stimulus. In contrast, spleen cells from castrated female mice that had been treated with testosterone produced more IL-10 (1284±36 pg/mL) and less IFN-γ (587±14 pg/mL) than cells from normal female. In conclusion, our results reveal that the sexual hormones had a profound effect on the biology of immune cells, and estradiol favours protective responses to P. brasiliensis infection. In addition, fungal components, such as paracoccin, may provide additional support to the gender dimorphic immunity that marks P. brasiliensis infection

Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.FAPESP[00/09333-2]FAPESP[06/60642-2]FAPESP[02/12725-5]FAPESP[05/00303-7]CNPq[350418/00-4]CAPE

Recall immune response induced by immunization with <i>Salmonella</i> expressing the VapA protein.

<p>BALB/c mice were orally immunized with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i>, or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 or PBS on days 0 and 14. Five months after the last immunization, all mice were intravenously challenged with 4×10⁶ CFU of virulent <i>R. equi</i>. On day 8 after infection, the spleen (<b>A</b> and <b>B</b>) and liver (not shown) were harvested and homogenized for bacterial burdens and NO quantification. Results are expressed as the mean of five mice per group ± SD. The experiment was performed twice with similar results. *p<0.05, **p<0.01, and ***p<0.001 compared to the control groups.</p

Transcription levels of T-bet and GATA-3 mRNA in spleen cells of mice vaccinated with <i>Salmonella</i> expressing the VapA protein.

<p>(A–B) Groups of BALB/c mice were orally immunized on days 0 and 14 with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i>, or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 or PBS. On day 14 after the second immunization, spleen cells were harvested and the total RNA was extracted and assessed by real-time PCR. (C–D) Immunized (pYA3137<i>vapA</i>) and control (pYA3137 or PBS) mice were intravenously challenged with 4×10⁶ CFU of virulent <i>R. equi</i> strain 14 days after the last immunization. Spleen cells were collected 8 days post-infection, and the total RNA was extracted and assessed by real-time PCR. The cDNA contents were normalized on the basis of predetermined β-actin levels. Data are mean ± SD of triplicate samples in one of the three similar experiments. *p<0.05 compared to the two control groups; ⁺⁺p<0.01 compared to the PBS control group.</p

Alteration of lymphocyte subpopulation in the spleen of mice.

<p>BALB/c mice orally administered with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i>, <i>S. enterica</i> Typhimurium χ3987-pYA3137, or PBS, on days 0 and 14, were intravenously challenged with 4×10⁶ CFU of virulent <i>R. equi</i> strain 14 days after the last immunization. Spleen cells were harvested 8 days post-infection and analyzed by flow cytometry. The cells were stained with specific anti-CD19 (<b>A</b>), anti-CD3 plus anti-CD4 (<b>B</b>), and anti-CD3 plus anti-CD8 (<b>C</b>) monoclonal antibodies. (<b>D</b>) CD4⁺ spleen T cells stained with specific anti-CD25 plus anti-Foxp-3 monoclonal antibodies. The average of the percentage of spleen cells positive for the respective CD markers in five samples is shown. Similar results were obtained in three independent experiments. (<b>E</b>) Total RNA was extracted from spleen cells harvested 8 days post-infection and assessed by real-time PCR for detection of TGF-β mRNA. The cDNA contents were normalized on the basis of predetermined β-actin levels. Data are mean ± SD of triplicate samples in one of the three similar experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups.</p

Humoral and cellular immune responses elicited by vaccination with <i>Salmonella</i> expressing the VapA protein.

<p>(A) Fecal IgA antibody response. Groups of BALB/c mice were orally immunized on days 0 and 14 with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i> (closed triangles/dashed line), or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). Fecal extracts were obtained on days 7, 14, 21, 28, 35, and 42 after the second immunization, for measurement of specific IgA by ELISA. The <i>R. equi</i> surface antigen (APTX) was used as the coating antigen (1 µg/mL). Results are expressed as the mean of OD values of five mice per group and are a representative experiment of three assays. ***p<0.001 compared to control groups. (B) Lymphocyte proliferative response. Spleen cells were harvested from immunized (pYA3137<i>vapA</i>) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or Concanavalin A (2 µg/mL) for 72 h. Cell proliferation was measured by [³H]-thymidine incorporation assay. Each bar represents the average of five mice per group ± SD and is representative of four independent experiments. ***p<0.001 compared to control groups. (C–F) Cytokines production. Whole spleen cells were collected from immunized (pYA3137<i>vapA</i>) and control mice (pYA3137 or PBS) on day 14 after the last immunization and cultured in the presence of medium alone, APTX (5 µg/mL), or LPS (1 µg/mL) plus IFN-γ (1 ng/mL) for 48 h. The concentration of IL-12 (C), IFN-γ (D), TNF-α (E), IL-10 (F), and IL-4 (not shown) cytokines in the supernatants of the cell cultures was measured by ELISA. Each bar represents the mean ± SD of triplicate samples and is a representative experiment of three assays. ***p<0.001 compared to the two control groups; ⁺⁺⁺p<0.001 compared to PBS-inoculated mice.</p

Cytokine levels in the organs of mice after <i>R. equi</i> infection.

<p>BALB/c mice were orally immunized on days 0 and 14 with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i> (closed triangles/dashed line), or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). All mice were intravenously challenged with 4×10⁶ CFU of <i>R. equi</i> ATCC33701 14 days after the last immunization. The lung (<b>A–E</b>), liver (<b>F–J</b>), and spleen (<b>K–O</b>) were harvested 2, 4, 8, and 10 days post-infection and homogenized for IL-12 (<b>A</b>, <b>F</b>, and <b>K</b>), IFN-γ (<b>B</b>, <b>G</b>, and <b>L</b>), TNF-α (<b>C</b>, <b>H</b>, and <b>M</b>), IL-10 (<b>D</b>, <b>I</b>, and <b>N</b>), and IL-4 (<b>E</b>, <b>J</b>, and <b>O</b>) detection. Results are expressed as means of five mice per group ± SD and are representative of three experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups; and <b>⁺</b>p<0.05, <b>⁺⁺</b>p<0.01, and <b>⁺⁺⁺</b>p<0.001 compared to the PBS control group.</p

Nitrite and hydrogen peroxide production in the organs of mice following <i>R. equi</i> infection.

<p>BALB/c mice were orally immunized on days 0 and 14 with <i>S. enterica</i> Typhimurium χ3987-pYA3137<i>vapA</i> (closed triangles/dashed line), or inoculated with either <i>S. enterica</i> Typhimurium χ3987-pYA3137 (closed squares) or PBS (open circles). All mice were intravenously challenged with 4×10⁶ CFU of <i>R. equi</i> ATCC33701 14 days after the last immunization. On days 2, 4, 8, and 10 after infection, the lung, liver, and spleen were harvested and homogenized for NO (<b>A</b>, <b>C</b>, and <b>E</b>) and H₂O₂ (<b>B</b>, <b>D</b>, and <b>F</b>) detection. The endogenous H₂O₂ release was measured by the horseradish peroxidase-dependent phenol red oxidation methods, whereas the nitrite levels were measured by Griess reaction. Data are expressed as means of five mice per group ± SD and are representative of three separate experiments. *p<0.05, **p<0.01, and ***p<0.001 compared to the two control groups.</p