Extracellular vesicles produced by immunomodulatory cells harboring OX40 ligand and 4-1BB ligand enhance antitumor immunity.

Genetically modified tumor cells harboring immunomodulators may be used as therapeutic vaccines to stimulate antitumor immunity. The therapeutic benefit of these tumor vaccines is extensively investigated and mechanisms by which they boost antitumor response may be further explored. Tumor cells are large secretors of extracellular vesicles (EVs). These EVs are able to vehiculate RNA and proteins to target cells, and engineered EVs also vehiculate recombinant proteins. In this study, we explore immunomodulatory properties of EVs derived from antitumor vaccines expressing the TNFSF ligands 4-1BBL and OX40L, modulating immune response mediated by immune cells and eliminating tumors. Our results suggest that the EVs secreted by genetically modified tumor cells harboring TNFSF ligands can induce T cell proliferation, inhibit the transcription factor FoxP3, associated with the maintenance of Treg phenotype, and enhance antitumor activity mediated by immune cells. The immunomodulatory extracellular vesicles have potential to be further engineered for developing new approaches for cancer therapy.

Association between IL-27 and Tr1 cells in severe form of paracoccidioidomycosis.

Interleukin-27, a cytokine of the IL-12 family, is secreted by antigen-presenting cells such as macrophages and dendritic cells (DCs). Recent studies suggest an anti-inflammatory role for IL-27 by inducing IL-10 producing Tr1 cells capable of inhibiting Th1 and Th17 type responses. Our study aimed to investigate the involvement of IL-27 and Tr1 cells in the immunomodulation of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Brazil. The presence of IL-27 was evaluated in serum and biopsies of patients with PCM by ELISA, immunohistochemistry, and immunofluorescence. The presence of Tr1 in peripheral blood was analyzed by flow cytometry. In vitro assays were performed to verify the ability of P. brasiliensis yeast to induce IL-27 production by DCs and macrophages, as well as the polarization of lymphocytes to the Tr1 phenotype. Patients with the acute form and severe chronic form, the most severe and disseminated forms of PCM, presented higher serum concentrations of IL-27 and higher percentage of Tr1 cells compared to patients with mild chronic form. IL-27 was also detected in lesions of patients with PCM and associated with DCs and macrophages. P. brasiliensis Pb18 yeasts were able to induce IL-27 production by both DCs and macrophages. We found that DCs pulsed with Pb18 were able to induce Tr1 lymphocytes in vitro. Our data suggest that IL-27 and Tr1 cells could contribute to the deficient immune response to P. brasiliensis that leads to severe and disseminated forms of the disease.

Paracoccin distribution supports its role in Paracoccidioides brasiliensis growth and dimorphic transformation.

Paracoccidioides brasiliensis yeast was reported to express paracoccin, a GlcNAc-binding protein that displays N-acetyl-ß-d-glucosaminidase (NAGase) activity. Highly specific anti-paracoccin antibodies have been previously used to examine the localization of paracoccin in yeast and inhibit its growth in vitro. In the present study, anti-paracoccin antibodies were used to characterize, by scanning confocal microscopy, the distribution of paracoccin in P. brasiliensis hyphae, transition forms from hyphae to yeast, and mature yeast. In the mycelial phase, paracoccin was detected mainly in the hyphae tips, where it demonstrated a punctate distribution, and was associated with the cell wall. During the first 48 hours after a temperature shift from 26°C to 37°C, paracoccin expression in the differentiating hyphae was mainly detected in the budding regions, i.e. lateral protrusions, and inside the new daughter cells. There was an increased number of chlamydoconidia that expressed a high concentration of paracoccin on their surfaces and/or in their interiors 72-96 hours after the temperature shift. After 120 hours, yeast cells were the predominant form and their cytoplasm stained extensively for paracoccin, whereas Wheat Germ Agglutinin (WGA) staining was predominant on their exterior walls. After 10 days at 37°C, the interior of both mother and daughter yeast cells, as well as the budding regions, stained intensely for paracoccin. The comparison of mRNA-expression in the different fungal forms showed that PCN transcripts, although detected in all evaluated morphological forms, were higher in hypha and yeast-to-hypha transition forms. In conclusion, the pattern of paracoccin distribution in all P. brasiliensis morphotypes supports prevalent beliefs that it plays important roles in fungal growth and dimorphic transformation.

Functional and phenotypic evaluation of eosinophils from patients with the acute form of paracoccidioidomycosis.

BACKGROUND: Eosinophilia is a typical finding of the acute/juvenile form of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. This clinical form is characterized by depressed cellular immune response and production of Th2 cytokines. Moreover, it has been shown that the increased number of eosinophils in peripheral blood of patients returns to normal values after antifungal treatment. However, the role of eosinophils in PCM has never been evaluated. This study aimed to assess the phenotypic and functional characteristics of eosinophils in PCM. METHODS/PRINCIPAL FINDINGS: In 15 patients with the acute form of the disease, we detected expression of MBP, CCL5 (RANTES) and CCL11 (eotaxin) in biopsies of lymph nodes and liver. In addition, there were higher levels of chemokines and granule proteins in the peripheral blood of patients compared to controls. Isolation of eosinophils from blood revealed a higher frequency of CD69+ and TLR2+ eosinophils in patients compared to controls, and a lower population of CD80+ cells. We also evaluated the fungicidal capacity of eosinophils in vitro. Our results revealed that eosinophils from PCM patients and controls exhibit similar ability to kill P. brasiliensis yeast cells, although eosinophils of patients were less responsive to IL-5 stimulation than controls. CONCLUSION/PRINCIPAL FINDINGS: In conclusion, we suggest that eosinophils might play a role in the host response to fungi and in the pathophysiology of PCM by inducing an intense and systemic inflammatory response in the initial phase of the infection.

Saccharomyces cerevisiae expressing Gp43 protects mice against Paracoccidioides brasiliensis infection.

The dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM). It is believed that approximately 10 million people are infected with the fungus and approximately 2% will eventually develop the disease. Unlike viral and bacterial diseases, fungal diseases are the ones against which there is no commercially available vaccine. Saccharomyces cerevisiae may be a suitable vehicle for immunization against fungal infections, as they require the stimulation of different arms of the immune response. Here we evaluated the efficacy of immunizing mice against PCM by using S. cerevisiae yeast expressing gp43. When challenged by inoculation of P. brasiliensis yeasts, immunized animals showed a protective profile in three different assays. Their lung parenchyma was significantly preserved, exhibiting fewer granulomas with fewer fungal cells than found in non-immunized mice. Fungal burden was reduced in the lung and spleen of immunized mice, and both organs contained higher levels of IL-12 and IFN-γ compared to those of non-vaccinated mice, a finding that suggests the occurrence of Th1 immunity. Taken together, our results indicate that the recombinant yeast vaccine represents a new strategy to confer protection against PCM.

Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

Nasal vaccination with attenuated Salmonella expressing VapA: TLR2 activation is not essential for protection against R. equi infection.

Virulent strains of Rhodococcus equi have a large plasmid of 80-90kb, which encodes several virulence-associated proteins (Vap), including VapA, a lipoprotein highly associated with disease. We have previously demonstrated that oral immunisation with attenuated Salmonella enterica Typhimurium strain expressing the antigen VapA (STM VapA+) induces specific and long-term humoral and cellular immunity against R. equi. It was shown that VapA activates Toll-like receptor 2 (TLR2) on macrophages by establishing an interaction that ultimately favours immunity against R. equi infection. The purpose of this study was to evaluate the immune response triggered by nasal immunisation with STM VapA+ and to determine whether TLR2 supports the vaccine effect. We developed an optimised protocol for a single nasal immunisation that conferred protection against R. equi infection in mice, which was manifested by efficient R. equi clearance in challenged animals. Nasal vaccination with STM VapA+ has also induced protection in Tlr2(-/-) mice and mice with non-functional TLR4. Moreover, spleen cells of vaccinated mice augmented T-bet expression, as well as the production of IL-12, IFN-γ, nitric oxide and hydrogen peroxide. Notably, the population of CD4(+) T cells with memory phenotype significantly increased in the spleens of vaccinated mice challenged 1 or 5 months after immunisation. In these animals, the spleen bacterial burden was also reduced. When similar experimental procedures were performed in TLR2 knockout mice, an increase in CD4(+) T cells with memory phenotype was not observed. Consequently, we conclude that nasal vaccination with attenuated Salmonella expressing the R. equi virulence factor VapA confers long-lasting protection against experimental rhodoccocosis and that TLR2 engagement was not crucial to induce this protection but may be required for a long-term immune response.

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-gamma (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-gamma (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.

Lack of galectin-3 drives response to Paracoccidioides brasiliensis toward a Th2-biased immunity.

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

Toxoplasma gondii infection reveals a novel regulatory role for galectin-3 in the interface of innate and adaptive immunity.

In attempts to investigate the role of galectin-3 in innate immunity, we studied galectin-3-deficient (gal3-/-) mice with regard to their response to Toxoplasma gondii infection, which is characterized by inflammation in affected organs, Th-1-polarized immune response, and accumulation of cysts in the central nervous system. In wild-type (gal3+/+) mice, infected orally, galectin-3 was highly expressed in the leukocytes infiltrating the intestines, liver, lungs, and brain. Compared with gal3+/+, infected gal3-/- mice developed reduced inflammatory response in all of these organs but the lungs. Brain of gal3-/- mice displayed a significantly reduced number of infiltrating monocytes/macrophages and CD8+ cells and a higher parasite burden. Furthermore, gal3-/- mice mounted a higher Th1-polarized response and had comparable survival rates on peroral T. gondii infection, even though they were more susceptible to intraperitoneal infection. Interestingly, splenic cells and purified CD11c+ dendritic cells from gal3-/- mice produced higher amounts of interleukin-12 than cells from gal3+/+ mice, possibly explaining the higher Th1 response verified in the gal3-/- mice. We conclude that galectin-3 exerts an important role in innate immunity, including not only a pro-inflammatory effect but also a regulatory role on dendritic cells, capable of interfering in the adaptive immune response.