RESUMO
BACKGROUND: Celecoxib, an anti-inflammatory drug, combined therapies using antimicrobials and immune modulator drugs are being studied. OBJECTIVE: To assess whether Celecoxib has direct in vitro antifungal effect against the Paracoccidioides brasiliensis, the causative agent of Paracoccidioidomycosis-(PCM) and also if it improves the in vivo activity of neutrophils-(PMN) in an experimental murine subcutaneous-(air pouch) model of the disease. METHODS: The antifungal activity of Celecoxib(6 mg/mL) on P. brasiliensis-(Pb18) was evaluated using the microdilution technique. Splenocytes co-cultured with Pb18 and treated with Celecoxib(6 mg/mL) were co-cultured for 24, 48 and 72-hours. Swiss mice were inoculated with Pb18 and treated with Celecoxib(6 mg/kg) in the subcutaneous air pouch. Neutrophils were collected from the air pouch. Mitochondrial activity, reactive oxygen production, catalase, peroxidase, cytokines and chemokines, nitrogen species, total protein, microbicidal activity of PMNs and viable Pb18 cells numbers were analyzed. RESULTS: Celecoxib had no cytotoxic effect on splenocytes co-cultured with Pb18, but had a marked direct antifungal effect, inhibiting fungal growth both in vitro and in vivo. Celecoxib interaction with immune system cells in the air pouch, it leads to activation of PMNs, as confirmed by several parameters (mitochondrial activity, reactive oxygen species, peroxidase, KC and IL-6 increase, killing constant and phagocytosis). Celecoxib was able to reduce IL-4, IL-10 and IL-12 cytokine production. The number of recovered viable Pb18 decreased dramatically. CONCLUSIONS: This is the first report of the direct antifungal activity of Celecoxib against P. brasiliensis. The use of Celecoxib opens a new possibility for future treatment of PCM.
Assuntos
Antifúngicos , Celecoxib , Neutrófilos , Paracoccidioides , Paracoccidioidomicose , Animais , Paracoccidioides/efeitos dos fármacos , Paracoccidioides/imunologia , Camundongos , Celecoxib/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/imunologia , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Citocinas/metabolismo , Células Cultivadas , Masculino , Baço/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Modelos Animais de Doenças , Espécies Reativas de Oxigênio/metabolismoRESUMO
Background: Paracoccidioidomycosis (PCM) is a systemic endemic fungal disease prevalent in Latin America. Previous studies revealed that host immunity against PCM is tightly regulated by several suppressive mechanisms mediated by tolerogenic plasmacytoid dendritic cells, the enzyme 2,3 indoleamine dioxygenase (IDO-1), regulatory T-cells (Tregs), and through the recruitment and activation of myeloid-derived suppressor cells (MDSCs). We have recently shown that Dectin-1, TLR2, and TLR4 signaling influence the IDO-1-mediated suppression caused by MDSCs. However, the contribution of these receptors in the production of important immunosuppressive molecules used by MDSCs has not yet been explored in pulmonary PCM. Methods: We evaluated the expression of PD-L1, IL-10, as well as nitrotyrosine by MDSCs after anti-Dectin-1, anti-TLR2, and anti-TLR4 antibody treatment followed by P. brasiliensis yeasts challenge in vitro. We also investigated the influence of PD-L1, IL-10, and nitrotyrosine in the suppressive activity of lung-infiltrating MDSCs of C57BL/6-WT, Dectin-1KO, TLR2KO, and TLR4KO mice after in vivo fungal infection. The suppressive activity of MDSCs was evaluated in cocultures of isolated MDSCs with activated T-cells. Results: A reduced expression of IL-10 and nitrotyrosine was observed after in vitro anti-Dectin-1 treatment of MDSCs challenged with fungal cells. This finding was further confirmed in vitro and in vivo by using Dectin-1KO mice. Furthermore, MDSCs derived from Dectin-1KO mice showed a significantly reduced immunosuppressive activity on the proliferation of CD4+ and CD8+ T lymphocytes. Blocking of TLR2 and TLR4 by mAbs and using MDSCs from TLR2KO and TLR4KO mice also reduced the production of suppressive molecules induced by fungal challenge. In vitro, MDSCs from TLR4KO mice presented a reduced suppressive capacity over the proliferation of CD4+ T-cells. Conclusion: We showed that the pathogen recognition receptors (PRRs) Dectin-1, TLR2, and TLR4 contribute to the suppressive activity of MDSCs by inducing the expression of several immunosuppressive molecules such as PD-L1, IL-10, and nitrotyrosine. This is the first demonstration of a complex network of PRRs signaling in the induction of several suppressive molecules by MDSCs and its contribution to the immunosuppressive mechanisms that control immunity and severity of pulmonary PCM.
Assuntos
Antígeno B7-H1 , Modelos Animais de Doenças , Interleucina-10 , Lectinas Tipo C , Camundongos Endogâmicos C57BL , Células Supressoras Mieloides , Paracoccidioidomicose , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Animais , Camundongos , Interleucina-10/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/imunologia , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Paracoccidioidomicose/imunologia , Paracoccidioides/imunologia , Tirosina/análogos & derivados , Tirosina/metabolismo , Linfócitos T Reguladores/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Transdução de Sinais , Masculino , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Camundongos KnockoutRESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis with serious clinical consequences in which the use of antifungal drugs requires long-term treatment. Therefore, we studied the effect of low-level LASER therapy (LLLT) to evaluate its prospects as a complementary treatment for PCM and improve the clinical response to the disease. OBJECTIVES: Our study focused on the resolution of lesions caused by fungal infection using a subcutaneous air pouch model of infection. METHODS: We evaluated cell profile and cytokines, fungi viability, and the presence of fibroblasts and fibrocytes at the site of infection. Inoculation of P. brasiliensis (Pb) was performed using a subcutaneous air pouch model and the LLLT irradiation was performed on alternate days on the rear paws of mice for 10 days, after which the cells from the air pouch were collected and analyzed. RESULTS: In animals irradiated with LLLT, the influx of cells to the air pouch was reduced, but they were more activated and produced pro-inflammatory (IL-12, IL-17 and TNF-α) and neutrophil (PMN) activating cytokines (IL-8, GM-CSF and γ-IFN). A better resolution of the infection, evidenced by the reduction in the number of viable fungi with preserved morphology in the air pouch, and an increase in the number of fibrocytes, indicating a healing profile were also observed. CONCLUSION: LLLT decreased the influx of PMN, but those presents were highly activated, with increased fungicidal activity. LLLT irradiation also resulted in earlier cicatrization at the site of infection, leading to a better outcome of the infection. These data are favorable to the use of LLLT as a complementary therapy in PCM.
Assuntos
Citocinas , Terapia com Luz de Baixa Intensidade , Paracoccidioidomicose , Células Th1 , Células Th2 , Animais , Camundongos , Citocinas/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Paracoccidioidomicose/radioterapia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/patologia , Células Th2/imunologia , Células Th2/metabolismo , Paracoccidioides/imunologia , Camundongos Endogâmicos BALB C , MasculinoRESUMO
BACKGROUND: Accurate diagnosis of paracoccidioidomycosis is crucial for improving patient outcomes. Paracoccidioides antibody detection by double immunodiffusion (DID) is a convenient diagnostic tool, but testing performance can vary based on certain factors. METHODS: We assessed DID performance using a commercially prepared Paracoccidioides reagents (IMMY, USA), involving 40 serum specimens, including 20 from patients with proven paracoccidioidomycosis and 20 from patients without the disease. The DID test demonstrated a sensitivity of 90% (95% CI=68%-99%) and a specificity of 100% (95% CI=83%-100%). CONCLUSIONS: Our findings suggest that DID using commercial reagents may provide a feasible tool with satisfactory testing performance for anti-Paracoccidioides antibody detection.
Assuntos
Anticorpos Antifúngicos , Imunodifusão , Paracoccidioides , Paracoccidioidomicose , Sensibilidade e Especificidade , Humanos , Anticorpos Antifúngicos/sangue , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/imunologia , Paracoccidioides/imunologia , Kit de Reagentes para Diagnóstico , Feminino , MasculinoRESUMO
Paracoccidioidomycosis (PCM) defines a broad spectrum of human and animal diseases caused by Paracoccidioides species (Onygenales). In the twenty-first century, Paracoccidioides advanced from a monotypic taxon to a genus that harbors seven species, including P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, P. lutzii, P. loboi, and P. cetii. Classic PCM, acquired upon inhalation of propagules from P. brasiliensis sensu stricto, P. americana, P. restrepiensis, P. venezuelensis, and P. lutzii, affects the human lungs and may progress to systemic granulomatous disease with tegumentary and visceral involvement. On the other hand, PCM loboi and PCM ceti caused by the unculturable P. loboi and P. cetii are subcutaneous mycoses, typically observed as keloid lesions in humans and dolphins. Such heterogeneity highlights the importance of recognizing species boundaries in Paracoccidioides to gain insights into the ecology, evolution, clinical features, and mitigation strategies to tackle the advance of PCM.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Animais , Humanos , Golfinhos/microbiologia , Genômica , Paracoccidioides/classificação , Paracoccidioides/genética , Paracoccidioides/isolamento & purificação , Paracoccidioidomicose/diagnóstico , Paracoccidioidomicose/epidemiologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , FilogeniaRESUMO
Fungal infection is an important health problem in Latin America, and in Brazil in particular. Paracoccidioides (mainly P. brasiliensis and P. lutzii) is responsible for paracoccidioidomycosis, a disease that affects mainly the lungs. The glycoprotein gp43 is involved in fungi adhesion to epithelial cells, which makes this protein an interesting target of study. A specific stretch of 15 amino acids that spans the region 181-195 (named P10) of gp43 is an important epitope of gp43 that is being envisioned as a vaccine candidate. Here we show that synthetic P10 forms typical amyloid aggregates in solution in very short times, a property that could hamper vaccine development. Seeds obtained by fragmentation of P10 fibrils were able to induce the aggregation of P4, but not P23, two other peptides derived from gp43. In silico analysis revealed several regions within the P10 sequence that can form amyloid with steric zipper architecture. Besides, in-silico proteolysis studies with gp43 revealed that aggregation-prone, P10-like peptides could be generated by several proteases, which suggests that P10 could be formed under physiological conditions. Considering our data in the context of a potential vaccine development, we redesigned the sequence of P10, maintaining the antigenic region (HTLAIR), but drastically reducing its aggregation propensity.
Assuntos
Amiloide/química , Antígenos de Neoplasias/química , Antígenos/química , Paracoccidioides/imunologia , Paracoccidioidomicose/prevenção & controle , Algoritmos , Animais , Antígenos de Fungos/imunologia , Dicroísmo Circular , Biologia Computacional/métodos , Simulação por Computador , Epitopos , Proteínas Fúngicas/química , Vacinas Fúngicas/imunologia , Glicoproteínas/química , Humanos , Técnicas In Vitro , Paracoccidioidomicose/imunologia , Peptídeos/química , Conformação Proteica , Dobramento de Proteína , Software , Solventes/química , Desenvolvimento de VacinasRESUMO
Monoclonal antibodies (mAbs) are promising alternatives to treat infectious diseases, especially given their potential for applications in combination therapies with antimicrobial drugs to enhance the antifungal efficacy. Protection mediated by mAbs used to treat experimental paracoccidioidomycosis (PCM) has been demonstrated previously. Our aim in the present work was to characterize a monoclonal antibody (mAbF1.4) raised against a cell wall glycoconjugate fraction of Paracoccidioides spp. and to analyze its efficacy combined with trimethoprim-sulfamethoxazole (TMP/SMX) as treatment for experimental PCM. We demonstrated that the epitope recognized by mAbF1.4 is consistent with branched glucose residues present on a cell wall ß-glucan polymer. In vitro, mAbF1.4 increased the phagocytic capacity and nitric oxide concentration induced by the macrophage cell line J774.1A, and this resulted in a significant reduction in the viability of the opsonophagocytized yeasts. In vivo, we detected a significant reduction in pulmonary fungal burdens of mice treated with mAbF1.4 in association with TMP/SMX, which correlated with increased pulmonary concentrations (determined by ELISA) of IFN- γ, TNF-α, IL-10 and IL-17. In parallel, we observed a decrease in IL-4, suggesting that the treatment was associated with a mixed Th1-Th17 type immune response. Histopathology of lung segments from mice receiving the combination therapy showed a significant reduction in granulomas, which were well-defined, and improved maintenance of lung architecture. These findings demonstrate that mAbF1.4 + TMP/SMX therapy is a promising approach to combat PCM as well as decrease disease sequelae and highlights the potential benefits of immune mediators in PCM combined immunotherapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Imunoterapia/métodos , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Combinação Trimetoprima e Sulfametoxazol/farmacologia , Animais , Antifúngicos/farmacologia , Antígenos de Fungos/imunologia , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Pulmão/microbiologia , Pulmão/patologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/microbiologiaRESUMO
Aim: We aimed to verify the frequency of CD8+ T cell subsets in patients with acute form and chronic form of paracoccidioidomycosis. Material & Methods: Mononuclear cells from paracoccidioidomycosis patients and healthy donors were isolated and phenotyped by flow cytometry. Dendritic cells were pulsed with Paracoccidioides brasiliensis yeast and co-cultures with lymphocytes. Cytokine production was measured by ELISA. Results: Acute form patients present a higher frequency of Tc1 and Tc10 cells, while chronic form patients have more Tc1 and Tc21 cells, compared with healthy controls. In vitro assays showed that P. brasiliensis induced polarization to the Tc17/Tc22 subsets. Conclusion: Our results suggest that CD8+ T cells can respond in a similar way to P. brasiliensis infection, regardless of the clinical presentation of the disease.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Paracoccidioidomicose , Humanos , Paracoccidioides , Paracoccidioidomicose/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Paracoccidioidomycosis (PCM) is a systemic disease caused by Paracoccidioides spp. PCM is endemic in Latin America and most cases are registered in Brazil. This mycosis affects mainly the lungs, but can also spread to other tissues and organs, including the liver. Several approaches have been investigated to improve treatment effectiveness and protection against the disease. Extracellular vesicles (EVs) are good antigen delivery vehicles. The present work aims to investigate the use of EVs derived from Paracoccidioides brasiliensis as an immunization tool in a murine model of PCM. For this, male C57BL/6 were immunized with two doses of EVs plus adjuvant and then infected with P. brasiliensis. EV immunization induced IgM and IgG in vivo and cytokine production by splenocytes ex vivo. Further, immunization with EVs had a positive effect on mice infected with P. brasiliensis, as it induced activated T lymphocytes and NKT cell mobilization to the infected lungs, improved production of proinflammatory cytokines and the histopathological profile, and reduced fungal burden. Therefore, the present study shows a new role for P. brasiliensis EVs in the presence of adjuvant as modulators of the host immune system, suggesting their utility as immunizing agents.
Assuntos
Antígenos de Fungos/imunologia , Vesículas Extracelulares/microbiologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Paracoccidioidomicose/microbiologia , Substâncias Protetoras/farmacologia , Animais , Anticorpos Antifúngicos/imunologia , Movimento Celular , Citocinas/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Imunização , Memória Imunológica , Pulmão/microbiologia , Pulmão/patologia , Ativação Linfocitária/imunologia , Masculino , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/imunologia , Padrões de ReferênciaRESUMO
In agreement with other fungal infections, immunoprotection in pulmonary paracoccidioidomycosis (PCM) is mediated by Th1/Th17 cells whereas disease progression by prevalent Th2/Th9 immunity. Treg cells play a dual role, suppressing immunity but also controlling excessive tissue inflammation. Our recent studies have demonstrated that the enzyme indoleamine 2,3 dioxygenase (IDO) and the transcription factor aryl hydrocarbon receptor (AhR) play an important role in the immunoregulation of PCM. To further evaluate the immunomodulatory activity of AhR in this fungal infection, Paracoccidioides brasiliensis infected mice were treated with two different AhR agonists, L-Kynurenin (L-Kyn) or 6-formylindole [3,2-b] carbazole (FICZ), and one AhR specific antagonist (CH223191). The disease severity and immune response of treated and untreated mice were assessed 96 hours and 2 weeks after infection. Some similar effects on host response were shared by FICZ and L-Kyn, such as the reduced fungal loads, decreased numbers of CD11c+ lung myeloid cells expressing activation markers (IA, CD40, CD80, CD86), and early increased expression of IDO and AhR. In contrast, the AhR antagonist CH223191 induced increased fungal loads, increased number of pulmonary CD11c+ leukocytes expressing activation markers, and a reduction in AhR and IDO production. While FICZ treatment promoted large increases in ILC3, L-Kyn and CH223191 significantly reduced this cell population. Each of these AhR ligands induced a characteristic adaptive immunity. The large expansion of FICZ-induced myeloid, lymphoid, and plasmacytoid dendritic cells (DCs) led to the increased expansion of all CD4+ T cell subpopulations (Th1, Th2, Th17, Th22, and Treg), but with a clear predominance of Th17 and Th22 subsets. On the other hand, L-Kyn, that preferentially activated plasmacytoid DCs, reduced Th1/Th22 development but caused a robust expansion of Treg cells. The AhR antagonist CH223191 induced a preferential expansion of myeloid DCs, reduced the number of Th1, Th22, and Treg cells, but increased Th17 differentiation. In conclusion, the present study showed that the pathogen loads and the immune response in pulmonary PCM can be modulated by AhR ligands. However, further studies are needed to define the possible use of these compounds as adjuvant therapy for this fungal infection.