RESUMEN
Paracoccidioidomycosis (PCM) is the main and most prevalent systemic mycosis in Latin America, that until recently, it was believed to be caused only by Paracoccidioides brasiliensis (P. brasiliensis). In 2006, researchers described three cryptic species: S1, PS2, PS3, and later, another one, PS4. In 2009, Paracoccidioides lutzii (Pb01-like) was described, and in 2017, a new nomenclature was proposed for the different agents: P. brasiliensis (S1), P. americana (PS2), P. restrepiensis (PS3), and P. venezuelensis (PS4). These species are not uniformly distributed throughout Latin America and, knowing that more than one cryptic species could coexist in some regions, we aimed to identify those species in patients' biopsy samples for a better understanding of the distribution and occurrence of these recently described species in Botucatu region. The Hospital of Medical School of Botucatu-UNESP, which is a PCM study pole, is located in São Paulo State mid-west region and is classified as a PCM endemic area. Genotyping analyses of clinical specimens from these patients that have been diagnosed and treated in our Hospital could favor a possible correlation between genetic groups and mycological and clinical characteristics. For this, molecular techniques to differentiate Paracoccidioides species in these biopsies, such as DNA extraction, PCR, and sequencing of three target genes (ITS, CHS2, and ARF) were conducted. All the sequences were analyzed at BLAST to testify the presence of P. brasiliensis. The phylogenetic trees were constructed using Mega 7.0 software and showed that 100% of our positive samples were from S1 cryptic species, therefore P. brasiliensis. This is important data, demonstrating the predominance of this species in the São Paulo State region.
RESUMEN
Paracoccidioidomycosis (PCM) is a neglected mycosis most commonly occurring in Latin America. The etiologic agents are thermo dimorphic fungi of the genus Paracoccidioides, and cause an important granulomatous response in affected tissues. The Botucatu Medical School, from São Paulo State University (UNESP), is a PCM study pole, located in São Paulo State Midwest region, which is classified as a hyperendemic area in the Southeast region in Brazil. This study aimed to perform a retrospective epidemiological, geographical, and clinical analysis by the information available in medical records. It was listed as socio-demographic data along with clinical characteristics from patients diagnosed and treated during a 10-year period in Botucatu, totaling 177 patients with Paracoccidioidomycosis confirmed by the histopathological test. It was observed that the main clinical presentation was the chronic type (76,3%), most commonly identified in white male individuals over the age of 29 years old, smokers, and alcoholics, providing evidences for the first time that white individuals were more affected by the disease, in comparison to non-white individuals that may be more resistant to infection. This data opens new avenues for study within ancestry, resistance and susceptibility in paracoccidioidomycosis.
Asunto(s)
Susceptibilidad a Enfermedades , Paracoccidioidomicosis/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Brasil/epidemiología , Etnicidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Paracoccidioides , Paracoccidioidomicosis/diagnóstico , Paracoccidioidomicosis/terapia , Embarazo , Estudios Retrospectivos , Adulto JovenRESUMEN
Paracoccidioidomycosis is a systemic fungal disease, considered endemic in Latin America. Its etiological agents, fungi of the Paracoccidioides complex, have restricted geographic habitat, conidia as infecting form, and thermo-dimorphic characteristics. Polymorphonuclear neutrophils (PMNs) are responsible for an important defense response against fungus, releasing Neutrophil Extracellular Traps (NETs), which can wrap and destroy the yeasts. However, it has been described that some pathogens are able to evade from these DNA structures by releasing DNase as an escape mechanism. As different NETs patterns have been identified in PMNs cultures challenged with different isolates of Paracoccidioides brasiliensis, the general objective of this study was to identify if different patterns of NETs released by human PMNs challenged with Pb18 (virulent) and Pb265 (avirulent) isolates would be correlated with fungal ability to produce a DNase-like protein. To this end, PMNs from healthy subjects were isolated and challenged in vitro with both fungal isolates. The production, release, and conformation of NETs in response to the fungi were evaluated by Confocal Microscopy, Scanning Microscopy, and NETs Quantification. The identification of fungal DNase production was assessed by DNase TEST Agar, and the relative gene expression for hypothetical proteins was investigated by RT-qPCR, whose genes had been identified in the fungal genome in the GenBank (PADG_11161 and PADG_08285). It was possible to verify the NETs release by PMNs, showing different NETs formation when in contact with different isolates of the fungus. The Pb18 isolate induced the release of looser, larger, and more looking like degraded NETs compared to the Pb265 isolate, which induced the release of denser and more compact NETs. DNase TEST Agar identified the production of a DNase-like protein, showing that only Pb18 showed the capacity to degrade DNA in these plates. Besides that, we were able to identify that both PADG_08528 and PADG_11161 genes were more expressed during interaction with neutrophil by the virulent isolate, being PADG_08528 highly expressed in these cultures, demonstrating that this gene could have a greater contribution to the production of the protein. Thus, we identified that the virulent isolate is inducing more scattered and loose NETs, probably by releasing a DNase-like protein. This factor could be an important escape mechanism used by the fungus to escape the NETs action.
Asunto(s)
Trampas Extracelulares , Paracoccidioides , Paracoccidioidomicosis , Desoxirribonucleasas , Humanos , Neutrófilos , Paracoccidioides/genéticaRESUMEN
The glycoprotein gp43 is the major antigenic/diagnostic component of Paracoccidioides brasiliensis, one of the etiologic agents of paracoccidioidomycosis (PCM). Gp43 has protective roles in mice, but due to adhesive properties, this glycoprotein has also been associated with immune evasion mechanisms. The present study evaluated gp43 interaction in vitro with Toll-like receptors 2 and 4 (TLR2 and TLR4) present in polymorphonuclear neutrophils (PMNs) from healthy human individuals and the consequent modulation of the immune response through the expression and release of cytokines and eicosanoids. PMNs were incubated in the absence or presence of monoclonal antibodies anti-TLR2 and anti-TLR4 (individually or in combination) before gp43 stimulation. Then, PMNs were analyzed for the expression of both surface receptors and the detection of intracytoplasmic IL-17A and IL-4 using flow cytometry, while the production of PGE2, LTB4, IL-6, IL-10, IL-12, IFN-γ, and TNF-α was evaluated in the supernatants by enzyme-linked immunosorbent assay (ELISA). Our results showed that gp43 increased TLR2 and TLR4 expression by PMNs and induced PGE2 and IL-17A via TLR4 and TLR2, respectively. Thus, our data suggest that gp43 from P. brasiliensis might modulate host susceptibility to the fungal infection by affecting PGE2 and IL-17A production.
Asunto(s)
Antígenos Fúngicos/inmunología , Dinoprostona/metabolismo , Proteínas Fúngicas/inmunología , Interleucina-17/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/metabolismo , Adulto , Biomarcadores , Citocinas/biosíntesis , Femenino , Expresión Génica , Humanos , Mediadores de Inflamación/metabolismo , Masculino , Paracoccidioidomicosis/genética , Paracoccidioidomicosis/microbiología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Adulto JovenRESUMEN
Paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America, occurs after inhalation of mycelial components of Paracoccidioides spp. When the fungus reaches the lungs and interacts with the alveolar macrophages and other cells, phagocytic cells such as neutrophils and monocytes are immediately recruited to the injured site. The interaction between surface molecules of pathogens and homologous receptors, present on the surface membrane of phagocytes, modulates the innate immune cell activation. Studies have shown the importance of fungal recognition by the Dectin-1 receptor, which can induce a series of cellular protective responses against fungi. The objective of the present study was to evaluate Dectin-1 receptor expression and the effector mechanisms of human monocytes and neutrophils activated or not with different cytokines, such as IFN-γ, TNF-α, and GM-CSF, followed by the challenge with Paracoccidioides brasiliensis (P. brasiliensis or Pb265). Therefore, analysis of Dectin-1 receptor expression was done by flow cytometry whereas the effector mechanisms were evaluated by fungal recovery by colony-forming unit (CFU) counting and hydrogen peroxide (H2O2) production. Our results showed that, after treatment with IFN-γ, TNF-α, and GM-CSF and challenge with Pb265, cells, especially monocytes, demonstrated an increase in Dectin-1 expression. Both types of cells treated with the cytokines exhibited a decreased fungal recovery and, conversely, an increased production of H2O2. However, when cultures were treated with an anti-Dectin-1 monoclonal antibody, to block the P. brasiliensis binding, a decrease in H2O2 production and an increase in fungal recovery were detected. This effect was observed in all cultures treated with the specific monoclonal antibody. These results show the involvement of the Dectin-1 receptor in fungal recognition and its consequent participation in the induction of the killing mechanisms against P. brasiliensis.
Asunto(s)
Citocinas/farmacología , Lectinas Tipo C/metabolismo , Paracoccidioidomicosis/inmunología , Fagocitos/inmunología , Recuento de Colonia Microbiana , Femenino , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Peróxido de Hidrógeno/análisis , Interferón gamma/farmacología , Lectinas Tipo C/genética , Masculino , Monocitos/efectos de los fármacos , Monocitos/inmunología , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , Paracoccidioides , Fagocitos/efectos de los fármacosRESUMEN
Fungal recognition by Dectin-1 receptor triggers a series of cellular mechanisms involved in a protective activation of the immune system. In this study, we aimed to evaluate the participation of Dectin-1 receptor in the induction of IL-8, TNF-α, IL-12, IL-10 and IL-17A secretion by human monocytes activated with different cytokines, and challenged in vitro with Paracoccidioides brasiliensis (P. brasiliensis). Our results show that monocytes challenged with P. brasiliensis (Pb265) are able to produce IL-12, IL-8, IL-17, IL-10 and TNF-α. Dectin-1 receptor blockage decreased the IL-12, IL-17, IL-10 and TNF-α levels indicating the participation of such receptor in the induction of these cytokines. Only IL-8 production was not affected by the blockage. Cells activation with different cytokines showed that GM-CSF was able to induce secretion of all cytokines and the receptor blockage prior to the challenge also decreased the cytokine secretion, except IL-8. Monocytes activated with TNF-α promoted IL-8, IL-10 and TNF-α production, whereas stimulation with IFN-γ promoted mainly IL-12 and TNF-α. Thus, these findings bring new and important knowledge about Dectin-1 participation in cytokines production by monocytes challenged with Pb265.
Asunto(s)
Citocinas/metabolismo , Lectinas Tipo C/metabolismo , Monocitos/inmunología , Monocitos/microbiología , Paracoccidioides/inmunología , Adulto , Células Cultivadas , Humanos , Persona de Mediana Edad , Adulto JovenRESUMEN
Paracoccidioidomycosis (PCM) is a systemic mycosis, endemic in most Latin American countries, especially in Brazil. It is caused by the thermo-dimorphic fungus of the genus Paracoccidioides (Paracoccidioides brasiliensis and Paracoccidioides lutzii). Innate immune response plays a crucial role in host defense against fungal infections, and neutrophils (PMNs) are able to combat microorganisms with three different mechanisms: phagocytosis, secretion of granular proteins, which have antimicrobial properties, and the most recent described mechanism called NETosis. This new process is characterized by the release of net-like structures called Neutrophil Extracellular Traps (NETs), which is composed of nuclear (decondensed DNA and histones) and granular material such as elastase. Several microorganisms have the ability of inducing NETs formation, including gram-positive and gram-negative bacteria, viruses and some fungi. We proposed to identify NETs in tegumentary lesions of patients with PCM and to analyze the interaction between two strains of P. brasiliensis and human PMNs by NETs formation in vitro. In this context, the presence of NETs in vivo was evidenced in tegumentary lesions of patients with PCM by confocal spectrum analyzer. Furthermore, we showed that the high virulent P. brasiliensis strain 18 (Pb18) and the lower virulent strain Pb265 are able to induce different patterns of NETs formation in vitro. The quantification of extracellular DNA corroborates the idea of the ability of P. brasiliensis in inducing NETs release. In conclusion, our data show for the first time the identification of NETs in lesions of patients with PCM and demonstrate distinct patterns of NETs in cultures challenged with fungi in vitro. The presence of NETs components both in vivo and in vitro open new possibilities for the detailed investigation of immunity in PCM.