Modulação da atividade dos macrófagos por diferentes formas do Fonsecaea pedrosoi

Dissertação (mestrado)—Universidade de Brasília, Programa de Pós-graduação em Patologia Molecular, 2014.A cromoblastomicose (CBM) é uma micose subcutânea, crônica, de distribuição cosmopolita, causada por vários fungos demáceos, pigmentados e dimórficos, sendo o Fonsecaea pedrosoi um dos agentes etiológicos mais recorrente. Estudos a respeito da importância dos receptores do tipo Toll no processo da internalização e reconhecimento de F. pedrosoi ainda são escassos de modo que o presente trabalho analisou a participação de alguns receptores de reconhecimento padrão dos macrófagos infectados com diferentes formas do fungo. Nossos resultados demonstraram que os conídeos são capazes de proliferar no interior dos macrófagos previamente estimulados com agonistas de TLR4, TLR2/TLR6 e Dectina-1. Já as células muriformes, apresentaram um resultado diferente, onde macrófagos infectados com essa forma fúngica e previamente estimulados com zymosan e LPS, apresentaram ação fungicida. Outros componentes da parede do fungo, que poderiam interferir na ativação correta dos macrófagos são os lipídeos, porém lipídeos totais extraídos das células muriformes de F. pedrosoi não modularam a ativação dos macrófagos quanto à produção das citocinas MCP-1, IL-6, IL-12 e NO. A ativação adequada dos macrófagos, para que possa eliminar o fungo internalizado, depende da ativação conjunta de mais de um receptor, como quando estimulado com zymosan e LPS, bem como da adequada maturação do endossomo, considerando que o fungo consegue sobreviver em meio ácido. ______________________________________________________________________________________________ ABSTRACTThe chromoblastomycosis is a chronic subcutaneous mycosis of cosmopolitan distribution, caused by several pigmented (dematiaceous) and dimorphic fungi. Fonsecaea pedrosoi is one of the most recurrent etiological agents. Studies on the importance of pattern recognition receptors (PRR) in the process of internalization and recognition F. pedrosoi are poor, so the objective of this work was examined the participation of some macrophage PRR during infection with different forms of this fungus. Our results showed a higher proliferation index of conidia within macrophages when macrophage were previously stimulated with TLR4, TLR2 / TLR6 and Dectin-1 agonists. Macrophages previously stimulated with zymosan and LPS and infected with muriformes cells showed a fungicidal action. Other components of the fungal wall, which could interfere with proper activation of macrophages are lipids, but total lipids extracted from F. pedrosoi muriformes cells did not modulate macrophage activation inducing production of MCP-1, IL-6, IL-12 and NO. Macrophages properly activated can eliminate internalized fungus and this activity is associated with a receptors crosstalk, as when stimulated with zymosan and LPS

Modulation of the immune response by Fonsecaea pedrosoi morphotypes in the course of experimental chromoblastomycosis and their role on inflammatory response chronicity

A common theme across multiple fungal pathogens is their ability to impair the establishment of a protective immune response. Although early inflammation is beneficial in containing the infection, an uncontrolled inflammatory response is detrimental and may eventually oppose disease eradication. Chromoblastomycosis (CBM), a cutaneous and subcutaneous mycosis, caused by dematiaceous fungi, is capable of inducing a chronic inflammatory response. Muriform cells, the parasitic form of Fonsecaea pedrosoi, are highly prevalent in infected tissues, especially in long-standing lesions. In this study we show that hyphae and muriform cells are able to establish a murine CBM with skin lesions and histopathological aspects similar to that found in humans, with muriform cells being the most persistent fungal form, whereas mice infected with conidia do not reach the chronic phase of the disease. Moreover, in injured tissue the presence of hyphae and especially muriform cells, but not conidia, is correlated with intense production of pro-inflammatory cytokines in vivo. Highthroughput RNA sequencing analysis (RNA-Seq) performed at early time points showed a strong up-regulation of genes related to fungal recognition, cell migration, inflammation, apoptosis and phagocytosis in macrophages exposed in vitro to muriform cells, but not conidia. We also demonstrate that only muriform cells required FcγR and Dectin-1 recognition to be internalized in vitro, and this is the main fungal form responsible for the intense inflammatory pattern observed in CBM, clarifying the chronic inflammatory reaction observed in most patients. Furthermore, our findings reveal two different fungal-host interaction strategies according to fungal morphotype, highlighting fungal dimorphism as an important key in understanding the bipolar nature of inflammatory response in fungal infections

Genetic diversity analysis of sporotrichosis agents using SSR markers

A esporotricose é a principal micose subcutânea mundialmente, transmitida por vetores animais ou vegetais, e frequentemente evolui para surtos ou epidemias. Atualmente a esporotricose transmitida por gatos, causada pelo Sporothrix brasiliensis, tornou-se um problema de saúde pública significativo na América do Sul. A dinâmica da transmissão permanece enigmática devido à falta de desenvolvimento de marcadores polimórficos para análise epidemiológica molecular. Este estudo usou uma estratégia de alto rendimento para caracterizar marcadores de repetição de sequência simples (SSR) nos genomas de Sporothrix spp. Um total de 118.140–143.912 loci SSR foram identificados (82.841–98.369 marcadores únicos), com uma densidade de 3651,55–3804,65 SSR/Mb e a maioria dos motivos encontrados são dinucleotídeos (GC/CG). Desenvolvemos um painel de 15 marcadores SSR altamente polimórficos adequados para genotipagem de S. brasiliensis, S. schenckii e S. globosa. A amplificação por PCR revelou 240 alelos em 180 isolados de Sporothrix com excelente conteúdo de informação polimórfica (PIC=0,9101), heterozigosidade esperada (H=0,9159) e poder de discriminação (D=0,7127), apoiando a eficácia dos marcadores SSR em revelar a diversidade genética críptica. O estudo sistemático de genética populacional estimou três agrupamentos, correspondendo a S. brasiliensis (população 1, n=97), S. schenckii (população 2, n=49) e S. globosa (população 3, n=34), com uma assinatura fraca de ancestralidade mista entre as populações 1 e 2 ou 3 e 2. A partição da variação genética via AMOVA revelou populações altamente estruturadas (ΦPT=0,539; Nm=0,213; P <0,0001), com variabilidade genética aproximadamente equivalente dentro (46%) e entre (54%) populações. A análise da diversidade SSR sugere o Rio de Janeiro (RJ) como o centro de origem das infecções contemporâneas por S. brasiliensis. O recente surgimento de esporotricose transmitida por gatos no nordeste do Brasil indica uma migração RJ-Nordeste resultando em efeitos fundadores durante a introdução de animais doentes em áreas livres de esporotricose. Nossos resultados demonstraram alta transferabilidade entre espécies, reprodutibilidade e informatividade dos marcadores genéticos SSR, ajudando a esclarecer estruturas genéticas profundas e em escala fina. Desta maneira, orientando a tomada de decisões para mitigar os efeitos nocivos da expansão da esporotricose transmitida por gatos.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoa de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP: 433276/2018-5CAPES: 88887.159096/2017-00CNPq: 433276/2018-

CBM progression in a Zymosan-induced inflammation model.

<p>After 15 days post infection with fungal propagules (FP), animals were treated intra lesionally (i.l.) in the footpad with 20μl of a suspension containing 5 mg/ml of zymosan (ZYM) or PBS, until 15 days post treatment start (d.p.t) (A). Animals treated with ZYM displayed intense inflammatory response up to 30 days post infection (d.p.i) (B). Animals facing prolonged inflammation showed no reduction in fungal load over time, as observed for those animals treated with PBS (C). **P<0.01 and ***P<0.001.</p

Phagocytosis gene expression in peritoneal macrophages co-culturing with conidia.

<p>Peritoneal macrophages (PM) co-culturing with conidia (FC) did not induce much gene expression related to phagocytosis.</p

Quantification of <i>F</i>. <i>pedrosoi</i> fungal cells in the tissue and production of pro-inflammatory cytokines in the course of murine CBM.

<p>Fungal cells were counted on twenty fields chosen at random in histopathological slides with the aid of a counting reticle (A-D). In all groups muriform cells (red arrows) could be identified after 15 days after infection. At the same time, hyphal fragments (brown arrow) were also present in animals infected either with hyphae (FH) (B) or muriform cells (MC) (C). Few conidia (black arrow) are observed after 15 days of infection in animals infected with <i>F</i>. <i>pedrosoi</i> conidia (FC) (A). Cell counts were expressed as cells per mm² of injured tissue. 1000x magnification (A-D). After 15 days of infection, high levels of TNF-α (E), IL-1β (F), IL-6 (G) and MCP-1 (H) were observed in groups with higher numbers of hyphae and muriform cells in the tissue. Cytokine production was measured by ELISA from homogenized footpad tissue. *P<0.05, **P<0.01 and ***P<0.001 compared to FC group.</p

<i>In vitro</i> cytokine and chemokine production.

<p>The production of cytokines and chemokines by macrophage in conidia (FC) or muriform cell (MC) co-culture supernatants was assessed by ELISA (A-E). NO₂ concentration in culture supernatants was used as an indicator of NO generation and measured using Griess reagent (F). High levels of TNF-α (A), IL-1β (B) and IL-6 (C) were observed in co-culture with muriform cells, but not with conidia. To assess IL-1β production after 24 hours, peritoneal macrophages required LPS co-stimulation (B). Muriform cells also induced higher levels of MCP-1 after 48h when compared to macrophages infected with conidia (E). Production of IL-12 (D) and NO₂ (F) was strongly inhibited by muriform cells. **P<0.01 and ***P<0.001.</p

Progression of murine chromoblastomycosis induced by different <i>F</i>. <i>pedrosoi</i> fungal forms.

<p>BALB/c mice were infected in the footpad with 1x10⁶ conidia (FC), hyphae (FH), muriform cells (MC) or a combination of hyphal fragments and conidia (FP) in the ratio of 3:1 (A). Morphometric (B) and CFU data (C) showed a fast clearance of inoculated conidia, while infection with MCs was reflected in the persistence of the fungus in the tissue up to 45 days after infection. 400x magnification (A). *P<0.05 and ***P<0.001 compared to FH group.</p