Investigação de potenciais sítios reservatórios para a infecção criptocócica latente e o papel dos macrófagos na dormência de Cryptococcus neoformans

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2020.Uma característica marcante da criptococose, doença causada pelo fungo Cryptococcus neoformans (Cn), é o estabelecimento de uma infecção latente, normalmente em granulomas pulmonares. Não obstante, acredita-se na existência de outros sítios anatômicos reservatórios de leveduras dormentes (D-Cn). Os macrófagos (MØ) desempenham um papel paradoxal na resposta à infecção, sendo importantes para o seu controle, mas também associados ao abrigo intracelular, persistência e disseminação de Cn no hospedeiro. Objetivamos aqui identificar potenciais sítios reservatórios da infecção criptocócica latente em modelo experimental murino, bem como esclarecer os mecanismos subjacentes à sua manutenção ou reativação com base na interação entre D-Cn e MØ in vitro. Primeiramente, caracterizamos e validamos linhagens fluorescentes de Cn quanto à expressão de determinados fenótipos relevantes, incluindo dormência, e aplicabilidade em experimentos de interação Cn-hospedeiro. Isso nos permitiu demonstrar características morfológicas e mecanismos regulatórios pós-transcricionais associados ao fenótipo dormente em Cn. Por meio da infecção experimental de camundongos, observamos que Cn foi capaz de disseminar para uma diversidade de sítios anatômicos, incluindo sítios extrapulmonares previamente associados à infecção latente, como o trato urogenital, e sítios nunca relatados na infecção murina mas associados à infecção latente em outros modelos de patógenos, como a medula óssea. A análise do transcriptoma do MØ incubado com Cn na fase exponencial, estacionária (S-Cn) ou dormente mostrou que D-Cn promoveu uma reduzida e específica modificação transcricional do macrófago, mas semelhante a S-Cn. D-Cn reteve a capacidade de modular no macrófago genes importantes para a resposta imune, como aqueles envolvidos em vias do inflamassoma. Inclusive, D-Cn foi incapaz de promover a secreção de IL-1β dependente do inflamassoma NLRP3 em macrófagos pré- ativados, diferentemente de Cn não dormente. Também avaliamos o papel do MØ, sob diferentes perfis de polarização, na manutenção/saída da dormência do fungo. Observamos um aprofundamento do fenótipo dormente na população de D-Cn intracelular independentemente do perfil fenotípico do MØ. Em adição, leveduras presumivelmente exocitadas que foram encontradas no sobrenadante de cultura reativaram. No entanto, a reativação foi frustrada por MØ classicamente ativados, por meio da produção de óxido nítrico. Em conclusão, nossos resultados suportam a hipótese da existência de sítios reservatórios extrapulmonares na infecção criptocócica. Ademais, indicam que os macrófagos atuam como sítios de indução e persistência de células dormentes na infecção criptocócica latente, sendo o fenótipo de polarização dessas células, reflexo do status da resposta imune do hospedeiro, determinante para a manutenção ou reativação dessa infecção.CNPq - FAP-DFA striking feature of cryptococcosis, a disease caused by the fungus Cryptococcus neoformans (Cn), is the establishment of a latent infection, usually in pulmonary granulomas. Nevertheless, it is believed that there are other anatomical reservoirs sites for dormant yeasts (D-Cn). Macrophages (MØ) play a paradoxical role in the response to infection, being important for its control, but also associated with the intracellular shelter, persistence and dissemination of Cn in the host. Our aim was to identify potential reservoir sites for latent cryptococcal infection in a murine experimental model, as well as to clarify the mechanisms underlying their maintenance or reactivation based on the interaction between D-Cn and MØ in vitro. First, we characterized and validated fluorescent Cn strains for the expression of certain relevant phenotypes, including dormancy, and applicability in Cn-host interaction experiments. This allowed us to demonstrate morphological characteristics and post-transcriptional regulatory mechanisms associated to the dormant phenotype in Cn. Through experimental infection in mice, we observed that Cn was able to spread across a variety of anatomical sites, including extrapulmonary sites previously associated with latent infection, such as the urogenital tract, and sites never reported in murine infection but associated with latent infection in others models of pathogens, such as the bone marrow. Transcriptome analysis of the MØ incubated with Cn at the exponential, stationary (S- Cn) or dormant phase showed that D-Cn promoted a reduced and specific transcriptional modification of the macrophage, but close to S-Cn condition. D-Cn retained the ability to modulate on the macrophage genes important to immune response, such as those involved in inflammasome pathways. In fact, unlike non-dormant Cn, D-Cn was unable to trigger a NLRP3- dependent IL-1β secretion by primed macrophages. We also evaluated the role of MØ, under different polarization profiles, on fungal maintenance/exit from dormancy. We observed a deepening of the dormant phenotype in the intracellular D-Cn population regardless of the phenotype of the MØ. Moreover, presumably exocytosed yeasts that were found in the cell supernatant reactivated. However, this phenomenon was thwarted by classically activated MØ, through the production of nitric oxide. In conclusion, our results support the hypothesis of existence of extrapulmonary reservoir sites in cryptococcal infection. Furthermore, we indicate that macrophages behavior as sites of induction and persistence of dormant cells in latent cryptococcal infection, with the polarization phenotype of these cells, which reflects the status of the host's immune response, determining for the maintenance or reactivation of this infection

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

Engineered fluorescent strains of cryptococcus neoformans : a versatile toolbox for studies of host-pathogen interactions and fungal biology, including the viable but nonculturable state

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hygr ] or neomycin resistance [Neor]). We validated their similarity to the parental strain in the stress response, the expression of virulence-related phenotypes, mating, virulence in Galleria mellonella, and survival within murine macrophages. PAB-GFP, the brightest transformant, was successfully applied for the analysis of phagocytosis by flow cytometry and fluorescence microscopy. Moreover, we demonstrated that an engineered fluorescent strain of C. neoformans was able to generate VBNC cells. GFP-tagged Pab1, a key regulator of the stress response, evidenced nuclear retention of Pab1 and the assembly of cytoplasmic stress granules, unveiling posttranscriptional mechanisms associated with dormant C. neoformans cells. Our results support that the PAB-GFP strain is a useful tool for research on C. neoformans

Peptides ToAP3 and ToAP4 decrease release of inflammatory cytokines through TLR-4 blocking

Antimicrobial peptides (AMPs) are small molecules with microbicidal and immunoregulatory activities. In this study we evaluated the anti-inflammatory and antimicrobial activities of peptides ToAP3 and ToAP4, AMPs from the venom of the Brazilian scorpion Tityus obscurus. To test the peptides’ activity, murine bone marrow-derived macrophages (BMDMs) or dendritic cells (BMDCs) were stimulated with peptides plus LPS to analyze their ability to modulate cytokine release as well as phenotypic markers. For antimicrobial analysis, we evaluated the indirect activity against macrophage-internalized Cryptococcus neoformans and direct activity against Mycobacterium massiliense. Our data demonstrate that they were able to reduce TNF-α and IL-1β transcript levels and protein levels for BMDM and BMDC. Furthermore, the reduction of TNF-α secretion, before LPS- inflammatory stimuli, is associated with peptide interaction with TLR-4. ToAP4 increased MHC-II expression in BMDC, while ToAP3 decreased co-stimulatory molecules such as CD80 and CD86. Although these peptides were able to modulate the production of cytokines and molecules associated with antigen presentation, they did not increase the ability of clearance of C. neoformans by macrophages. In antimicrobial analysis, only ToAP3 showed potent action against bacteria. Altogether, these results demonstrate a promising target for the development of new immunomodulatory and anti-bacterial therapies

O papel do inflamassoma na infecção induzida pelas formas saprofíticas do fungo Fonsecaea pedrosoi

O Fonsecaea pedrosoi é um fungo dimórfico e o principal agente etiológico da cromoblastomicose, infecção crônica da pele e tecidos subcutâneos iniciada pela inoculação transcutânea das formas saprofíticas do fungo. No hospedeiro, essas células transformam-se em células muriformes, tida como a forma parasítica do fungo e grandes produtoras de melanina, considerada um importante fator de virulência. Estudos recentes têm proposto que a cronicidade da cromoblastomicose decorre de falhas, tanto no reconhecimento inato do fungo, levando à baixa produção de citocinas pró-inflamatórias, quanto na montagem de uma resposta imune adequada para a contenção da infecção. Nesse contexto, considerando o inflamassoma como um importante sistema de reconhecimento inato que compõe respostas imunes antifúngicas, este trabalho foi conduzido visando avaliar a ativação do inflamassoma por F. pedrosoi, bem como o seu papel na infecção induzida pelas formas saprofíticas do fungo. Foi observado, inicialmente, que hifas, ao contrário de conídios, induzem a secreção de IL-1 provendo os 2 sinais necessários à ativação do inflamassoma, tanto em BMDMs, quanto em BMDCs. O fornecimento exógeno do primeiro ou segundo sinal não só incrementou a secreção da citocina durante a infecção com hifas, como possibilitou a secreção de IL-1β por BMDCs infectadas com conídios. Utilizando inibidores ou células nocautes, verificou-se que a secreção de IL-1β é dependente do receptor NLRP3, da caspase-1, e influenciada pela caspase-8. Em adição, observamos que a secreção de IL-1 mediada pelo inflamassoma NLRP3 é dependente: da sinalização via NF-κB independente de MyD88; dos mecanismos de efluxo de K+; da liberação de catepsina B; e, de forma não essencial, da geração de espécies reativas de oxigênio. Ademais, foi mostrado que a melanina presente na parede do fungo, mas não a secretada, contribui para a secreção da IL-1. Foi demonstrado, ainda, que a caspase-1, caspase-11 e o receptor NLRP3, não foram capazes de promover a atividade fungicida de macrófagos em ensaios de co-cultura com o fungo. Entretanto, dados dos ensaios de infecções in vivo sugerem que as caspases-1 e -11, assim como o NLRP3, auxiliam na contenção da infecção por propágulos fúngicos de F. pedrosoi. No entanto, eles são dispensáveis na resposta à infecção com o conídio, confirmando o papel desta forma fúngica na persistência do F. pedrosoi no hospedeiro como resultado da ativação inadequada da resposta imune e seus mecanismos efetores. _________________________________________________________________________________________ ABSTRACTFonsecaea pedrosoi is a dimorphic fungus, and the main etiologic agent of chromoblastomycosis, a chronic infection of the skin and subcutaneous tissue initiated after transcutaneous inoculation of saprophytic forms of the fungus. Inside the host, these cells turn into muriformes cells, considered the parasitic form, which produce large amount of melanin, an important virulence factor. Recent studies have suggested that the chronicity observed in chromoblastomycosis is due to both, failure in innate recognition of the fungus, leading to low production of pro-inflammatory cytokines, and development of an inappropriate immune response unable to contain the disease. In this context, and considering the inflammasome as an important innate recognition system that comprises antifungal immune responses, this study was conducted to evaluate inflammasome activation by F. pedrosoi, as well as its role in the infection induced by saprophytic forms of the fungus. First, it was observed that, in contrast to conidia, hyphae induced the secretion of IL-1, and provides both first and second signals for inflammasome activation in BMDMs and BMDCs. Exogenous stimulation of the first or second signal not only increased IL-1 secretion during infection with hyphae, but also enabled the secretion of IL-1 by BMDCs infected with conidia. Using inhibitors or knockout cells, it was found that IL-1β secretion is dependent on NLRP3, caspase-1 and influenced by caspase-8. In addition, we observed that IL-1 secretion mediated by NLRP3 inflammasome is dependent: on a NF-kB signaling pathway independent of MyD88; on efflux of K+; on cathepsin B release; and, in an unessential way, on generation of reactive oxygen species. Furthermore, it was demonstrated that melanin present in fungal cell wall, but not the secreted one, contribute to IL-1β secretion. It was further demonstrated that caspase-1, caspase-11 and NLRP3 receptor did not promote in vitro fungicidal activity of macrophages. However, data from in vivo infection studies suggest that caspase-1 and -11, as well as NLRP3, assist in the disease containment caused by infection with fungal propagules of F. pedrosoi. However, they are dispensable in response to infection with conidia, confirming the role of conidia in F. pedrosoi conidial form persistence in the host through the inappropriate activation of the immune response and its effectors mechanisms

Early immune response against Fonsecaea pedrosoi requires Dectin-2-mediated Th17 activity, whereas Th1 response, aided by Treg cells, is crucial for fungal clearance in later stage of experimental chromoblastomycosis.

Chromoblastomycosis (CBM) is a chronic worldwide subcutaneous mycosis, caused by several dimorphic, pigmented dematiaceous fungi. It is difficult to treat patients with the disease, mainly because of its recalcitrant nature. The correct activation of host immune response is critical to avoid fungal persistence in the tissue and disease chronification. CD4+ T cells are crucial for the development of protective immunity to F. pedrosoi infection. Here, we investigated T helper cell response dynamics during experimental CBM. Following footpad injection with F. pedrosoi hyphae and conidia, T cells were skewed towards a Th17 and Th1 phenotype. The Th17 population was the main Th cell subset found in the infected area during the early stages of experimental murine CBM, followed by Th1 predominance in the later stages, coinciding with the remission phase of the disease in this experimental model. Depletion of CD25+ cells, which leads to a reduction of Treg cells in the draining lymph node, resulted in decline in fungal burden after 14 days of infection. However, fungal cells were not cleared in the later stages of the disease, prolonging CBM clinical features in those animals. IL-17A and IFN-γ neutralization hindered fungal cell elimination in the course of the disease. Similarly, in dectin-2 KO animals, Th17 contraction in the course of experimental CBM was accompanied by fungal burden decrease in the first 14 days of infection, although it did not affect disease resolution. In this study, we gained insight into T helper subsets' dynamics following footpad injections of F. pedrosoi propagules and uncovered their contribution to disease resolution. The Th17 population proved to be important in eliminating fungal cells in the early stages of infection. The Th1 population, in turn, closely assisted by Treg cells, proved to be relevant not only in the elimination of fungal cells at the beginning of infection but also essential for their complete elimination in later stages of the disease in a mouse experimental model of CBM

Kicking sleepers out of bed: Macrophages promote reactivation of dormant Cryptococcus neoformans by extracellular vesicle release and non-lytic exocytosis.

Macrophages play a key role in disseminated cryptococcosis, a deadly fungal disease caused by Cryptococcus neoformans. This opportunistic infection can arise following the reactivation of a poorly characterized latent infection attributed to dormant C. neoformans. Here, we investigated the mechanisms underlying reactivation of dormant C. neoformans using an in vitro co-culture model of viable but non-culturable (VBNC; equivalent of dormant) yeast cells with bone marrow-derived murine macrophages (BMDMs). Comparative transcriptome analysis of BMDMs incubated with log, stationary phase or VBNC cells of C. neoformans showed that VBNC cells elicited a reduced transcriptional modification of the macrophage but retaining the ability to regulate genes important for immune response, such as NLRP3 inflammasome-related genes. We further confirmed the maintenance of the low immunostimulatory capacity of VBNC cells using multiplex cytokine profiling, and analysis of cell wall composition and dectin-1 ligands exposure. In addition, we evaluated the effects of classic (M1) or alternative (M2) macrophage polarization on VBNC cells. We observed that intracellular residence sustained dormancy, regardless of the polarization state of macrophages and despite indirect detection of pantothenic acid (or its derivatives), a known reactivator for VBNC cells, in the C. neoformans-containing phagolysosome. Notably, M0 and M2, but not M1 macrophages, induced extracellular reactivation of VBNC cells by the secretion of extracellular vesicles and non-lytic exocytosis. Our results indicate that VBNC cells retain the low immunostimulatory profile required for persistence of C. neoformans in the host. We also describe a pro-pathogen role of macrophage-derived extracellular vesicles in C. neoformans infection and reinforce the impact of non-lytic exocytosis and the macrophage profile on the pathophysiology of cryptococcosis

The Major Chromoblastomycosis Etiologic Agent Fonsecaea pedrosoi Activates the NLRP3 Inflammasome

Fonsecaea pedrosoi is the main etiologic agent of chromoblastomycosis (CBM), one of the most prevalent subcutaneous mycosis in tropical and subtropical countries. CBM is a poorly characterized chronic infection that commonly starts after transcutaneous inoculation of conidia and saprophytic hyphae of F. pedrosoi. Recently, we have shown that unlike conidia, hyphae and muriform cells (the parasitic morphotype) of F. pedrosoi promotes an intense inflammatory response pattern in vivo, which comprises the production of an inflammasome-derived cytokine, IL-1β. Nonetheless, the mechanisms underlying IL-1β production and maturation upon F. pedrosoi infection and its functional output in the course of CBM remains unknown. We show here that F. pedrosoi hyphae, differently from conidia, induce IL-1β secretion in both bone marrow-derived dendritic cells and macrophages. Using inhibitors and knockout cells, we demonstrated that the mechanisms underlying IL-1β production by hyphae-infected macrophages were dependent on dectin-1, -2, and -3 receptors and the Syk-NF-kB signaling pathway. Furthermore, F. pedrosoi promoted a NLRP3-dependent inflammasome activation, which required potassium efflux, reactive oxygen species production, phagolysosomal acidification, and cathepsin B release as triggers. IL-1β processing and release was mediated primarily by caspase-1 and, to a lesser extent, by caspase-8-dependent cleavage. Finally, we showed using a murine CBM model that F. pedrosoi elicits a NLRP3-regulated IL-1β and interleukin-18 release in vivo, but without NLRP3 inflammasome activation interfering in the course of the experimental infection

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