Turning up the heat : inflammasome activation by fungal pathogens

Since its first description in 2002 [1], the inflammasome has been implicated in the mechanisms underlying a growing number of infectious, autoimmune, and metabolic diseases [2]. Regarding infectious processes, several studies have shown the involvement of this critical component of innate immunity in the outcome of infection with nearly every class of microbe, including fungi [3]. Innate immunity is the frontline of defense against infection and relies on the ability of its main players (phagocytes and epithelial barriers) to detect conserved components of microbes or pathogen-associated molecular patterns (PAMPs). In fungi, the carbohydrate polymers of the cell wall, such as chitin, β-glucan, and mannan are the major PAMPs recognized by the host’s innate immune cells; this recognition occurs via germline-encoded receptors termed pattern recognition receptors (PRRs) [4]. In addition to PAMPs, endogenous molecules associated with damaged host cells, or damage-associated molecular patterns (DAMPs), are released during tissue injury and activate PRRs. This innate detection system includes the Toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and AIM2-like receptors (ALRs). Although the main fungal- recognition PRRs (CLRs and TLRs) are bound to the cytoplasmic membrane of innate immune cells [4], fungal sensing by PRRs located in the cytosol, such as the NLRs and ALRs, is becoming increasingly evident. A number of NLRs and ALRs can assemble into the inflammasome, a multiprotein complex consisted of PRRs such as NLRP3 (NLR family, pyrin domain-containing 3), NLRC4, or AIM2, adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD), and procaspase-1 [3]. Upon formation of the complex, procaspase-1 is cleaved into an active cysteine protease, which further cleaves the proinflammatory cytokines IL-1β and IL-18 into their mature forms, followed by unconventional secretion. IL-1β and IL-18 mediate several innate antimicrobial responses and are critical to direct adaptive Th17/Th1 cellular responses [5]. In addition, inflammasome activation causes pyroptosis, a lytic inflammatory form of cell death [2,5]

NLRP3 inflammasome activation by Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is the etiologic agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis that is geographically confined to Latin America. The pro-inflammatory cytokine IL-1b that is mainly derived from the activation of the cytoplasmic multiprotein complex inflammasome is an essential host factor against opportunistic fungal infections; however, its role in infection with a primary fungal pathogen, such as P. brasiliensis, is not well understood. In this study, we found that murine bone marrow-derived dendritic cells responded to P. brasiliensis yeast cells infection by releasing IL-1b in a spleen tyrosine kinase (Syk), caspase-1 and NOD-like receptor (NLR) family member NLRP3 dependent manner. In addition, P. brasiliensis-induced NLRP3 inflammasome activation was dependent on potassium (K+) efflux, reactive oxygen species production, phagolysosomal acidification and cathepsin B release. Finally, using mice lacking the IL- 1 receptor, we demonstrated that IL-1b signaling has an important role in killing P. brasiliensis by murine macrophages. Altogether, our results demonstrate that the NLRP3 inflammasome senses and responds to P. brasiliensis yeast cells infection and plays an important role in host defense against this fungus

Effects of metoclopramide on the expression of metalloproteinases and interleukins in left colonic anastomoses : an experimental study

PURPOSE: To evaluate the effects of metoclopramide on metalloproteinases (MMP) and interleukins (IL) gene expression in colonic anastomoses in rats. METHODS: Eighty rats were divided into two groups for euthanasia on the 3rd or 7th postoperative day (POD), then into two subgroups for sepsis induction or not, and then into subgroups to receive either metoclopramide or saline solution. Left colonic anastomosis were performed and then analyzed. RESULTS : On the 3rd POD, metoclopramide was associated with increased expression of MMP-1a, MMP-13, and TNF-α. On the 7th POD, the transcripts of all MMPs, TNF-α, IL-1β, IFN-γ, and IL-10 of the treated animals became negatively modulated. In the presence of sepsis, metoclopramide did not change MMPs and decreased IL-6, IL-1β, IFN-γ and IL-10 gene expression on the 3rd POD. On the 7th POD, increased expression of all MMPs, IFN-γ and IL-10 and negative modulated TNF-α and IL-6 gene expression. CONCLUSION: Administration of metoclopramide increased metalloproteinases and interleukins gene expression on the 3rd postoperative day and negatively modulated them on the 7th POD. In the presence of abdominal sepsis, metoclopramide did not change MMPs and decreased ILs gene expression on the 3rd POD. On the 7th POD, the drug increased expression of all MMPs

ScFv from antibody that mimics gp43 modulates the cellular and humoral immune responses during Experimental Paracoccidioidomycosis

Paracoccidioidomycosis (PCM), caused by Paracoccidioides species is a prevalent systemic and progressive mycosis that occurs in Latin America. It is caused by Paracoccidioides species. Immunization with dendritic cells transfected with a plasmid encoding the scFv (pMAC/PS-scFv) that mimics the main antigen of P. brasiliensis (gp43) confers protection in experimental PCM. DCs link innate and adaptive immunity by recognizing invading pathogens and selecting the type of effector T cell to mediate the immune response. Here, we showed that DC-pMAC/PS-scFv induces the activation of CD4+ and CD8+ T cells. Moreover, our results demonstrated that BALB/c mice infected with P. brasiliensis and treated with DC-pMAC/PS-scFv showed the induction of specific IgG production against gp43 and IFN-γ, IL-12 and IL-4 cytokines. Analysis of regional lymph nodes revealed increases in the expression of clec7a, myd88, tlr2, gata3 and tbx21, which are involved in the immune response. Taken together, our results indicate that the scFv modulates the humoral and cellular immune responses and presents epitopes to CD4+ and CD8+ T cells

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

Activity of scorpion venom-derived antifungal peptides against planktonic cells of Candida spp. and Cryptococcus neoformans and Candida albicans Biofilms

The incidence of fungal infections has been increasing in the last decades, while the number of available antifungal classes remains the same. The natural and acquired resistance of some fungal species to available therapies, associated with the high toxicity of these drugs on the present scenario and makes an imperative of the search for new, more efficient and less toxic therapeutic choices. Antimicrobial peptides (AMPs) are a potential class of antimicrobial drugs consisting of evolutionarily conserved multifunctional molecules with both microbicidal and immunomodulatory properties being part of the innate immune response of diverse organisms. In this study, we evaluated 11 scorpion-venom derived non-disulfide-bridged peptides against Cryptococcus neoformans and Candida spp., which are important human pathogens. Seven of them, including two novel molecules, showed activity against both genera with minimum inhibitory concentration values ranging from 3.12 to 200 μM and an analogous activity against Candida albicans biofilms. Most of the peptides presented low hemolytic and cytotoxic activity against mammalian cells. Modifications in the primary peptide sequence, as revealed by in silico and circular dichroism analyses of the most promising peptides, underscored the importance of cationicity for their antimicrobial activity as well as the amphipathicity of these molecules and their tendency to form alpha helices. This is the first report of scorpion-derived AMPs against C. neoformans and our results underline the potential of scorpion venom as a source of antimicrobials. Further characterization of their mechanism of action, followed by molecular optimization to decrease their cytotoxicity and increase antimicrobial activity, is needed to fully clarify their real potential as antifungals

Transcriptional remodeling patterns in murine dendritic cells infected with Paracoccidioides brasiliensis : more is not necessarily better

Most people infected with the fungus Paracoccidioides spp. do not get sick, but approximately 5% develop paracoccidioidomycosis. Understanding how host immunity determinants influence disease development could lead to novel preventative or therapeutic strategies; hence, we used two mouse strains that are resistant (A/J) or susceptible (B10.A) to P. brasiliensis to study how dendritic cells (DCs) respond to the infection. RNA sequencing analysis showed that the susceptible strain DCs remodeled their transcriptomes much more intensely than those from the resistant strain, agreeing with a previous model of more intense innate immunity response in the susceptible strain. Contrastingly, these cells also repress genes/processes involved in antigen processing and presentation, such as lysosomal activity and autophagy. After the interaction with P. brasiliensis, both DCs and macrophages from the susceptible mouse reduced the autophagy marker LC3-II recruitment to the fungal phagosome compared to the resistant strain cells, confirming this pathway’s repression. These results suggest that impairment in antigen processing and presentation processes might be partially responsible for the inefficient activation of the adaptive immune response in this model

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

Transcriptional profiles of the human pathogenic fungus paracoccidioides brasiliensis in mycelium and yeast cells

This work was supported by MCT, CNPq, CAPES, FUB, UFG, and FUNDECT-MS. PbGenome Network: Alda Maria T. Ferreira, Alessandra Dantas, Alessandra J. Baptista, Alexandre M. Bailão, Ana Lídia Bonato, André C. Amaral, Bruno S. Daher, Camila M. Silva, Christiane S. Costa, Clayton L. Borges, Cléber O. Soares, Cristina M. Junta, Daniel A. S. Anjos, Edans F. O. Sandes, Eduardo A. Donadi, Elza T. Sakamoto-Hojo, Flábio R. Araújo, Flávia C. Albuquerque, Gina C. Oliveira, João Ricardo M. Almeida, Juliana C. Oliveira, Kláudia G. Jorge, Larissa Fernandes, Lorena S. Derengowski, Luís Artur M. Bataus, Marcus A. M. Araújo, Marcus K. Inoue, Marlene T. De-Souza, Mauro F. Almeida, Nádia S. Parachin, Nadya S. Castro, Odair P. Martins, Patrícia L. N. Costa, Paula Sandrin-Garcia, Renata B. A. Soares, Stephano S. Mello, and Viviane C. B. ReisParacoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets