Neutrophil-suppressive activity over T-cell proliferation and fungal clearance in a murine model of Fonsecaea pedrosoi infection.

Neutrophils are essential to control several fungal infections. These cells are commonly known for their pro-inflammatory activities. However, some studies have demonstrated the anti-inflammatory properties of neutrophils during certain infectious diseases, culminating in the inhibition of T cell proliferation. Chromoblastomycosis (CBM) is a deep and progressive mycosis that affects thousands of people worldwide. Although neutrophil infiltrates are observed in the lesion histopathology, the fungus can overtake the immune system response and destroy the host-infected tissue. The present study demonstrated that neutropenic animals had an increase in the IL-6 production in the spleen and liver, followed by a lower fungal burden in these organs up to 14 days of infection. Neutropenic animals also showed a lower F. pedrosoi-specific antibody production 14-days post infection and higher T-cell proliferation in the in vitro experiments after stimulation with F. pedrosoi-purified proteins. Taken together, our results suggest that the presence of regulatory neutrophils in the mouse model of F. pedrosoi infection could act favoring the spread of the fungus and the chronicity of the infection. These findings shed light on the CBM treatment, which might target neutrophil polarization as a new therapy approach to treat CBM lesions.

The importance of Toll-like receptor 4 during experimental Sporothrix brasiliensis infection.

Here we investigated the importance of Toll-like receptor 4 (TLR-4) in innate immune response to Sporothrix brasiliensis, a virulent fungus of Sporothrix spp. In vitro assays, using C57Bl/6 (wild type [WT]) bone marrow-derived macrophages (BMDMs), and TLR-4 knockout (TLR-4-/-) showed that the absence of TLR-4 resulted in impaired phagocytosis and lower levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and nitric oxide. In vivo assays were also performed, and the mice (WT and TLR-4-/-) were intraperitoneally infected with S. brasiliensis yeast ATCC MyA-4831 and euthanized on days 7, 14, and 28 postinfection, with the following parameters evaluated: fungal burden in liver, spleen, kidney, and brain, and the production of cytokines interferon γ (IFN-γ), TNF-α, IL-2, IL-4, IL-6, and IL-10. The results demonstrate the macrophages dependency on TLR-4 for inflammatory activation and in the absence of TLR-4 during experimental S. brasiliensis infection enhanced dissemination occurred after 14 and 28 days. These data show that TLR-4 signals are important for the recognition of S. brasiliensis by macrophages, and their absence promotes the persistence of the infection.

The impact of the absence of Toll-like receptor-2 during Sporothrix brasiliensis infection.

PURPOSE: Sporothrix brasiliensis, a member of the Sporothrix schenckii complex, is a major cause of epidemic outbreaks of sporotrichosis due to its greater virulence and ability to evade the immune system. The absence of studies about this species led to this study, with the aim to evaluate the importance of Toll-like receptor-2 (TLR-2) during S. brasiliensis infection. METHODOLOGY: In vitro assays were performed using bone marrow-derived macrophages from both wild-type (C57BL/6) and TLR-2 knockout (-/-) mice. In vivo assays were also performed, on which the mice (C57BL/6 and TLR-2-/-) were intraperitoneally infected with S. brasiliensis yeast American Type Culture Collection MYA-4831 and euthanized on days 7, 14 and 28 post infection. The following parameters were then evaluated: fungal burden in spleen, liver, kidney and brain; the production of cytokines TNF-α, IFN-γ, IL-4, IL-2, IL-6 and IL-10. RESULTS: The in vitro results showed that the absence of TLR-2 resulted in impaired phagocytosis, microbicide mechanisms utilizing the production of nitric oxide, and the cytokine production (TNF-α, IL-6 and IL-10). The in vivo results demonstrated that the absence of TLR-2 during experimental S. brasiliensis infection promoted increased dissemination after 14 and 28 days and suggests a polarized Th17 response in an attempt to control the infection. CONCLUSIONS: TLR-2 signalling appears to be important in the innate immune response against S. brasiliensis.

An In Vitro Model for the Study of the Macrophage Response Upon Trichophyton rubrum Challenge.

Diversity in the macrophage models currently employed in immunology studies may lead to opposed results and interpretations. In this study, we aimed to analyze the suitability of J774 macrophage-like cells as a model for the interaction between the dermatophyte Trichophyton rubrum and macrophages. J774 cells were competent in fungal phagocytosis, but succumbed to hyphal growth. Nevertheless, they could also secrete IL-1ß in response to the dermatophyte. On the opposite direction, inflammatory, thioglycollate-induced peritoneal macrophages did not succumb to fungal growth and showed no significant IL-1ß production. The proteomic profiling of these cells uncovered vimentin and plastin-2 as proteins whose abundance was altered by the fungal interaction. Our study indicates that this cell line could be an interesting tool in the investigation of T. rubrum infection biology.

IL-1 signaling inhibits Trichophyton rubrum conidia development and modulates the IL-17 response in vivo.

Dermatophytosis are one of the most common fungal infections in the world. They compromise keratinized tissues and the main etiological agent is Trichophyton rubrum. Macrophages are key cells in innate immunity and prominent sources of IL-1ß, a potent inflammatory cytokine whose main production pathway is by the activation of inflammasomes and caspase-1. However, the role of inflammasomes and IL-1 signaling against T.rubrum has not been reported. In this work, we observed that bone marrow-derived macrophages produce IL-1ß in response to T.rubrum conidia in a NLRP3-, ASC- and caspase-1-dependent fashion. Curiously, lack of IL-1 signaling promoted hyphae development, uncovering a protective role for IL-1ß in macrophages. In addition, mice lacking IL-1R showed reduced IL-17 production, a key cytokine in the antifungal defense, in response to T.rubrum. Our findings point to a prominent role of IL-1 signaling in the immune response to T.rubrum, opening the venue for the study of this pathway in other fungal infections.

O papel da flagelina e do sistema de secreção de Escherichia coli enteroinvasora na resposta imune inata dos macrófagos / The role of flagellin and secretion system of enteroinvasive Escherichia coli in the immune response innate macrophages

Escherichia coli enteroinvasora (EIEC) é um dos agentes etiológicos da disenteria bacilar. Seu processo fisiopatológico é desencadeado pela expressão de fatores de virulência, que proporcionam sua invasão e sobrevivência nas células do hospedeiro, ativando o sistema imune inato e adaptativo da mucosa intestinal. Trabalhos recentes têm salientado a importância do sistema de secreção e da flagelina bacteriana como agonista de receptores da imuninade inata dos macrófagos, em especial alguns dos receptores do tipo NLR. Uma vez que esta espécie de E. coli também é capaz de expressar flagelina e fazer a montagem completa do flagelo e do sistema de secreção do tipo III, a nossa proposta foi avaliar o papel da flagelina e do sistema de secreção de EIEC na resposta imune dos macrófagos murinos. Para isso, utilizamos três cepas de EIEC: a cepa selvagem; a cepa mutante no gene responsável pela síntese da flagelina; e a cepa sem o plasmídio de virulência plnv, deficiente no sistema de secreção, para a infecção de macrófagos peritoniais de camundongos C57BI/6, caspase-1-/-, IPAF-/- e ASC-/-. Neste estudo foi possível observar que o escape bacteriano e a morte dos macrófagos infectados por EIEC, assim como a ativação da caspase-1 e posterior secreção de IL-1ß é independente da flagelina bacteriana, mas dependente do sistema de secreção, além disso, a ativação da caspase-1 de macrófagos infectados por EIEC é dependente do receptor IPAF e parcialmente da proteína adaptadora ASC. Assim, no nosso modelo, a ativação da caspase-1 dos macrófagos infectados por EIEC parece estar envolvida com o processamento e secreção de IL-1ß e, possivelmente na secreção de IL-18, mas não na morte celular. No modelo de infecção in vivo, o sistema de secreção bacteriano foi importante para a sobrevivência bacteriana no hospedeiro, assim como para a indução de uma resposta inflamatória no local da infecção. Ainda, a caspase-1 parece ter um papel importante para o controle da infecção in vivo por EIEC, podendo assim contribuir para uma resposta imune protetora do hospedeiro

Enteroinvasive Escherichia coli (EIEC) is one of the etiologic agents responsible for bacillary dysentery. The pathophysiological process induced by this bacteria is triggered by the expression of virulence factors that provide the invasion and survival in host cells, resulting in activation of innate and adaptive immune system present on intestinal mucosa. Recent studies have emphasized the importance of the secretion system and bacterial flagellin as agonist of innate immune receptors present in macrophage, especially NLR (Nod like receptors). Then, our proposal was evaluate the role of flagellin (f1iC) and secretion system of EIEC in the induction of immune response of murine macrophages using the EIEC strains wild type (WT), mutant flagellin gene (f1iC), and a strain deficient in secretion system (DSS) for infection of peritoneal macrophages of C57Bl/6, caspase-1-/-, IPAF-/- and ASC-/-- mice. In this study we observed that the bacterial escape and death of infected macrophages with EIEC, the caspase-1 activation and subsequent IL-1ß secretion is independent of bacterial flagellin, but dependent of secretion system, moreover, the caspase-1 activation in infected macrophages is IPAF-dependent and partially dependent of the adapter protein ASC. Thus, in our model, the caspase-1 activation in EIEC infected macrophages seems to be involved with the processing and secretion of IL-1ß and possibly with the secretion of IL-18, but not involved with cell death. In the infection model in vivo, bacterial secretion system was important for bacterial survival in the host, as well as for the inflammatory response induction at the infection site. In addition, caspase-1 seems to have an important role to the control of in vivo infection by EIEC and can contribute to a protective immune response of the host

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states.

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Enteroinvasive Escherichia coli vs. Shigella flexneri: how different patterns of gene expression affect virulence.

Important features of the enteroinvasive Escherichia coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.