An Alpha-Glucan from Lomentospora prolificans Mediates Fungal-Host Interaction Signaling through Dectin-1 and Mincle.

Scedosporium and Lomentospora are a group of filamentous fungi with some clinically relevant species causing either localized, invasive, or disseminated infections. Understanding how the host immune response is activated and how fungi interact with the host is crucial for a better management of the infection. In this context, an α-glucan has already been described in S. boydii, which plays a role in the inflammatory response. In the present study, an α-glucan has been characterized in L. prolificans and was shown to be exposed on the fungal surface. The α-glucan is recognized by peritoneal macrophages and induces oxidative burst in activated phagocytes. Its recognition by macrophages is mediated by receptors that include Dectin-1 and Mincle, but not TLR2 and TLR4. These results contribute to the understanding of how Scedosporium's and Lomentospora's physiopathologies are developed in patients suffering with scedosporiosis and lomentosporiosis.

Peptidorhamnomannan negatively modulates the immune response in a scedosporiosis murine model.

In this study, we analyzed the impact of immunization with the peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium (Lomentospora) prolificans in a murine model of invasive scedosporiosis. Immunization with PRM decreased the survival of mice infected with S. prolificans. Immunization of mice with PRM led to decreased secretion of pro-inflammatory cytokines and chemokines but did not affect the secretion of IL-10. Mice immunized with PRM showed an increase in IgG1 secretion, which is an immunoglobulin linked to a nonprotective response. Splenocytes isolated from mice infected with S. prolificans and immunized with PRM showed no differences in the percentages of Th17 cells and no increase in the frequency of the CD4(+)CD62L(Low) T cell population. PRM-immunized mice showed a significant increase in the percentage of Treg cells. In summary, our results indicated that immunization with PRM did not assist or improve the immunological response against S. prolificans infection. PRM exacerbated the infection process by reducing the inflammatory response, thereby facilitating colonization, virulence and dissemination by the fungus.

O-glycosylation in cell wall proteins in Scedosporium prolificans is critical for phagocytosis and inflammatory cytokines production by macrophages.

In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines.

High in vitro immune reactivity to Escherichia coli in neuromyelitis optica patients is correlated with both neurological disabilities and elevated plasma lipopolysaccharide levels.

The pathogenesis of neuromyelitis optica (NMO) is influenced by a combination of genetic and environmental factors, including infectious agents. Several infectious diseases can both trigger or exacerbate autoimmunity. The objective of the present work was to evaluate the in vitro immune responsiveness to Escherichia coli (EC), Staphylococcus aureus (SA) and Candida albicans (CA) in remittent-recurrent NMO patients, and correlate it with the level of neurological disability. Our results revealed that the extent of lymphoproliferation and cytokine profile in response to SA- and CA-stimulated PBMC cultures was similar between NMO patients and healthy individuals. Nevertheless, a higher in vitro CD4(+) T cell proliferation associated with elevated IL-1ß, IL-6 and IL-17 release was observed in NMO-derived EC-stimulated cell cultures. Additionally, in these last cultures, the IL-10 production was significantly lower as compared with control group. The in vitro EC-induced levels of IL-6 and IL-17 were positively related with neurological disabilities. This higher tendency to produce Th17-related cytokines was proportional to the production of IL-23 and IL-6 by LPS-activated monocytes. Interestingly, elevated LPS levels were quantified in the plasma of NMO patients. The results suggest that a higher Th17-responsiveness to E. coli could be involved in the NMO pathogenesis.

Low sensitivity to glucocorticoid inhibition of in vitro Th17-related cytokine production in multiple sclerosis patients is related to elevated plasma lipopolysaccharide levels.

Exogenous glucocorticoid plays an important role in controlling clinical relapses of multiple sclerosis (MS), but the response to this treatment differs among patients. In this study, T-cell proliferation and IL-17 production were less sensitive to hydrocortisone (HC) inhibition in MS patients than healthy individuals, mainly in CD8(+) compartment. Furthermore, in vitro IL-17 production was positively related with neurological disability and its release was proportional to IL-23 and IL-6 productions by LPS-activated monocytes. Interestingly, elevated LPS levels were quantified in the plasma of MS patients, and their levels were directly related to in vivo IL-6 production. Finally, HC-resistance in reducing IL-17 production by polyclonally-activated CD8(+) T cells was particularly observed among MS patients with higher in vivo LPS levels. In summary, the results indicate that T-cells derived from MS patients show an enhanced Th17-like phenotype that is directly associated with neurological disability, resistance to glucocorticoid inhibition and elevated bacterial translocation.

The impact of maternal anti-retroviral therapy on cytokine profile in the uninfected neonates.

The number of HIV-infected young women has been increasing since the beginning of the AIDS epidemic. The objective of the present study was to investigate the impact of anti-retroviral treatment (ART) of HIV-1-infected pregnant women (PW) on cytokine profile of uninfected neonates. Our results demonstrated that higher levels of IL-1ß and TNF-α associated with lower IL-10 production were detected in the plasma obtained from neonates born from ART-treated PW. Furthermore, the production of TNF- α and IFN-γ was also significantly higher in polyclonally-activated T cells from those neonates. This elevated pro-inflammatory pattern detected by these activated-T cells was not associated to HIV-1 antigens sensitization. Finally, ART-exposed neonates showed to be born with lower weight, and it was inversely correlated with maternal peripheral TNF-a level. In summary, the data presented here suggest a significant disturbance in cytokine network of HIV-1-uninfected neonates exposed to potent anti-retroviral schemes during pregnancy.

The ex vivo production of IL-6 and IL-21 by CD4+ T cells is directly associated with neurological disability in neuromyelitis optica patients.

Neuromyelitis optica (NMO), also known as Devic's disease, is an autoimmune, inflammatory disorder of the central nervous system (CNS) in which the immune system attacks myelin of the neurons located at the optic nerves and spinal cord, thus producing a simultaneous or sequential optic neuritis and myelitis. The objective of this study was evaluated the background T-cell function of patients suffering from neuromyelitis optica (NMO), an autoimmune disorder of the central nervous system. In our study, the in vitro T cell proliferation and the production of Th1 cytokines were significantly lower in cell cultures from NMO patients, as compared with healthy individuals. In contrast, a dominant Th17-like phenotype, associate with higher IL-23 and IL-6 production by LPS-activated monocytes, was observed among NMO patients. The release of IL-21 and IL-6 by polyclonally activated CD4+ T cells was directly correlated to neurological disability. In addition, the in vitro release of IL-21, IL-6 and IL-17 was significantly more resistant to glucocorticoid inhibition in NMO patients. In conclusion, the results indicate dominant Th17-related response in NMO patients that was directly proportional to neurological disability. Furthermore, our results can help to explain why NMO patients trend to be more refractory to corticoid treatment.

Toll-like receptors (TLR2 and TLR4) recognize polysaccharides of Pseudallescheria boydii cell wall.

Pseudallescheria boydii is an opportunistic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections in both immunocompromised and immunocompetent hosts. The host response to fungi is in part dependent on the activation of evolutionary conserved receptors including Toll-like receptors and phagocytic receptors. This review will discuss the isolation and structural characterization of α-glucans and rhamnomannans from P. boydii cell wall and their roles in the induction of innate immune response.

Glycoconjugates and polysaccharides from the Scedosporium/Pseudallescheria boydii complex: structural characterisation, involvement in cell differentiation, cell recognition and virulence.

Peptidorhamnomannans (PRMs), rhamnomannans and α-glucans are especially relevant for the architecture of the Scedosporium/Pseudallescheria boydii cell wall, but many of them are immunologically active, with great potential as regulators of pathogenesis and the immune response of the host. In addition, some of them can be specifically recognised by antibodies from the sera of patients, suggesting that they could also be useful in diagnosis of fungal infections. Their primary structures have been determined, based on a combination of techniques including gas chromatography, electrospray ionization - mass spectrometry (ESI-MS), (1)H-COSY and TOCSY, (13)C and (1)H/(13)C NMR spectroscopy. Using monoclonal antibodies to PRM, we showed that it is involved in germination and viability of P. boydii conidia, in the phagocytosis of P. boydii conidia by macrophages and non-phagocytic cells and in the survival of mice with P. boydii infection. Also, components of the fungal cell wall, such as α-glucans, are involved. Rhamnomannans are immunostimulatory and participate in the recognition and uptake of fungal cells by the immune system. These glycosylated polymers, being present in the fungal cell wall, are mostly absent from mammalian cells, and are excellent targets for the design of new agents capable of inhibiting fungal growth and differentiation of pathogens.

Dopamine up-regulates Th17 phenotype from individuals with generalized anxiety disorder.

Our objective was to evaluate the effect of stress-related dose of dopamine (DA) on the in vitro proliferation and cytokine production in polyclonally-activated T cells from healthy individuals or individuals with generalized anxiety disorder (GAD). Our results demonstrated that cell cultures from GAD group proliferated less following T cell activation, as compared with control group. The addition of DA reduced the proliferative response in cell cultures from healthy but not from GAD individuals. The cytokine profile in GAD individuals revealed Th1 and Th2 deficiencies associated with a dominant Th17 phenotype, which was enhanced by DA. A similar DA-induced immunomodulation was also observed in PPD-activated cell cultures from GAD individuals. Unlike the control, DA-enhanced Th17 cytokine production in GAD individuals was not affected by glucocorticoid. In conclusion, our results show that the T cell functional dysregulation in GAD individuals is significantly amplified by DA. These immune abnormalities can have impact in increasing the susceptibility of individuals with anxiety disorders to infectious diseases and inflammatory/autoimmune disorders.

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans.

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Monoclonal antibodies against peptidorhamnomannans of Scedosporium apiospermum enhance the pathogenicity of the fungus.

Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs) are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1) to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis.

ß-Galactofuranose-containing structures present in the cell wall of the saprophytic fungus Cladosporium (Hormoconis) resinae.

A peptidogalactomannan was isolated from mycelia of Cladosporium (Hormoconis) resinae and characterized using methylation-fragmentation analysis, partial acid hydrolysis and ¹H and ¹³C-NMR spectroscopy. The galactomannan component was a branched structure and consisted of a main chain containing (1→6)-linked α-d-Manp residues substituted at O-2 by side chains containing (1→2)-linked α-D-Manp residues. ß-D-Galf residues were present as side chains of 3-4 units that are (1→5)-interlinked. This structure is very similar to a pGM isolated from Aspergillus fumigatus and differs from that of Cladosporium werneckii (currently named Hortaea werneckii), with this pGM and other fungal galactomannans having single terminal (1→6)-linked ß-Galf residues. The importance of the carbohydrate moiety of Cladosporium resinae pGM in immunoassays was also demonstrated. On FACS examination, a decrease (60%) in rabbit serum anti- C. resinae binding to C. resinae conidia occurred when this serum had been previously incubated with pGMs from C. resinae and A. fumigatus or with mannoprotein from Candida parapsilosis, suggesting the presence of cross-reactive determinants in these fungi.

Biochemical characterization of potential virulence markers in the human fungal pathogen Pseudallescheria boydii.

The ubiquitous Pseudallescheria boydii (anamorph Scedosporium apiospermum) is a saprophytic filamentous fungus recognized as a potent etiologic agent of a wide variety of infections in immunocompromised as well as in immunocompetent patients. Very little is known about the virulence factors expressed by this fungal pathogen. The present review provides an overview of recent discoveries related to the identification and biochemical characterization of potential virulence attributes produced by P. boydii, with special emphasis on surface and released molecules. These structures include polysaccharides (glucans), glycopeptides (peptidorhamnomannans), glycolipids (glucosylceramides) and hydrolytic enzymes (proteases, phosphatases and superoxide dismutase), which have been implicated in some fundamental cellular processes in P. boydii including growth, differentiation and interaction with host molecules. Elucidation of the structure of cell surface components as well as the secreted molecules, especially those that function as virulence determinants, is of great relevance to understand the pathogenic mechanisms of P. boydii.

An alpha-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and Toll-like receptor activation.

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an alpha-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that alpha-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked alpha-D-Glcp residues substituted at position 6 with alpha-D-Glcp branches. Soluble alpha-glucan, but not beta-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when alpha-glucan from conidial surface was removed by enzymatic treatment with alpha-amyloglucosidase, thus indicating an essential role of alpha-glucan in P. boydii internalization by macrophages. alpha-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with alpha-amyloglucosidase. Finally, alpha-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and alpha-glucan could be a target for intervention during fungal infections.

Beta-galactofuranose-containing O-linked oligosaccharides present in the cell wall peptidogalactomannan of Aspergillus fumigatus contain immunodominant epitopes.

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, 1H COSY and TOCSY, and 1H-13C HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5]3-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.