Phagocytosis Of Conidia Research Articles

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

Surfactant protein D inhibits growth, alters cell surface polysaccharide exposure and immune activation potential of Aspergillus fumigatus

Humoral immunity plays a defensive role against invading microbes. However, it has been largely overlooked with respect to Aspergillus fumigatus, an airborne fungal pathogen. Previously, we have demonstrated that surfactant protein D (SP-D), a major humoral component in human lung-alveoli, recognizes A. fumigatus conidial surface exposed melanin pigment. Through binding to melanin, SP-D opsonizes conidia, facilitates conidial phagocytosis, and induces the expression of protective pro-inflammatory cytokines in the phagocytic cells. In addition to melanin, SP-D also interacts with galactomannan (GM) and galactosaminogalactan (GAG), the cell wall polysaccharides exposed on germinating conidial surfaces. Therefore, we aimed at unravelling the biological significance of SP-D during the germination process. Here, we demonstrate that SP-D exerts direct fungistatic activity by restricting A. fumigatus hyphal growth. Conidial germination in the presence of SP-D significantly increased the exposure of cell wall polysaccharides chitin, α-1,3-glucan and GAG, and decreased β-1,3-glucan exposure on hyphae, but that of GM was unaltered. Hyphae grown in presence of SP-D showed positive immunolabelling for SP-D. Additionally, SP-D treated hyphae induced lower levels of pro-inflammatory cytokine, but increased IL-10 (anti-inflammatory cytokine) and IL-8 (a chemokine) secretion by human peripheral blood mononuclear cells (PBMCs), compared to control hyphae. Moreover, germ tube surface modifications due to SP-D treatment resulted in an increased hyphal susceptibility to voriconazole, an antifungal drug. It appears that SP-D exerts its anti-A. fumigatus functions via a range of mechanisms including hyphal growth-restriction, hyphal surface modification, masking of hyphal surface polysaccharides and thus altering hyphal immunostimulatory properties.

Platelets are critical for survival and tissue integrity during murine pulmonary Aspergillus fumigatus infection.

Beyond their canonical roles in hemostasis and thrombosis, platelets function in the innate immune response by interacting directly with pathogens and by regulating the recruitment and activation of immune effector cells. Thrombocytopenia often coincides with neutropenia in patients with hematologic malignancies and in allogeneic hematopoietic cell transplant recipients, patient groups at high risk for invasive fungal infections. While neutropenia is well established as a major clinical risk factor for invasive fungal infections, the role of platelets in host defense against human fungal pathogens remains understudied. Here, we examined the role of platelets in murine Aspergillus fumigatus infection using two complementary approaches to induce thrombocytopenia without concurrent neutropenia. Thrombocytopenic mice were highly susceptible to A. fumigatus challenge and rapidly succumbed to infection. Although platelets regulated early conidial phagocytosis by neutrophils in a spleen tyrosine kinase (Syk)-dependent manner, platelet-regulated conidial phagocytosis was dispensable for host survival. Instead, our data indicated that platelets primarily function to maintain hemostasis and lung integrity in response to exposed fungal antigens, since thrombocytopenic mice exhibited severe hemorrhage into the airways in response to fungal challenge in the absence of overt angioinvasion. Challenge with swollen, heat-killed, conidia was lethal in thrombocytopenic hosts and could be reversed by platelet transfusion, consistent with the model that fungus-induced inflammation in platelet-depleted mice was sufficient to induce lethal hemorrhage. These data provide new insights into the role of platelets in the anti-Aspergillus host response and expand their role to host defense against filamentous molds.

Trichophyton rubrum Elicits Phagocytic and Pro-inflammatory Responses in Human Monocytes Through Toll-Like Receptor 2.

Dermatophytosis is a superficial fungal infection mostly restricted to keratinized tissues such as skin, hair, and nails but with potential to cause invasive or even systemic disease in immunocompromised patients. Trichophyton rubrum is the main etiologic agent, accounting for approximately 80% of the cases. Mononuclear phagocytes respond to pathogens through phagocytosis followed by production of several antimicrobial molecules, such as reactive oxygen and nitrogen species, and failure in doing so may contribute to development of chronic fungal infections. Toll-like receptors (TLRs) located on the surface of phagocytic cells bind either directly to target particles or through opsonizing ligands and trigger an actin-mediated ingestion. Even though the mechanisms involved in TLR-mediated cytokine responses are well established, the contribution of TLR in the recognition of T. rubrum by adherent monocytes remains unclear. Here, we report that phagocytosis of T. rubrum conidia by adherent monocytes is mediated by TLR2. Blockade of TLR2 by neutralizing antibodies impaired the fungicidal activity of monocytes as well their secretion of tumor necrosis factor (TNF)-α, but neither nitric oxide (NO) production nor interleukin (IL)-10 secretion was disturbed. So far, our data suggest that TLR2 is required for efficient conidial phagocytosis, and the absence of TLR2 signaling in human monocytes may impair the subsequent inflammatory response. These findings expand our understanding of phagocyte modulation by this important fungal pathogen and may represent a potential target for interventions aiming at enhancing antifungal immune responses.

Macrophages protect Talaromyces marneffei conidia from myeloperoxidase-dependent neutrophil fungicidal activity during infection establishment in vivo.

Neutrophils and macrophages provide the first line of cellular defence against pathogens once physical barriers are breached, but can play very different roles for each specific pathogen. This is particularly so for fungal pathogens, which can occupy several niches in the host. We developed an infection model of talaromycosis in zebrafish embryos with the thermally-dimorphic intracellular fungal pathogen Talaromyces marneffei and used it to define different roles of neutrophils and macrophages in infection establishment. This system models opportunistic human infection prevalent in HIV-infected patients, as zebrafish embryos have intact innate immunity but, like HIV-infected talaromycosis patients, lack a functional adaptive immune system. Importantly, this new talaromycosis model permits thermal shifts not possible in mammalian models, which we show does not significantly impact on leukocyte migration, phagocytosis and function in an established Aspergillus fumigatus model. Furthermore, the optical transparency of zebrafish embryos facilitates imaging of leukocyte/pathogen interactions in vivo. Following parenteral inoculation, T. marneffei conidia were phagocytosed by both neutrophils and macrophages. Within these different leukocytes, intracellular fungal form varied, indicating that triggers in the intracellular milieu can override thermal morphological determinants. As in human talaromycosis, conidia were predominantly phagocytosed by macrophages rather than neutrophils. Macrophages provided an intracellular niche that supported yeast morphology. Despite their minor role in T. marneffei conidial phagocytosis, neutrophil numbers increased during infection from a protective CSF3-dependent granulopoietic response. By perturbing the relative abundance of neutrophils and macrophages during conidial inoculation, we demonstrate that the macrophage intracellular niche favours infection establishment by protecting conidia from a myeloperoxidase-dependent neutrophil fungicidal activity. These studies provide a new in vivo model of talaromycosis with several advantages over previous models. Our findings demonstrate that limiting T. marneffei’s opportunity for macrophage parasitism and thereby enhancing this pathogen’s exposure to effective neutrophil fungicidal mechanisms may represent a novel host-directed therapeutic opportunity.

Fungal melanin stimulates surfactant protein D–mediated opsonization of and host immune response to Aspergillus fumigatus spores

Surfactant protein D (SP-D), a C-type lectin and pattern-recognition soluble factor, plays an important role in immune surveillance to detect and eliminate human pulmonary pathogens. SP-D has been shown to protect against infections with the most ubiquitous airborne fungal pathogen, Aspergillus fumigatus, but the fungal surface component(s) interacting with SP-D is unknown. Here, we show that SP-D binds to melanin pigment on the surface of A. fumigatus dormant spores (conidia). SP-D also exhibited an affinity to two cell-wall polysaccharides of A. fumigatus, galactomannan (GM) and galactosaminogalactan (GAG). The immunolabeling pattern of SP-D was punctate on the conidial surface and was uniform on germinating conidia, in accordance with the localization of melanin, GM, and GAG. We also found that the collagen-like domain of SP-D is involved in its interaction with melanin, whereas its carbohydrate-recognition domain recognized GM and GAG. Unlike un-opsonized conidia, SP-D-opsonized conidia were phagocytosed more efficiently and stimulated the secretion of proinflammatory cytokines by human monocyte-derived macrophages. Furthermore, SP-D-/- mice challenged intranasally with wildtype conidia or melanin ghosts (i.e. hollow melanin spheres) displayed significantly reduced proinflammatory cytokines in the lung compared with wildtype mice. In summary, SP-D binds to melanin present on the dormant A. fumigatus conidial surface, facilitates conidial phagocytosis, and stimulates the host immune response.

Fucose-specific lectin of Aspergillus fumigatus: binding properties and effects on immune response stimulation.

Aspergillus fumigatus is the major causative fungus of aspergillosis, and many studies have explored the relationship between A. fumigatus and pathogenicity. In the current study, we focused on a fucose-specific lectin, FleA, as a novel molecule which related to the pathogenicity of A. fumigatus. The disruption of the fleA gene did not lead to clear morphological changes compared to parental strain under several stress conditions in culture, but germination become earlier. In comparison with parental strain, the pathogenicity of disruptant was enhanced in a mouse infection model. The pattern of conidial phagocytosis and adhesion to cultured cells did not explain this enhanced pathogenicity. FleA was reported to contain six conserved fucose-binding sites; the analysis of constructed FleA point mutants revealed nonequivalent contribution of the fucose-binding sites to fucose binding. Based on the immune response induced in the cultured cells upon exposure to wild-type and mutant FleA, we propose a model of the FleA molecule in A. fumigatus infection.

Impact of human mesenchymal stromal cells on antifungal host response against Aspergillus fumigatus.

Mesenchymal stromal cells (MSCs) are increasingly given as immunotherapy to hematopoietic stem cell transplant (HSCT) recipients with refractory graft-versus-host disease (GvHD). Whereas the immunosuppressive properties of MSCs seem to be beneficial in GvHD, there is, at the same time, major concern that MSCs increase the risk for infection. We therefore investigated the interplay of human MSCs with Aspergillus fumigatus and the impact of MSCs on different arms of the anti-Aspergillus host response in vitro. Although A. fumigatus hyphae increase mRNA levels of IL6 in MSCs, the extracellular availability of IL-6 and other pro-inflammatory cytokines remains unaffected. Human MSCs are able to phagocyte Aspergillus conidia, but phagocytosis of conidia is not associated with an alteration of the cytokine production by MSCs. In addition, human MSCs do not affect activation and function of A. fumigatus specific CD4+ T cells, and MSCs do not negatively impact the oxidative burst activity of phagocytes. Our in vitro data indicate that administration of human MSCs is not associated with a negative impact on the host response against A. fumigatus and that the fungus does not stimulate MSCs to increase the release of those cytokines which play a central role in the pathophysiology of GvHD.

Infection-Mediated Priming of Phagocytes Protects against Lethal Secondary Aspergillus fumigatus Challenge.

Phagocytes restrict the germination of Aspergillus fumigatus conidia and prevent the establishment of invasive pulmonary aspergillosis in immunecompetent mice. Here we report that immunecompetent mice recovering from a primary A. fumigatus challenge are protected against a secondary lethal challenge. Using RAGγc knock-out mice we show that this protection is independent of T, B and NK cells. In protected mice, lung phagocytes are recruited more rapidly and are more efficient in conidial phagocytosis and killing. Protection was also associated with an enhanced expression of CXCR2 and Dectin-1 on bone marrow phagocytes. We also show that protective lung cytokine and chemokine responses are induced more rapidly and with enhanced dynamics in protected mice. Our findings support the hypothesis that following a first encounter with a non-lethal dose of A. fumigatus conidia, the innate immune system is primed and can mediate protection against a secondary lethal infection.

Flow Cytometry of Lung and Bronchoalveolar Lavage Fluid Cells from Mice Challenged with Fluorescent Aspergillus Reporter (FLARE) Conidia

Aspergillus fumigatus is a ubiquitous fungal pathogen that forms airborne conidia. The process of restricting conidial germination into hyphae by lung leukocytes is critical in determining infectious outcomes. Tracking the outcome of conidia-host cell encounters in vivo is technically challenging and an obstacle to understanding the molecular and cellular basis of antifungal immunity in the lung. Here, we describe a method that utilizes a genetically engineered Aspergillus strain [called FLARE (Jhingran et al., 2012; Espinosa et al., 2014; Heung et al., 2015)] to monitor conidial phagocytosis and killing by leukocytes within the lung environment at single encounter resolution.

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.

Methylprednisolone impairs conidial phagocytosis but does not attenuate hyphal damage by neutrophils against Exserohilum rostratum.

Exserohilum rostratum caused a multistate fungal meningitis outbreak following iatrogenic inoculation of contaminated methylprednisolone in the United States. To gain insight into the immunopathogenesis of this infection, we studied the innate host responses of human neutrophils against E. rostratum conidia and hyphae with or without methylprednisolone. The neutrophil-induced percentage fungal damage against conidia and hyphae was effector-to-target ratio dependent (≤55%). While methylprednisolone did not affect neutrophil-induced fungal damage by treatment of Exserohilum or neutrophils, it compromised phagocytosis of conidia (P < 0.05). These findings suggest that methylprednisolone-treated neutrophils may have altered phagocytic clearance of Exserohilum conidia, reducing host capacity to contain the invasive process.

Elimination of Aspergillus fumigatus conidia from the airways of mice with allergic airway inflammation

BackgroundAspergillus fumigatus conidia can exacerbate asthma symptoms. Phagocytosis of conidia is a principal component of the host antifungal defense. We investigated whether allergic airway inflammation (AAI) affects the ability of phagocytic cells in the airways to internalize the resting fungal spores.MethodsUsing BALB/c mice with experimentally induced AAI, we tested the ability of neutrophils, macrophages, and dendritic cells to internalize A. fumigatus conidia at various anatomical locations. We used light microscopy and differential cell and conidium counts to determine the ingestion potential of neutrophils and macrophages present in bronchoalveolar lavage (BAL). To identify phagocyte-conidia interactions in conducting airways, conidia labeled with tetramethylrhodamine-(5-(and-6))-isothiocyanate were administered to the oropharyngeal cavity of mice. Confocal microscopy was used to quantify the ingestion potential of Ly-6G+ neutrophils and MHC II+ antigen-presenting cells located in the intraepithelial and subepithelial areas of conducting airways.ResultsAllergen challenge induced transient neutrophil recruitment to the airways. Application of A. fumigatus conidia at the acute phase of AAI provoked recurrent neutrophil infiltration, and consequently increased the number and the ingestion potential of the airway neutrophils. In the absence of recurrent allergen or conidia provocation, both the ingestion potential and the number of BAL neutrophils decreased. As a result, conidia were primarily internalized by alveolar macrophages in both AAI and control mice at 24 hours post-inhalation. Transient influx of neutrophils to conducting airways shortly after conidial application was observed in mice with AAI. In addition, the ingestion potential of conducting airway neutrophils in mice with induced asthma exceeded that of control mice. Although the number of neutrophils subsequently decreased, the ingestion capacity remained elevated in AAI mice, even at 24 hours post-conidia application.ConclusionsAspiration of allergen to sensitized mice enhanced the ingestion potential of conducting airway neutrophils. Such activation primes neutrophils so that they are sufficient to control dissemination of non-germinating A. fumigatus conidia. At the same time, it can be a reason for the development of sensitivity to fungi and subsequent asthma exacerbation.

Phagocytosis of melanized Aspergillus conidia by macrophages exerts cytoprotective effects by sustained PI3K/Akt signalling

Host cell death is a critical component of innate immunity and often determines the progression and outcome of infections. The opportunistic human pathogen Aspergillus fumigatus can manipulate the immune system either by inducing or by inhibiting host cell apoptosis dependent on its distinct morphological form. Here, we show that conidia of Aspergillus ssp. inhibit apoptosis of macrophages induced via the intrinsic (staurosporine) and extrinsic (Fas ligand) pathway. Hence, mitochondrial cytochrome c release and caspase activation were prevented. We further found that the anti-apoptotic effect depends on both host cell de novo protein synthesis and phagocytosis of conidia by macrophages. Moreover, sustained PI3K/Akt signalling in infected cells is an important determinant to resist apoptosis. We demonstrate that pigmentless pksP mutant conidia of A. fumigatus failed to trigger protection against apoptosis and provide evidence that the sustained survival of infected macrophages depends on the presence of the grey-green conidial pigment consisting of dihydroxynaphthalene-melanin. In conclusion, we revealed a novel potential function of melanin in the pathogenesis of A. fumigatus. For the first time, we show that melanin itself is a crucial component to inhibit macrophage apoptosis which may contribute to dissemination of the fungus within the host.

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.

The Aspergillus fumigatus cspA Gene Encoding a Repeat-Rich Cell Wall Protein Is Important for Normal Conidial Cell Wall Architecture and Interaction with Host Cells

cspA (for cell surface protein A) encodes a repeat-rich glycophosphatidylinositol (GPI)-anchored cell wall protein (CWP) in the pathogenic fungus Aspergillus fumigatus. The number of repeats in cspA varies among isolates, and this trait is used for typing closely related strains of A. fumigatus. We have previously shown that deletion of cspA is associated with rapid conidial germination and reduced adhesion of dormant conidia. Here we show that cspA can be extracted with hydrofluoric acid (HF) from the cell wall, suggesting that it is a GPI-anchored CWP. The cspA-encoded CWP is unmasked during conidial germination and is surface expressed during hyphal growth. Deletion of cspA results in weakening of the conidial cell wall, whereas its overexpression increases conidial resistance to cell wall-degrading enzymes and inhibits conidial germination. Double mutant analysis indicates that cspA functionally interacts with the cell wall protein-encoding genes ECM33 and GEL2. Deletion of cspA together with ECM33 or GEL2 results in strongly reduced conidial adhesion, increased disorganization of the conidial cell wall, and exposure of the underlying layers of chitin and beta-glucan. This is correlated with increasing susceptibility of the DeltacspA, DeltaECM33, and DeltacspA DeltaECM33 mutants to conidial phagocytosis and killing by human macrophages and hyphal damage induced by neutrophils. However, these strains did not exhibit altered virulence in mice with infected lungs. Collectively, these results suggest a role for cspA in maintaining the strength and integrity of the cell wall.

Characterisation of the phagocytic uptake of Aspergillus fumigatus conidia by macrophages

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for severe, life-threatening infections in immunocompromised patients. Airborne conidia are the infectious agent and can reach the lower parts of the respiratory system. In the lung, phagocytes represent the first line of defence. Resident macrophages are able to track down, engulf and kill the invading spores. Phagocytosis of the conidia is therefore a prerequisite for their efficient elimination. Using human and murine macrophages we analysed the phagocytic uptake of A. fumigatus conidia. We found that conidial phagocytosis is an actin-depending process that additionally requires myosin motor, phosphoinositide-3-phosphate kinase and tyrosine kinase activity. Both broad range tyrosine kinase inhibitors and inhibitors that specifically block src kinases had a strong impact on the conidial uptake. Immunofluorescence data demonstrate the recruitment of tyrosine-phosphorylated proteins to the vicinity of engulfed conidia. Uptake of the conidia was accompanied by a strong and local reorganisation of the actin cytoskeleton, whereas no prominent reorganisation was apparent for the microtubules. Both confocal immunofluorescence and electron microscopic data revealed the presence of large ruffle-like structures engaged in the uptake of conidia. This suggests that the internalisation of A. fumigatus spores can be mediated by a process resembling macropinocytosis, which is furthermore supported by the detection of intracellular conidia within spacious vacuoles. Taken together, our data provide new insights into the internalisation of A. fumigatus spores by macrophages, a key process in the early immune defence against an Aspergillus infection.

Influence of amphotericin B and amphotericin B colloidal dispersion on the functions of human phagocytes in defence against Aspergillus species

This study investigated the in vitro effects of amphotericin B and amphotericin B colloidal dispersion (ABCD) on phagocytosis and inhibition of germination of clinical isolates of Aspergillus spp. by monocyte-derived macrophages (MDMs). Both amphotericin B and ABCD caused significant reductions in uptake of conidia of Aspergillus spp. by MDMs (p < 0.01). The inhibition of germination was superior with conidia of Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus isolates. Aspergillus flavus growth was significantly less inhibited of either antimycotic as compared to A. fumigatus and A. terreus (p < 0.01).We demonstrate that amphotericin B or ABCD acts as a potent inhibitor of Aspergillus germination. By contrast, treatment of MDMs with these antimycotics diminished phagocytosis of conidia in vitro.

The Role of CC Chemokine Receptor 6 in Host Defense in a Model of Invasive Pulmonary Aspergillosis

Invasive aspergillosis is a severe fungal infection afflicting immunocompromised patients, particularly patients with neutrophil defects. CCR6, a beta-chemokine receptor, mediates migration of dendritic cells (DCs) and several lymphocyte subsets to sites of epithelial inflammation, but its role in infections has not been examined extensively. To test the hypothesis that CCR6-mediated leukocyte recruitment is necessary for effective host defense in neutropenic hosts with invasive pulmonary aspergillosis. Neutropenic wild-type mice and mice with targeted deletion of CCR6 were infected with Aspergillus fumigatus. The host responses to the infection were compared in vivo and leukocyte responses to the fungus were examined in vitro. In the context of infection, immature myeloid DCs were the major population of CCR6-expressing cells in the lungs. As compared with wild-type animals, CCR6-deficient mice developed a more severe infection when challenged with A. fumigatus conidia, as documented by a higher mortality rate and greater lung fungal burden. This was associated with reduced accumulation of DCs in the lungs. CCR6-deficient and wild-type DCs did not differ in their phagocytosis of conidia, cytokine response, or maturation in vitro. In adoptive transfer experiments, however, DCs from CCR6-deficient donors showed lesser accumulation in the lungs of infected mice as compared with wild-type cells, and transfer of wild-type, but not CCR6-deficient, DCs resulted in attenuated severity of infection in CCR6-deficient recipients. Taken together, these results implicate CCR6-mediated DC influx into the lung in the initial host defense in invasive aspergillosis.

Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.