Fungal spore swelling and germination are restricted by the macrophage phagolysosome.

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.

Nature of ß-1,3-Glucan-Exposing Features on Candida albicans Cell Wall and Their Modulation.

Candida albicans exists as a commensal of mucosal surfaces and the gastrointestinal tract without causing pathology. However, this fungus is also a common cause of mucosal and systemic infections when antifungal immune defenses become compromised. The activation of antifungal host defenses depends on the recognition of fungal pathogen-associated molecular patterns (PAMPs), such as ß-1,3-glucan. In C. albicans, most ß-1,3-glucan is present in the inner cell wall, concealed by the outer mannan layer, but some ß-1,3-glucan becomes exposed at the cell surface. In response to host signals, such as lactate, C. albicans induces the Xog1 exoglucanase, which shaves exposed ß-1,3-glucan from the cell surface, thereby reducing phagocytic recognition. We show here that ß-1,3-glucan is exposed at bud scars and punctate foci on the lateral wall of yeast cells, that this exposed ß-1,3-glucan is targeted during phagocytic attack, and that lactate-induced masking reduces ß-1,3-glucan exposure at bud scars and at punctate foci. ß-1,3-Glucan masking depends upon protein kinase A (PKA) signaling. We reveal that inactivating PKA, or its conserved downstream effectors, Sin3 and Mig1/Mig2, affects the amounts of the Xog1 and Eng1 glucanases in the C. albicans secretome and modulates ß-1,3-glucan exposure. Furthermore, perturbing PKA, Sin3, or Mig1/Mig2 attenuates the virulence of lactate-exposed C. albicans cells in Galleria. Taken together, the data are consistent with the idea that ß-1,3-glucan masking contributes to Candida pathogenicity. IMPORTANCE Microbes that coexist with humans have evolved ways of avoiding or evading our immunological defenses. These include the masking by these microbes of their "pathogen-associated molecular patterns" (PAMPs), which are recognized as "foreign" and used to activate protective immunity. The commensal fungus Candida albicans masks the proinflammatory PAMP ß-1,3-glucan, which is an essential component of its cell wall. Most of this ß-1,3-glucan is hidden beneath an outer layer of the cell wall on these microbes, but some can become exposed at the fungal cell surface. Using high-resolution confocal microscopy, we examine the nature of the exposed ß-1,3-glucan at C. albicans bud scars and at punctate foci on the lateral cell wall, and we show that these features are targeted by innate immune cells. We also reveal that downstream effectors of protein kinase A (Mig1/Mig2, Sin3) regulate the secretion of major glucanases, modulate the levels of ß-1,3-glucan exposure, and influence the virulence of C. albicans in an invertebrate model of systemic infection. Our data support the view that ß-1,3-glucan masking contributes to immune evasion and the virulence of a major fungal pathogen of humans.

Immune cells fold and damage fungal hyphae.

Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans-macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, ß2-integrin, VASP, actin-myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.

Epitope Shaving Promotes Fungal Immune Evasion.

The cell wall provides a major physical interface between fungal pathogens and their mammalian host. This extracellular armor is critical for fungal cell homeostasis and survival. Fungus-specific cell wall moieties, such as ß-1,3-glucan, are recognized as pathogen-associated molecular patterns (PAMPs) that activate immune-mediated clearance mechanisms. We have reported that the opportunistic human fungal pathogen Candida albicans masks ß-1,3-glucan following exposure to lactate, hypoxia, or iron depletion. However, the precise mechanism(s) by which C. albicans masks ß-1,3-glucan has remained obscure. Here, we identify a secreted exoglucanase, Xog1, that is induced in response to lactate or hypoxia. Xog1 functions downstream of the lactate-induced ß-glucan "masking" pathway to promote ß-1,3-glucan "shaving." Inactivation of XOG1 blocks most but not all ß-1,3-glucan masking in response to lactate, suggesting that other activities contribute to this phenomenon. Nevertheless, XOG1 deletion attenuates the lactate-induced reductions in phagocytosis and cytokine stimulation normally observed for wild-type cells. We also demonstrate that the pharmacological inhibition of exoglucanases undermines ß-glucan shaving, enhances the immune visibility of the fungus, and attenuates its virulence. Our study establishes a new mechanism underlying environmentally induced PAMP remodeling that can be manipulated pharmacologically to influence immune recognition and infection outcomes.IMPORTANCE The immune system plays a critical role in protecting us against potentially fatal fungal infections. However, some fungal pathogens have evolved evasion strategies that reduce the efficacy of our immune defenses. Previously, we reported that the fungal pathogen Candida albicans exploits specific host-derived signals (such as lactate and hypoxia) to trigger an immune evasion strategy that involves reducing the exposure of ß-glucan at its cell surface. Here, we show that this phenomenon is mediated by the induction of a major secreted exoglucanase (Xog1) by the fungus in response to these host signals. Inactivating XOG1-mediated "shaving" of cell surface-exposed ß-glucan enhances immune responses against the fungus. Furthermore, inhibiting exoglucanase activity pharmacologically attenuates C. albicans virulence. In addition to revealing the mechanism underlying a key immune evasion strategy in a major fungal pathogen of humans, our work highlights the potential therapeutic value of drugs that block fungal immune evasion.

A novel 4-dimensional live-cell imaging system to study leukocyte-endothelial dynamics in ANCA-associated vasculitis.

Neutrophils, monocytes and the endothelium are critical to ANCA-associated vasculitis (AAV) pathogenesis. This study aimed to develop a 4-dimensional (4D) live-cell imaging system that would enable investigation of spatial and temporal dynamics of these cells in health and disease. We further aimed to validate this system using autologous donor serum from AAV patients and polyclonal ANCA IgG, as well as exploring its potential in the pre-clinical testing of putative therapeutic compounds. Neutrophils and monocytes were isolated from peripheral venous blood of AAV patients or healthy controls and co-incubated on an endothelial monolayer in the presence of autologous serum. Alternatively, polyclonal ANCA IgG was used, following TNF-α priming, and imaged in 4-dimensions for 3 h using a spinning disc confocal microscope. Volocity 6.3® analysis software was used for quantification of leukocyte dynamics. The use of autologous serum resulted in increased neutrophil degranulation (p = .002), transmigration (p = .0096) and monocyte transcellular transmigration (p = .0013) in AAV patients. Polyclonal MPO-ANCA IgG induced neutrophil degranulation (p < .001) in this system. C5aR1 antagonism reduced neutrophil degranulation (p < .0002). We have developed a novel 4D in vitro system that allows accurate quantification of multiple neutrophil- and monocyte-endothelial interactions in AAV in a single assay. This system has the potential to highlight dynamics key to pathophysiology of disease, as well investigating the impact of potential therapeutics on these functions.

The pattern recognition receptors dectin-2, mincle, and FcRγ impact the dynamics of phagocytosis of Candida, Saccharomyces, Malassezia, and Mucor species.

Phagocytosis is a receptor-mediated process critical to innate immune clearance of pathogens. It proceeds in a regulated sequence of stages: (a) migration of phagocytes towards pathogens, (b) recognition of PAMPs and binding through PRRs, (c) engulfment and internalisation into phagosomes, (d) phagosome maturation, and (e) killing of pathogen or host cells. However, little is known about the role that individual receptors play in these discrete stages in the recognition of fungal cells. In a previous study, we found that dectin-2 deficiency impacted some but not all stages of macrophage-mediated phagocytosis of Candida glabrata. Because the C-type lectin receptor dectin-2 critically requires coupling to the FcRγ chain for signalling, we hypothesised that this coupling may be important for regulating phagocytosis of fungal cargo. We therefore examined how deficiency in FcRγ itself or two receptors to which it couples (dectin-2 and mincle) impacts phagocytosis of six fungal organisms representing three different fungal taxa. Our data show that deficiency in these proteins impairs murine bone marrow-derived macrophage migration, engulfment, and phagosome maturation, but not macrophage survival. Therefore, FcRγ engagement with selective C-type lectin receptors (CLRs) critically affects the spatio-temporal dynamics of fungal phagocytosis.

Hypoxia Promotes Immune Evasion by Triggering ß-Glucan Masking on the Candida albicans Cell Surface via Mitochondrial and cAMP-Protein Kinase A Signaling.

Organisms must adapt to changes in oxygen tension if they are to exploit the energetic benefits of reducing oxygen while minimizing the potentially damaging effects of oxidation. Consequently, organisms in all eukaryotic kingdoms display robust adaptation to hypoxia (low oxygen levels). This is particularly important for fungal pathogens that colonize hypoxic niches in the host. We show that adaptation to hypoxia in the major fungal pathogen of humans Candida albicans includes changes in cell wall structure and reduced exposure, at the cell surface, of ß-glucan, a key pathogen-associated molecular pattern (PAMP). This leads to reduced phagocytosis by murine bone marrow-derived macrophages and decreased production of IL-10, RANTES, and TNF-α by peripheral blood mononuclear cells, suggesting that hypoxia-induced ß-glucan masking has a significant effect upon C. albicans-host interactions. We show that hypoxia-induced ß-glucan masking is dependent upon both mitochondrial and cAMP-protein kinase A (PKA) signaling. The decrease in ß-glucan exposure is blocked by mutations that affect mitochondrial functionality (goa1Δ and upc2Δ) or that decrease production of hydrogen peroxide in the inner membrane space (sod1Δ). Furthermore, ß-glucan masking is enhanced by mutations that elevate mitochondrial reactive oxygen species (aox1Δ). The ß-glucan masking defects displayed by goa1Δ and upc2Δ cells are suppressed by exogenous dibutyryl-cAMP. Also, mutations that inactivate cAMP synthesis (cyr1Δ) or PKA (tpk1Δ tpk2Δ) block the masking phenotype. Our data suggest that C. albicans responds to hypoxic niches by inducing ß-glucan masking via a mitochondrial cAMP-PKA signaling pathway, thereby modulating local immune responses and promoting fungal colonization.IMPORTANCE Animal, plant, and fungal cells occupy environments that impose changes in oxygen tension. Consequently, many species have evolved mechanisms that permit robust adaptation to these changes. The fungal pathogen Candida albicans can colonize hypoxic (low oxygen) niches in its human host, such as the lower gastrointestinal tract and inflamed tissues, but to colonize its host, the fungus must also evade local immune defenses. We reveal, for the first time, a defined link between hypoxic adaptation and immune evasion in C. albicans As this pathogen adapts to hypoxia, it undergoes changes in cell wall structure that include masking of ß-glucan at its cell surface, and it becomes better able to evade phagocytosis by innate immune cells. We also define the signaling mechanisms that mediate hypoxia-induced ß-glucan masking, showing that they are dependent on mitochondrial signaling and the cAMP-protein kinase pathway. Therefore, hypoxia appears to trigger immune evasion in this fungal pathogen.

Differential Kinetics of Aspergillus nidulans and Aspergillus fumigatus Phagocytosis.

Invasive aspergillosis mainly occurs in immunocompromised patients and is commonly caused by Aspergillus fumigatus, while A.nidulans is rarely the causative agent. However, in chronic granulomatous disease (CGD) patients, A. nidulans is a frequent cause of invasive aspergillosis and is associated with higher mortality. Immune recognition of A. nidulans was compared to A. fumigatus to offer an insight into why A. nidulans infections are prevalent in CGD. Live cell imaging with J774A.1 macrophage-like cells and LC3-GFP-mCherry bone marrow-derived macrophages (BMDMs) revealed that phagocytosis of A. nidulans was slower compared to A. fumigatus. This difference could be attributed to slower migration of J774A.1 cells and a lower percentage of migrating BMDMs. In addition, delayed phagosome acidification and LC3-associated phagocytosis was observed with A. nidulans. Cytokine and oxidative burst measurements in human peripheral blood mononuclear cells revealed a lower oxidative burst upon challenge with A. nidulans. In contrast, A. nidulans induced significantly higher concentrations of cytokines. Collectively, our data demonstrate that A. nidulans is phagocytosed and processed at a slower rate compared to A. fumigatus, resulting in reduced fungal killing and increased germination of conidia. This slower rate of A. nidulans clearance may be permissive for overgrowth within certain immune settings.

Immunoregulatory soluble CTLA-4 modifies effector T-cell responses in systemic lupus erythematosus.

BACKGROUND: The inhibitory CTLA-4 molecule is a crucial regulator of immune responses and a target for therapeutic intervention in both autoimmunity and cancer. In particular, CTLA-4 is important in controlling antigen-specific immunity, including responses to autoantigens associated with autoimmune disease. Here, we investigate cytokine responses to a range of lupus-associated autoantigens and assess whether the alternatively spliced isoform of CTLA-4, soluble CTLA-4 (sCTLA-4), contributes to immune regulation of autoantigen-specific immunity in systemic lupus erythematosus (SLE). METHODS: The cell culture supernatant production of sCTLA-4 as well as the cytokines IL-10, IFN-γ, and IL-17 from peripheral blood mononuclear cells (PBMC) from lupus patients and age- and sex-matched healthy volunteer donors were measured in response to previously identified histone and small nuclear ribonucleoprotein (snRNP) autoantigen-derived peptides (H391-105, H471-93, and U170K131-151) by ELISA. We also examined the functional contribution of sCTLA-4 to immune regulation in the context of these autoantigenic peptides following blockade of sCTLA-4 with a selective anti-sCTLA-4 monoclonal antibody, JMW-3B3. RESULTS: We identified responses to autoantigenic peptides, which revealed qualitative differences in cytokine (IL-10, IL-17, and IFN-γ) profiles between SLE patients and healthy donors. PBMC from healthy donors responded to each of the lupus peptides by secreting IFN-γ and IL-17, but PBMC from SLE patients produced IL-10. Although we did not observe differences in the levels of serum or PBMC culture supernatant sCTLA-4 in either cohort, blockade of sCTLA-4 in PBMC cultures responding to antigen enhanced the cytokine profiles associated with each group. CONCLUSION: The results show that lupus autoantigen-derived peptides display varied immunogenicity in lupus versus healthy volunteer donors, while sCTLA-4 acts to regulate the T-cell activity independently of response profile.

Pho4 mediates phosphate acquisition in Candida albicans and is vital for stress resistance and metal homeostasis.

During interactions with its mammalian host, the pathogenic yeast Candida albicans is exposed to a range of stresses such as superoxide radicals and cationic fluxes. Unexpectedly, a nonbiased screen of transcription factor deletion mutants revealed that the phosphate-responsive transcription factor Pho4 is vital for the resistance of C. albicans to these diverse stresses. RNA-Seq analysis indicated that Pho4 does not induce stress-protective genes directly. Instead, we show that loss of Pho4 affects metal cation toxicity, accumulation, and bioavailability. We demonstrate that pho4Δ cells are sensitive to metal and nonmetal cations and that Pho4-mediated polyphosphate synthesis mediates manganese resistance. Significantly, we show that Pho4 is important for mediating copper bioavailability to support the activity of the copper/zinc superoxide dismutase Sod1 and that loss of Sod1 activity contributes to the superoxide sensitivity of pho4Δ cells. Consistent with the key role of fungal stress responses in countering host phagocytic defenses, we also report that C. albicans pho4Δ cells are acutely sensitive to macrophage-mediated killing and display attenuated virulence in animal infection models. The novel connections between phosphate metabolism, metal homeostasis, and superoxide stress resistance presented in this study highlight the importance of metabolic adaptation in promoting C. albicans survival in the host.

SOCS3 is a modulator of human macrophage phagocytosis.

Suppressor of cytokine signaling (SOCS) proteins are recognized as key feedback inhibitors modulating the inflammatory activities of macrophages, but comparatively little is known about whether and how they affect phagocytosis. Here, we evaluated the role of SOCS3 in driving the inflammatory phenotype and phagocytic uptake of apoptotic cells by human macrophages and the signaling pathways that are necessary for efficient phagocytosis. In M1-activated human monocyte-derived macrophages, SOCS3 silencing, using short interfering RNA technology, resulted in a decreased expression of proinflammatory markers and an increased expression of M2 macrophage markers. Strikingly, we demonstrated for the first time that SOCS3 knockdown significantly enhances the phagocytic capacity of M1 macrophages for carboxylate-modified beads and apoptotic neutrophils. With the use of live-cell video microscopy, we showed that SOCS3 knockdown radically affects the temporal dynamics of particle engulfment, enabling more rapid uptake of a second target and delaying postengulfment processing, as evidenced by deferred acquisition of phagosome maturation markers. SOCS3 knockdown impacts on phagocytosis through increased PI3K and Ras-related C3 botulinum toxin substrate 1 (Rac1) activity, pathways essential for engulfment and clearance of apoptotic cells. Enhanced phagocytosis in SOCS3-silenced cells was reversed by pharmacological PI3K inhibition. Furthermore, we revealed that actin polymerization, downstream of PI3K/Rac1 activation, was significantly altered in SOCS3-silenced cells, providing a mechanism for their greater phagocytic activity. The findings support a new model, whereby SOCS3 not only plays an important role in driving macrophage inflammatory responses but modulates key signaling pathways organizing the actin cytoskeleton to regulate the efficiency of phagocytic processes.

Calcineurin orchestrates dimorphic transitions, antifungal drug responses and host-pathogen interactions of the pathogenic mucoralean fungus Mucor circinelloides.

Calcineurin plays essential roles in virulence and growth of pathogenic fungi and is a target of the natural products FK506 and Cyclosporine A. In the pathogenic mucoralean fungus Mucor circinelloides, calcineurin mutation or inhibition confers a yeast-locked phenotype indicating that calcineurin governs the dimorphic transition. Genetic analysis in this study reveals that two calcineurin A catalytic subunits (out of three) are functionally diverged. Homology modeling illustrates modes of resistance resulting from amino substitutions in the interface between each calcineurin subunit and the inhibitory drugs. In addition, we show how the dimorphic transition orchestrated by calcineurin programs different outcomes during host-pathogen interactions. For example, when macrophages phagocytose Mucor yeast, subsequent phagosomal maturation occurs, indicating host cells respond appropriately to control the pathogen. On the other hand, upon phagocytosis of spores, macrophages fail to form mature phagosomes. Cytokine production from immune cells differs following exposure to yeast versus spores (which germinate into hyphae). Thus, the morphogenic transition can be targeted as an efficient treatment option against Mucor infection. In addition, genetic analysis (including gene disruption and mutational studies) further strengthens the understanding of calcineurin and provides a foundation to develop antifungal agents targeting calcineurin to deploy against Mucor and other pathogenic fungi.

Candida albicans hypha formation and mannan masking of ß-glucan inhibit macrophage phagosome maturation.

UNLABELLED: Candida albicans is a major life-threatening human fungal pathogen in the immunocompromised host. Host defense against systemic Candida infection relies heavily on the capacity of professional phagocytes of the innate immune system to ingest and destroy fungal cells. A number of pathogens, including C. albicans, have evolved mechanisms that attenuate the efficiency of phagosome-mediated inactivation, promoting their survival and replication within the host. Here we visualize host-pathogen interactions using live-cell imaging and show that viable, but not heat- or UV-killed C. albicans cells profoundly delay phagosome maturation in macrophage cell lines and primary macrophages. The ability of C. albicans to delay phagosome maturation is dependent on cell wall composition and fungal morphology. Loss of cell wall O-mannan is associated with enhanced acquisition of phagosome maturation markers, distinct changes in Rab GTPase acquisition by the maturing phagosome, impaired hyphal growth within macrophage phagosomes, profound changes in macrophage actin dynamics, and ultimately a reduced ability of fungal cells to escape from macrophage phagosomes. The loss of cell wall O-mannan leads to exposure of ß-glucan in the inner cell wall, facilitating recognition by Dectin-1, which is associated with enhanced phagosome maturation. IMPORTANCE: Innate cells engulf and destroy invading organisms by phagocytosis, which is essential for the elimination of fungal cells to protect against systemic life-threatening infections. Yet comparatively little is known about what controls the maturation of phagosomes following ingestion of fungal cells. We used live-cell microscopy and fluorescent protein reporter macrophages to understand how C. albicans viability, filamentous growth, and cell wall composition affect phagosome maturation and the survival of the pathogen within host macrophages. We have demonstrated that cell wall glycosylation and yeast-hypha morphogenesis are required for disruption of host processes that function to inactivate pathogens, leading to survival and escape of this fungal pathogen from within host phagocytes. The methods employed here are applicable to study interactions of other pathogens with phagocytic cells to dissect how specific microbial features impact different stages of phagosome maturation and the survival of the pathogen or host.

Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

The characterisation and determinants of quality of life in ANCA associated vasculitis.

OBJECTIVES: To contextualise and identify the determinants of poor health related quality of life (QOL) among patients with antineutrophil cytoplasm antibody (ANCA) associated vasculitis (AAV). METHODS: A multicentre AAV case-control study was conducted using two matched groups of population and chronic disease controls. Measures of physical and mental QOL as well as putative bio-psychosocial determinants of QOL impairment were collected. Concurrently, putative clinical QOL determinants were recorded. Conditional logistic regression analyses characterised group differences while multivariable logistic regression identified within-case QOL associations which were further quantified using population attributable risks (PAR). RESULTS: Cases (n=410) experienced similar QOL to chronic disease controls (n=318) (physical QOL: OR 0.7, 95% CI 0.4 to 1.1; mental QOL: OR 1.1, 95% CI 0.8 to 1.6). However, they were substantially more likely to report poor QOL compared to general population controls (n=470) (physical QOL: OR 7.0, 95% CI 4.4 to 11.1; mental QOL: OR 2.5, 95% CI 1.7 to 3.6). A few clinical, but many more bio-psychosocial factors were independently associated with poor QOL. In population terms, fatigue was found to be of principal importance (physical QOL: PAR 24.6%; mental QOL: PAR 47.4%). CONCLUSIONS: AAV patients experienced significant QOL impairment compared to the general population, but similar to those with other chronic diseases whose considerable needs are already recognised. Potentially modifiable clinical determinants have been identified; however bio-psychosocial interventions are likely to provide the greater QOL gains in this patient population.

Role of Dectin-2 for host defense against systemic infection with Candida glabrata.

Although Candida glabrata is an important pathogenic Candida species, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2(-/-)) mice were found to be more susceptible to C. glabrata infections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2(-/-) mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2(-/-) macrophages and neutrophils. Neutrophils of Dectin-2(-/-) mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrata or C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrata and provides new insights into the host defense mechanisms against this important fungal pathogen.

Altered dynamics of Candida albicans phagocytosis by macrophages and PMNs when both phagocyte subsets are present.

UNLABELLED: An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection. IMPORTANCE: Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.

Explaining fatigue in ANCA-associated vasculitis.

OBJECTIVES: To identify the determinants of fatigue among patients with ANCA-associated vasculitis (AAV). METHODS: A multicentre cross-sectional study was conducted. Subjects fulfilling the European Medicines Agency criteria for granulomatosis with polyangiitis (Wegener's), microscopic polyangiitis and eosinophilic granulomatosis with polyangiitis (Churg-Strauss) were approached according to consecutive clinic attendance and invited to complete a questionnaire assessing fatigue and putative biopsychosocial determinants of this symptom. Concurrently, potential clinical determinants were recorded. Independent associations of fatigue were identified using forward stepwise logistic regression modelling and their overall impact expressed as population attributable risk (PAR). RESULTS: The majority (74.8%) of participants (n = 410) reported high levels of fatigue that were found to be significantly associated with numerous biopsychosocial and clinical factors. Sleep disturbance [odds ratio (OR) 5.3, 95% CI 2.7, 10.5] and pain (OR 3.8, 95% CI 2.0, 7.3) were the strongest independent associations of fatigue and, on a population level, each was more than twice as important as any other putative determinant (PAR 18.1% and 16.5%, respectively). Female gender (OR 2.1, 95% 1.1, 4.0), elevated CRP (OR 3.7, 95% CI 1.7, 8.1) and the dysfunctional coping strategies of behavioural disengagement (OR 2.4, 95% CI 1.04, 5.6) and denial (OR 2.4, 95% CI 0.9, 6.7) were also independently associated with fatigue. CONCLUSION: The data suggest that AAV-related fatigue is multifactorial in origin. Sleep disturbance and pain were found to be most important, although inflammation, as measured by CRP, was also associated. This study has identified potentially modifiable determinants that will inform future interventions aimed at alleviating fatigue.

Sialoadhesin promotes rapid proinflammatory and type I IFN responses to a sialylated pathogen, Campylobacter jejuni.

Sialoadhesin (Sn) is a macrophage (Mφ)-restricted receptor that recognizes sialylated ligands on host cells and pathogens. Although Sn is thought to be important in cellular interactions of Mφs with cells of the immune system, the functional consequences of pathogen engagement by Sn are unclear. As a model system, we have investigated the role of Sn in Mφ interactions with heat-killed Campylobacter jejuni expressing a GD1a-like, sialylated glycan. Compared to Sn-expressing bone marrow-derived macrophages (BMDM) from wild-type mice, BMDM from mice either deficient in Sn or expressing a non-glycan-binding form of Sn showed greatly reduced phagocytosis of sialylated C. jejuni. This was accompanied by a strong reduction in MyD88-dependent secretion of TNF-α, IL-6, IL-12, and IL-10. In vivo studies demonstrated that functional Sn was required for rapid TNF-α and IFN-ß responses to i.v.-injected sialylated C. jejuni. Bacteria were captured within minutes after i.v. injection and were associated with Mφs in both liver and spleen. In the spleen, IFN-ß-reactive cells were localized to Sn⁺ Mφs and other cells in the red pulp and marginal zone. Together, these studies demonstrate that Sn plays a key role in capturing sialylated pathogens and promoting rapid proinflammatory cytokine and type I IFN responses.

Interactions between macrophages and cell wall oligosaccharides of Candida albicans.

The fungal cell wall is the armour that protects the cell from changes in the external environment. The wall of Candida albicans, an opportunistic human pathogen, is also the immediate point of contact with the host immune system and contains most of the pathogen-associated molecular patterns recognised by innate immune cells. Along with the use of mutants altered in cell wall composition, the isolation and purification of cell wall components has proven useful in the identification of receptors involved in the sensing of these molecules, and assessment of the relative relevance of ligand-receptor interactions during the sensing of C. albicans by the immune system. Here, we describe protocols for the isolation of cell wall chitin, N-linked and O-linked mannans from C. albicans, and how they can subsequently be used to assess immunological activities such as phagocytosis and cytokine production by myeloid cells.