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.

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.

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.

Live-cell video microscopy of fungal pathogen phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells; and how engulfment leads to alteration of the cell cycle and behavior of macrophages. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

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.

Candida albicans infection inhibits macrophage cell division and proliferation.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to inhibit effective destruction by host phagocytes. Using live cell video microscopy, we show here for the first time that C. albicans inhibits cell division in macrophages undergoing mitosis. Inhibition of macrophage cell division is dependent on the ability of C. albicans to form hyphae, as it is rarely observed following phagocytosis of UV-killed or morphogenesis-defective mutant Candida. Interestingly, failed cell division following phagocytosis of hyphal C. albicans is surprisingly common, and leads to the formation of large multinuclear macrophages. This raises question as to whether inhibition of macrophage cell division is another virulence attribute of C. albicans or enables host macrophages to contain the pathogen.

Stage specific assessment of Candida albicans phagocytosis by macrophages identifies cell wall composition and morphogenesis as key determinants.

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.

Non-lytic expulsion/exocytosis of Candida albicans from macrophages.

Candida albicans is an opportunistic pathogen and is recognised and phagocytosed by macrophages. Using live-cell imaging, non-lytic expulsion/exocytosis of C. albicans from macrophages is demonstrated for the first time. Following complete expulsion, both the phagocyte and pathogen remain intact and viable. Partial engulfment of hyphal C. albicans without macrophage lysis is also demonstrated. These observations underpin the complexity of interactions between C. albicans and innate immune cells.

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.