An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections

Fluorescent Tracking of Yeast Division Clarifies the Essential Role of Spleen Tyrosine Kinase in the Intracellular Control of Candida glabrata in Macrophages

Macrophages play a critical role in the elimination of fungal pathogens. They are sensed via cell surface pattern-recognition receptors and are phagocytosed into newly formed organelles called phagosomes. Phagosomes mature through the recruitment of proteins and lysosomes, resulting in addition of proteolytic enzymes and acidification of the microenvironment. Our earlier studies demonstrated an essential role of Dectin-1-dependent activation of spleen tyrosine kinase (Syk) in the maturation of fungal containing phagosomes. The absence of Syk activity interrupted phago-lysosomal fusion resulting in arrest at an early phagosome stage. In this study, we sought to define the contribution of Syk to the control of phagocytosed live Candida glabrata in primary macrophages. To accurately measure intracellular yeast division, we designed a carboxyfluorescein succinimidyl ester (CFSE) yeast division assay in which bright fluorescent parent cells give rise to dim daughter cells. The CFSE-labeling of C. glabrata did not affect the growth rate of the yeast. Following incubation with macrophages, internalized CFSE-labeled C. glabrata were retrieved by cellular lysis, tagged using ConA-647, and the amount of residual CFSE fluorescence was assessed by flow cytometry. C. glabrata remained undivided (CFSE bright) for up to 18 h in co-culture with primary macrophages. Treatment of macrophages with R406, a specific Syk inhibitor, resulted in loss of intracellular control of C. glabrata with initiation of division within 4 h. Delayed Syk inhibition after 8 h was less effective indicating that Syk is critically required at early stages of macrophage–fungal interaction. In conclusion, we demonstrate a new method of tracking division of C. glabrata using CFSE labeling. Our results suggest that early Syk activation is essential for macrophage control of phagocytosed C. glabrata

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan

International audienceAspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients

Phagocyte functions of PMN-DCs.

<p>(<b>A-B</b>) Mice were challenged with GFP <i>B</i>. <i>dermatitidis</i>, and lungs were harvested 7 dpi and stained with calcofluor white to mark extracellular yeast. (A) Representative flow plots showing association of gated leukocyte populations with extracellular (Calcfluor⁺, blue) and phagocytosed (Calcofluor^-, green) yeast (GFP⁺). (B) Absolute number (histogram bars) of phagocytosed yeast by moDCs, canonical neutrophils or PMN-DCs with inset pie chart indicating the proportion of all yeast phagocytosis by leukocyte populations in the lung. (<b>C</b>) Surface expression of fungal-recognizing pattern recognition receptors on neutrophil populations and moDCs at 7 dpi with <i>B</i>. <i>dermatitidis</i>. Histograms show FMO controls (gray) and stained populations (red); relative expression of each receptor on each population. (<b>E</b>–<b>F</b>) <i>Ex vivo</i> staining of neutrophil populations and moDCs with ROS-indicator DHR-123 (C) or NO-indicator DAF-FM (D). Representative histograms shown on top; proportions of ROS⁺ and NO⁺ on the bottom left and ROS/NO production indicate by MFI on the right. All experiments are representative of at least two independent experiments. N = 5 mice. Means ± SEM indicated.</p

Presentation of fungal antigen (calnexin) to transgenic Tg1807 T cells by PMN-DCs generated <i>in vitro</i> from ER-HoxB8 GMP cells.

<p>(<b>A</b>–<b>D</b>) PMN-DCs or bone-marrow DCs (BMDCs) were incubated overnight with or without recombinant fungal calnexin, and the next day 5 x 10⁴ cells were injected subcutaneously into CD90.2 mice that had received adoptive transfer of Tg1807 cells (CD90.1⁺, calnexin-specific CD4⁺ T cells). One week later skin draining lymph nodes were harvested. <b>(A)</b> IL-6 in supernatants from overnight culture of PMN-DCs or BMDCs with or without calnexin, n.d,:not detected. <b>(B)</b> Experimental design of delivering calnexin-loaded APCs into mice that had received congenic Tg1807 cells. <b>(C)</b> Proportion of Tg1807 cells activated in lymph nodes (indicated by CD44⁺ and CD62L^-). <b>(D)</b> The absolute number of Tg1807 cells and activated Tg1807 cells for each treatment group (N = 5–6 mice). (<b>E</b>) Lymph node cells (as shown in C-D) were stimulated <i>ex vivo</i> for 3 days with calnexin, and IFN-γ and IL-17 assayed in culture supernatants, Ag: antigen pulsed. (<b>F-G</b>) PMN-DCs or BMDCs were incubated overnight with fungal calnexin or heat-killed <i>Blastomyces</i> yeast before enriched CD4⁺ Tg1807 cells were added. (F) Presence of IFN-γ and IL-17 in supernatants collected after 3 days was determined by ELISA. (G) Expression of MHC class II from PMN-DCs at the end of the assay (Day 11) shown with comparison of MHC class II expression before mixing with T cells (Day 7) and before PMN-DC differentiation (Day 0), MFI indicated (±SEM). Both <i>in vivo</i> and <i>in vitro</i> experiments were completed independently twice. Statistics: for C-E, statistical significance between calnexin-loaded PMN-DCs and no antigen controls; for F-G, statistical significance between samples with fungal antigen and unstimulated.</p

Differentiation of granulocyte/macrophage progenitor (GMP) cell line into PMN-DCs <i>in vitro</i>.

<p>(<b>A</b>) ER-HoxB8 GMP cells (GFP⁺) were cultured for 4–5 days in the presence or absence of estrogen and then cultured for an additional 5 days with or without GM-CSF or IL-4 in the presence of bone marrow feeder cells; differentiation of GFP⁺ cells into PMN-DCs was tracked by CD11c and MHC class II expression. <b>(B-C)</b> GMP cells were matured into neutrophils (Day 0) or further differentiated into PMN-DCs (after 5 days with GM-CSF and IL-4 and feeder cells). (<b>B</b>) Undifferentiated (Day 0) or differentiated (Day 5) neutrophils (upper panel) were incubated overnight with DsRed <i>A</i>. <i>fumigatus</i> spores stained with Uvitex and then analyzed by flow cytometry; the association rate with live (DsRed⁺) or dead (DsRed^-) <i>A</i>. <i>fumigatus</i> spores with each population from above plots is shown in the lower panel. (<b>C</b>) Undifferentiated neutrophils or differentiated PMN-DCs were incubated for 1 hour with β-glucan-coated AlexaFluor647 beads and analyzed by flow cytometry for association with cells. (<b>D</b>) Expression of CD11c and MHC class II on starting neutrophils (Day 0) vs. neutrophils differentiated for 5 days with or without feeder cells (MFI indicated). (<b>E</b>) GMP cells were matured into neutrophils (Day 0) and differentiated 5–7 days without feeder cells; representative flow plots and images of cells at day 0, 5 or 7. Mean ± SEM shown.</p

Appearance of PMN-DCs and fungal killing by the cells during pulmonary aspergillosis and systemic candidiasis.

<p>(<b>A</b>–<b>C</b>) Mice were infected IT with Uvitex-stained DsRed <i>A</i>. <i>fumigatus</i> spores, and lungs were harvest 48 hours later. (<b>A</b>) Proportions and absolute numbers of canonical and MHCII⁺ only neutrophils, PMN-DCs (MHCII⁺ CD11c⁺) and moDCs in the lung. (<b>B</b>) Representative plots showing association of leukocyte populations with live (DsRed⁺) and killed (DsRed^-) <i>Aspergillus</i> spores (Uvitex⁺). (<b>C</b>) Proportion of leukocytes associated with live and killed spores (top) and killing rate (% DsRed^-/Uvitex⁺) (bottom) of spores by leukocytes in the lungs. (<b>D</b>–<b>E</b>) Mice were challenged IV with <i>C</i>. <i>albicans</i> yeast; kidneys, spleens, and peripheral blood were harvested at day 1 or 3 or from naïve mice (day 0). (<b>D</b>) Representative plots showing neutrophils (CD11b⁺, Ly6G⁺, Ly6G^int, Siglec F^-) with inset plots indicating the proportion of CD11c⁺ neutrophils expressing MHC class II. (<b>E</b>) Time course showing absolute numbers of canonical (MHCII^- CD11c^-), MHCII⁺ CD11c^-, CD11c⁺ and MHCII⁺ CD11c⁺ neutrophils in tissues during systemic candidiasis. All data are representative of at least three independent experiments. N = 3–5 mice. Means±SEM indicated. For C, statistical comparisons were among leukocyte populations; for D, statistical comparisons were with Day 0 control.</p

Direct fungal killing by PMN-DCs and protection by adoptive transfer of PMN-DCs.

<p>Canonical neutrophils or PMN-DCs were generated <i>in vitro</i> from the GMP cell line and co-cultured <i>in vitro</i> with fungi (A-D) or transferred IV into mice (E). (<b>A</b>) Fungal killing by canonical neutrophils or PMN-DCs during <i>in vitro</i> culture with <i>C</i>. <i>albicans</i> yeast (top) or <i>B</i>. <i>dermatitidis</i> yeast (bottom). (<b>B</b>) Scanning electron microscopy (SEM) of canonical neutrophils or PMN-DCs alone, with <i>C</i>. <i>albicans</i> or <i>B</i>. <i>dermatitidis</i>, or stimulated with PMA. Higher magnification images of highlighted boxes are shown to the right of wider image. For interactions with fungi, top images show interactions between cells and fungi highlighting phagocytosis and attempted phagocytosis; bottom images show NETs. (<b>C</b>) SEM comparison of NET structure and thickness released by canonical neutrophils or PMN-DCs in response to <i>C</i>. <i>albicans</i> or <i>B</i>. <i>dermatitidis</i>. (<b>D</b>) <i>C</i>. <i>albicans</i> was incubated for 2 hours to become filamentous before neutrophils or PMN-DCs were added in killing assays in the presence or absence of 50 μg/ml DNase I. (<b>E</b>) WT mice were infected IV with 10⁵ <i>C</i>. <i>albicans</i> yeast and received 2 x 10⁶ canonical neutrophils or PMN-DCs (or PBS vehicle) IV 24 hours later. Burden in kidneys shown at 3 dpi. (A, D) <i>C</i>. <i>albicans</i> viability was determined by XTT assay and compared with neutrophil-absent control to calculate percent killing. (A) <i>B</i>. <i>dermatitidis</i> was plated on BHI agar after to determine number of remaining viable yeast. Means ± SEM are shown. Data are representative of at least two independent experiments.</p

PMN-DCs associate with yeast and kill yeast at higher frequencies than canonical neutrophils.

<p>Mice were challenged with DsRed <i>B</i>. <i>dermatitidis</i>, and lungs were harvested at 7 dpi. (<b>A</b>) Representative plots showing canonical neutrophils, PMN-DCs and moDCs indicating the cells of each population associated with yeast (% Uvitex⁺). Inset histograms show Uvitex⁺ events indicating live (DsRed⁺) and dead (DsRed^-, % noted) yeast associated with each population. (<b>B</b>) The proportion of each population associated with yeast as indicated by Uvitex staining. (<b>C</b>) The killing rate–proportion of Uvitex⁺ yeast that are DsRed^-. (<b>D</b>) The proportion of total yeast (all Uvitex⁺ events) associated with indicated leukocytes. (<b>E</b>) The contribution to yeast killing is indicated by the proportion of total killed yeast in the lung (all DsRed^-Uvitex⁺ events) associated with each population. Panel A shows a single representative experiment of 4 independent experiments; N = 5 mice; means ± SEM indicated.</p

Biguanides enhance antifungal activity against Candida glabrata

Candida spp. are the fourth leading cause of nosocomial blood stream infections in North America. Candida glabrata is the second most frequently isolated species, and rapid development of antifungal resistance has made treatment a challenge. In this study, we investigate the therapeutic potential of metformin, a biguanide with well-established action for diabetes, as an antifungal agent against C. glabrata. Both wild type and antifungal-resistant isolates of C. glabrata were subjected to biguanide and biguanide-antifungal combination treatment. Metformin, as well as other members of the biguanide family, were found to have antifungal activity against C. glabrata, with MIC50 of 9.34 ± 0.16 mg/mL, 2.09 ± 0.04 mg/mL and 1.87 ± 0.05 mg/mL for metformin, phenformin and buformin, respectively. We demonstrate that biguanides enhance the activity of several antifungal drugs, including voriconazole, fluconazole, and amphotericin, but not micafungin. The biguanide-antifungal combinations allowed for additional antifungal effects, with fraction inhibition concentration indexes ranging from 0.5 to 1. Furthermore, metformin was able to lower antifungal MIC50 in voriconazole and fluconazole-resistant clinical isolates of C. glabrata. We also observed growth reduction of C. glabrata with rapamycin and an FIC of 0.84 ± 0.09 when combined with metformin, suggesting biguanide action in C. glabrata may be related to inhibition of the mTOR complex. We conclude that the biguanide class has direct antifungal therapeutic potential and enhances the activity of select antifungals in the treatment of resistant C. glabrata isolates. These data support the further investigation of biguanides in the combination treatment of serious fungal infections