Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans

Behnsen, Judith; Narang, Priyanka; Hasenberg, Mike; Gunzer, Frank; Bilitewski, Ursula; Klippel, Nina; Rohde, Manfred; Brock, Matthias; Brakhage, Axel A.; Gunzer, Matthias

doi:10.1371/journal.ppat.0030013

Cited by 94 publications

(105 citation statements)

References 59 publications

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“…Neutrophils were also obtained by positive selection from mouse BM, as described previously. 15 BM cells were isolated as above after intravenous injection for 2 hours with either hG-CSF or phosphate-buffered saline (PBS) as below and then cell tested for TPO production in the supernatant by ELISA (R&D Systems) without further incubation.…”

Section: Cells and Cell Linesmentioning

confidence: 99%

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

Köhler

Filippo²,

Hasenberg

et al. 2011

Blood

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186

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Emergency mobilization of neutrophil granulocytes (neutrophils) from the bone marrow (BM) is a key event of early cellular immunity. The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) stimulates this process, but it is unknown how individual neutrophils respond in situ. We show by intravital 2-photon microscopy that a systemic dose of human clinical-grade G-CSF rapidly induces the motility and entry of neutrophils into blood vessels within the tibial BM of mice. Simultaneously, the neutrophil-attracting chemokine KC (Cxcl1) spikes in the blood. In mice lacking the KC receptor Cxcr2, G-CSF fails to mobilize neutrophils and antibody blockade of Cxcr2 inhibits the mobilization and induction of neutrophil motility in the BM. KC is expressed by megakaryocytes and endothelial cells in situ and is released in vitro by megakaryocytes isolated directly from BM. This production of KC is strongly increased by thrombopoietin (TPO). Systemic G-CSF rapidly induces the increased production of TPO in BM. Accordingly, a single injection of TPO mobilizes neutrophils with kinetics similar to G-CSF, and mice lacking the TPO receptor show impaired neutrophil mobilization after short-term G-CSF administration. Thus, a network of signaling molecules, chemokines, and cells controls neutrophil release from the BM, and their mobilization involves rapidly induced Cxcr2-mediated motility controlled by TPO as a pacemaker. IntroductionNeutrophils are the most abundant and, arguably, the most important leukocyte in the vertebrate immune system. Under normal conditions, human bone marrow (BM) produces ϳ 10 11 neutrophils per day. 1 In "danger situations" such as peripheral infections, the constant release of neutrophils can be dramatically increased within hours, a process termed danger or stress mobilization. 2 The hematopoietic cytokine granulocyte-colony stimulating factor (G-CSF) is central to the danger mobilization of neutrophils in both humans and mice. 3 However, although recombinant G-CSF has been used in clinical hematology for 20 years, the molecular mechanisms by which it mobilizes neutrophils are still not well understood.Neutrophils are restrained in the BM by the binding of their chemokine receptor Cxcr4 to the chemokine Cxcl12, which is expressed in a membrane-associated fashion by BM stromal cells. 4 There is evidence that G-CSF breaks the Cxcr4-Cxcl12 bond by activating neutrophil proteases, 5 thereby releasing neutrophils from the BM into the bloodstream. 3,6 However, several findings cannot be explained by the Cxcr4-Cxcl12 breakage concept alone. First, G-CSF can mobilize neutrophils in protease-deficient mice, arguing against the need for protease activation for this process. 7,8 Second, because neutrophil-specific deletion of Cxcr4 in mice results in much higher numbers of circulating neutrophils compared with wild-type animals, factors other than Cxcr4 must be involved in steering neutrophils into the BM blood sinuses (unless the process is passive). Finally, the specific Cxcr4-antagonist AMD3100 a...

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Section: Cells and Cell Linesmentioning

confidence: 99%

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

Köhler

Filippo²,

Hasenberg

et al. 2011

Blood

Self Cite

186

174

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“…Alveolar macrophages form the first barrier of the immune defence against Aspergillus, phagocytozing inhaled conidia Behnsen et al, 2007). Therefore, we studied the effect of propionate, as a propionyl-CoA-generating carbon source, on the killing efficiency of macrophages against conidia derived from both wild-type and a methylcitrate synthase mutant.…”

Section: In Vitro Susceptibility Of Conidia Against Alveolar Macrophagesmentioning

confidence: 99%

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis

et al. 2007

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SummaryInvasive aspergillosis is a life-threatening disease mainly caused by the fungus Aspergillus fumigatus. In immunocompromised individuals conidia are not efficiently inactivated, which can end in invasive fungal growth. However, the metabolic requirements of the fungus are hardly known. Earlier investigations revealed an accumulation of toxic propionyl-CoA in a methylcitrate synthase mutant, when grown on propionyl-CoA-generating carbon sources. During invasive growth propionyl-CoA could derive from proteins, which are released from infected host tissues. We therefore assumed that a methylcitrate synthase mutant might display an attenuated virulence. Here we show that the addition of propionate to cell culture medium enhanced the ability of alveolar macrophages to kill methylcitrate synthase mutant but not wild-type conidia. When tested in a murine infection model, the methylcitrate synthase mutant displayed attenuated virulence and, furthermore, was cleared from tissues when mice survived the first phase of acute infection. The amplification of cDNA from infected mouse lungs confirmed the transcription of the methylcitrate synthase gene during invasion, which leads to the suggestion that amino acids indeed serve as growth-supporting nutrients during invasive growth of A. fumigatus. Thus, blocking of methylcitrate synthase activity abrogates fungal growth and provides a suitable target for new antifungals.

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“…In a 3D microenvironment, cells need to either degrade the extracellular matrix (ECM) or squeeze through small pores in amoeboid fashion (Wolf and Friedl 2008), and they may experience chemokine gradients differently, since many are matrix-binding (Patel et al 2001). As a result, cells behave very differently in 3D vs. 2D environments (Behnsen et al 2007;Cukierman et al 2001;Griffith and Swartz 2006;Pedersen and Swartz 2005). Thus, to examine cell migration in a physiologically realistic setting, migration assays need to accurately recapitulate the 3D microenvironment.…”

Section: Introductionmentioning

confidence: 99%

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

Haessler

Kalinin

Swartz

et al. 2009

Biomed Microdevices

158

164

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The current state-of-art in 3D microfluidic chemotaxis device (μFCD) is limited by the inherent coupling of the fluid flow and chemical concentration gradients. Here, we present an agarose-based 3D μFCD that decouples these two important parameters, in that the flow control channels are separated from the cell compartment by an agarose gel wall. This decoupling is enabled by the transport property of the agarose gel, which-in contrast to the conventional microfabrication material such as polydimethylsiloxane (PDMS)-provides an adequate physical barrier for convective fluid flow while at the same time readily allowing protein diffusion. We demonstrate that in this device, a gradient can be pre-established in an agarose layer above the cell compartment (a gradient buffer) before adding the 3D cell-containing matrix, and the dextran (10 kDa) concentration gradients can be re-established within 10 min across the cell-containing matrix and remain stable indefinitely. We successfully quantified the chemotactic response of murine dendritic cells to a gradient of CCL19, an 8.8 kDa lymphoid chemokine, within a type I collagen matrix. This model system is easy to set up, highly reproducible, and will benefit research on 3D chemoinvasion studies, for example with cancer cells or immune cells. Because of its gradient buffering capacity, it is particularly suitable for studying rapidly migrating cells like mature dendritic cells and neutrophils.

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Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus fumigatus and Candida albicans

Cited by 94 publications

References 59 publications

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

G-CSF–mediated thrombopoietin release triggers neutrophil motility and mobilization from bone marrow via induction of Cxcr2 ligands

Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis

An agarose-based microfluidic platform with a gradient buffer for 3D chemotaxis studies

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