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In JoVE (1)
Other Publications (9)
Articles by Mike Hasenberg in JoVE
JoVE Immunology and Infection
Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy
1Institute for Molecular and Clinical Immunology, Otto-von-Guericke University Magdeburg, 2Department of Immunoregulation, Helmholtz Center for Infection Research
We show, how to use 2-photon microscopy for the observation of the dynamics of neutrophil granulocytes in infected lungs while they phagocytose pathogens or produce neutrophil extracellular traps (NETs).
Other articles by Mike Hasenberg on PubMed
Environmental Dimensionality Controls the Interaction of Phagocytes with the Pathogenic Fungi Aspergillus Fumigatus and Candida Albicans
The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why "delocalized" Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.
Basal Expression of the Aspergillus Fumigatus Transcriptional Activator CpcA is Sufficient to Support Pulmonary Aspergillosis
Aspergillosis is a disease determined by various factors that influence fungal growth and fitness. A conserved signal transduction cascade linking environmental stress to amino acid homeostasis is the Cross-Pathway Control (CPC) system that acts via phosphorylation of the translation initiation factor eIF2 by a sensor kinase to elevate expression of a transcription factor. Ingestion of Aspergillus fumigatus conidia by macrophages does not trigger this stress response, suggesting that their phagosomal microenvironment is not deficient in amino acids. The cpcC gene encodes the CPC eIF2alpha kinase, and deletion mutants show increased sensitivity towards amino acid starvation. CpcC is specifically required for the CPC response but has limited influence on the amount of phosphorylated eIF2alpha. Strains deleted for the cpcC locus are not impaired in virulence in a murine model of pulmonary aspergillosis. Accordingly, basal expression of the Cross-Pathway Control transcriptional activator appears sufficient to support aspergillosis in this disease model.
Identification of a Putative Crf Splice Variant and Generation of Recombinant Antibodies for the Specific Detection of Aspergillus Fumigatus
Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA) in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA.
Production of Extracellular Traps Against Aspergillus Fumigatus in Vitro and in Infected Lung Tissue is Dependent on Invading Neutrophils and Influenced by Hydrophobin RodA
Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.
G-CSF-mediated Thrombopoietin Release Triggers Neutrophil Motility and Mobilization from Bone Marrow Via Induction of Cxcr2 Ligands
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.
Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies in Vitro and in Vivo
Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (~1 h), has a high yield (5-10*10⁶ PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo.
Shaping the Fungal Adaptome--stress Responses of Aspergillus Fumigatus
Aspergillus fumigatus as prime pathogen to cause aspergillosis has evolved as a saprophyte, but is also able to infect and colonise immunocompromised hosts. Based on the 'dual use' hypothesis of fungal pathogenicity, general characteristics have to be considered as unspecific virulence determinants, among them stress adaptation capacities. The susceptible, warm-blooded mammalian host represents a specific ecological niche that poses several kinds of stress conditions to the fungus during the course of infection. Detailed knowledge about the cellular pathways and adaptive traits that have evolved in A. fumigatus to counteract situations of stress and varying environmental conditions is crucial for the identification of novel and specific antifungal targets. Comprehensive profiling data accompanied by mutant analyses have shed light on such stressors, and nutritional deprivation, oxidative stress, hypoxia, elevated temperature, alkaline pH, extensive secretion, and, in particular during treatment with antifungals, cell membrane perturbations appear to represent the major hazards A. fumigatus has to cope with during infection. Further efforts employing innovative approaches and advanced technologies will have to be made to expand our knowledge about the scope of the A. fumigatus adaptome that is relevant for disease.
Phagocyte Responses Towards Aspergillus Fumigatus
The saprophytic fungus Aspergillus fumigatus is a mold which is ubiquitously present in the environment. It produces large numbers of spores, called conidia that we constantly inhale with the breathing air. Healthy individuals normally do not suffer from true fungal infections with this pathogen. A normally robust resistance against Aspergillus is based on the presence of a very effective immunological defense system in the vertebrate body. Inhaled conidia are first encountered by lung-resident alveolar macrophages and then by neutrophil granulocytes. Both cell types are able to effectively ingest and destroy the fungus. Although some responses of the adaptive immune system develop, the key protection is mediated by innate immunity. The importance of phagocytes for defense against aspergillosis is also supported by large numbers of animal studies. Despite the production of aggressive chemicals that can extracellularly destroy fungal pathogens, the main effector mechanism of the innate immune system is phagocytosis. Very recently, the production of extracellular neutrophil extracellular traps (NETs) consisting of nuclear DNA has been added to the armamentarium that innate immune cells use against infection with Aspergillus. Phagocyte responses to Aspergillus are very broad, and a number of new observations have added to this complexity in recent years. To summarize established and newer findings, we will give an overview on current knowledge of the phagocyte system for the protection against Aspergillus.
Surface Display of Gaussia Princeps Luciferase Allows Sensitive Fungal Pathogen Detection During Cutaneous Aspergillosis
Non-invasive imaging techniques in microbial disease models have delivered valuable insights in the intimate pathogen-host interplay during infection. Here we describe evaluation and validation of a transgenic bioluminescence reporter strain of the human pathogenic mold Aspergillus fumigatus, one of the main fungal pathogens affecting immunocompromised individuals. Expression and surface display of the Gaussia princeps luciferase allowed sensitive and rapid detection of luminescence emitted from this strain after substrate addition, with photon fluxes strongly correlating to the amounts of fungal conidia or germlings. The reporter strain allowed spatio-temporal monitoring of infection in a cutaneous model of aspergillosis, where neutropenic mice maintained the fungal burden while immunocompetent ones were able to clear it entirely. Most importantly, antifungal therapy could be followed in this type of disease model making use of the bioluminescent A. fumigatus strain. In conclusion, combining sensitivity of the Gaussia luciferase with a surface display expression system in the fungal host allows longitudinal infection studies on cutaneous forms of aspergillosis, providing perspective on drug screening approaches at high-throughput.
