Publications by authors named "Matthias Gunzer"

136 Publications

Antibody-guided in vivo imaging of Aspergillus fumigatus lung infections during antifungal azole treatment.

Nat Commun 2021 03 17;12(1):1707. Epub 2021 Mar 17.

Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University, Tübingen, Germany.

Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA and its response to azole treatment is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo in a neutropenic mouse model and 3D light sheet fluorescence microscopy ex vivo in the infected mouse lungs to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.
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http://dx.doi.org/10.1038/s41467-021-21965-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969596PMC
March 2021

SOCS-1 inhibition of type I interferon restrains Staphylococcus aureus skin host defense.

PLoS Pathog 2021 Mar 10;17(3):e1009387. Epub 2021 Mar 10.

Department of Medicine, Division of Infectious Diseases, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

The skin innate immune response to methicillin-resistant Staphylococcus aureus (MRSA) culminates in the formation of an abscess to prevent bacterial spread and tissue damage. Pathogen recognition receptors (PRRs) dictate the balance between microbial control and injury. Therefore, intracellular brakes are of fundamental importance to tune the appropriate host defense while inducing resolution. The intracellular inhibitor suppressor of cytokine signaling 1 (SOCS-1), a known JAK/STAT inhibitor, prevents the expression and actions of PRR adaptors and downstream effectors. Whether SOCS-1 is a molecular component of skin host defense remains to be determined. We hypothesized that SOCS-1 decreases type I interferon production and IFNAR-mediated antimicrobial effector functions, limiting the inflammatory response during skin infection. Our data show that MRSA skin infection enhances SOCS-1 expression, and both SOCS-1 inhibitor peptide-treated and myeloid-specific SOCS-1 deficient mice display decreased lesion size, bacterial loads, and increased abscess thickness when compared to wild-type mice treated with the scrambled peptide control. SOCS-1 deletion/inhibition increases phagocytosis and bacterial killing, dependent on nitric oxide release. SOCS-1 inhibition also increases the levels of type I and type II interferon levels in vivo. IFNAR deletion and antibody blockage abolished the beneficial effects of SOCS-1 inhibition in vivo. Notably, we unveiled that hyperglycemia triggers aberrant SOCS-1 expression that correlates with decreased overall IFN signatures in the infected skin. SOCS-1 inhibition restores skin host defense in the highly susceptible hyperglycemic mice. Overall, these data demonstrate a role for SOCS-1-mediated type I interferon actions in host defense and inflammation during MRSA skin infection.
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http://dx.doi.org/10.1371/journal.ppat.1009387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984627PMC
March 2021

Stroke increases the expression of ACE2, the SARS-CoV-2 binding receptor, in murine lungs.

Brain Behav Immun 2021 05 20;94:458-462. Epub 2021 Feb 20.

Institute for Experimental Immunology and Imaging, University Hospital Essen, University of Duisburg-Essen, DE-45147 Essen, Germany; Leibniz-Institut für Analytische Wissenschaften - ISAS -e.V., Dortmund, Germany. Electronic address:

Background: The newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused a worldwide pandemic of human respiratory disease. Angiotensin-converting enzyme (ACE) 2 is the key receptor on lung epithelial cells to facilitate initial binding and infection of SARS-CoV-2. The binding to ACE2 is mediated via the spike glycoprotein present on the viral surface. Recent clinical data have demonstrated that patients with previous episodes of brain injuries are a high-risk group for SARS-CoV-2 infection. An explanation for this finding is currently lacking. Sterile tissue injuries including stroke induce the release of several inflammatory mediators that might modulate the expression levels of signaling proteins in distant organs. Whether systemic inflammation following brain injury can specifically modulate ACE2 expression in different vital tissues has not been investigated.

Methods: For the induction of brain stroke, mice were subjected to a surgical procedure for transient interruption of blood flow in the middle cerebral artery for 45 min and sacrificed after 1 and 3 days for analysis of brain, lung, heart, and kidney tissues. Gene expression and protein levels of ACE2, ACE, IL-6 and IL1β were measured by quantitative PCR and Western blot, respectively. The level of soluble ACE2 in plasma and bronchial alveolar lavage (BAL) was measured using an immunoassay. Immune cell populations in lymphoid organs were analyzed by flow cytometry. Post-stroke pneumonia in mice was examined by bacterial cultures from lung homogenates and whole blood.

Results: Strikingly, 1 day after surgery, we observed a substantial increase in the protein levels of ACE2 in the lungs of stroke mice compared to sham-operated mice. However, the protein levels of ACE2 were found unchanged in the heart, kidney, and brain of these animals. In addition, we found increased transcriptional levels of alveolar ACE2 after stroke. The increased expression of ACE2 was significantly associated with the severity of behavioral deficits after stroke. The higher protein levels of alveolar ACE2 persisted until 3 days of stroke. Interestingly, we found reduced levels of soluble ACE2 in plasma but not in BAL in stroke-operated mice compared to sham mice. Furthermore, stroke-induced parenchymal and systemic inflammation was evident with the increased expression of IL-6 and IL-1β. Reduced numbers of T-lymphocytes were present in the blood and spleen as an indicator of sterile tissue injury-induced immunosuppression.

Conclusions: We demonstrate specific augmented alveolar ACE2 levels and inflammation in murine lungs after experimental stroke. These pre-clinical findings suggest that patients with brain injuries may have increased binding affinity to SARS-CoV-2 in their lungs which might explain why stroke is a risk factor for higher susceptibility to develop COVID-19.
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http://dx.doi.org/10.1016/j.bbi.2021.01.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896496PMC
May 2021

Modulating Microglial Cells for Promoting Brain Recovery and Repair.

Front Cell Neurosci 2020 11;14:627987. Epub 2021 Jan 11.

Institute of Experimental Immunology and Imaging, University of Duisburg-Essen, University Hospital Essen, Essen, Germany.

Representing the brain's innate immune cells that interact vividly with blood-derived immune cells and brain parenchymal cells, microglia set the stage for successful brain remodeling and repair in the aftermath of brain damage. With the development of pharmacological colony-stimulating factor-1 receptor inhibitors, which allow inhibiting or depleting microglial cells, and of transgenic mice, allowing the inducible depletion of microglial cells, experimental tools have become available for studying roles of microglia in neurodegenerative and neurorestorative processes. These models open fundamental insights into roles of microglia in controlling synaptic plasticity in the healthy and the injured brain. Acting as a switch from injury to repair, microglial cells might open opportunities for promoting neurological recovery in human patients upon brain injury.
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http://dx.doi.org/10.3389/fncel.2020.627987DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829249PMC
January 2021

Neutrophil granulocytes promote flow stagnation due to dynamic capillary stalls following experimental stroke.

Brain Behav Immun 2021 Mar 22;93:322-330. Epub 2021 Jan 22.

Department of Neurology, Otto-von-Guericke University, Magdeburg, Germany. Electronic address:

Flow stagnation of peri-ischemic capillaries due to dynamic leukocyte stalls has been described to be a contributor to ongoing penumbral injury in transient brain ischemia, but has not been investigated in permanent experimental stroke so far. Moreover, it is discussed that obstructing neutrophils are involved in this process; however, their contribution has not yet been proven. Here, we characterize the dynamics of neutrophil granulocytes in two models of permanent stroke (photothrombosis and permanent middle cerebral artery occlusion) using intravital two-photon fluorescence microscopy. Different to previous studies on LysM-eGFP cells we additionally apply a transgenic mouse model with tdTomato-expressing neutrophils to avoid interference from additional immune cell subsets. We identify repetitively occurring capillary stalls of varying duration promoted by neutrophils in both models of permanent cerebral ischemia, validating the suitability of our new transgenic mouse model in determining neutrophil occlusion formation in vivo. Flow cytometric analysis of peripheral blood (PB) and brain tissue from mice subjected to photothrombosis reveal an increase in the total proportion of neutrophils, with selective upregulation of endothelial adherence markers in the PB. In conclusion, the dynamic microcirculatory stall phenomenon that is described after transient ischemia followed by reperfusion also occurs after permanent small- or large-vessel stroke and is clearly attributable to neutrophils.
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http://dx.doi.org/10.1016/j.bbi.2021.01.011DOI Listing
March 2021

Light Sheet Microscopy Using FITC-Albumin Followed by Immunohistochemistry of the Same Rehydrated Brains Reveals Ischemic Brain Injury and Early Microvascular Remodeling.

Front Cell Neurosci 2020 5;14:625513. Epub 2021 Jan 5.

Department of Neurology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Until recently, the visualization of cerebral microvessels was hampered by the fact that only short segments of vessels could be evaluated in brain sections by histochemistry. These limitations have been overcome by light sheet microscopy, which allows the 3D analysis of microvasculature in cleared brains. A major limitation of light sheet microscopy is that antibodies do not sufficiently penetrate cleared brains. We herein describe a technique of reverse clearing and rehydration, which after microvascular network analysis allows brain sectioning and immunohistochemistry employing a broad set of antibodies. Performing light sheet microscopy on brains of mice exposed to intraluminal middle cerebral artery occlusion (MCAO), we show that in the early phase of microvascular remodeling branching point density was markedly reduced, more strongly than microvascular length. Brain infarcts in light sheet microscopy were sharply demarcated by their autofluorescence signal, closely corresponding to brain infarcts revealed by Nissl staining. Neuronal survival, leukocyte infiltration, and astrocytic reactivity could be evaluated by immunohistochemistry in rehydrated brains, as shown in direct comparisons with non-cleared brains. Immunohistochemistry revealed microthrombi in ischemic microvessels that were likely responsible for the marked branching point loss. The balance between microvascular thrombosis and remodeling warrants further studies at later time-points after stroke.
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http://dx.doi.org/10.3389/fncel.2020.625513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813928PMC
January 2021

Platelet endothelial cell adhesion molecule-1 is a gatekeeper of neutrophil transendothelial migration in ischemic stroke.

Brain Behav Immun 2021 Mar 31;93:277-287. Epub 2020 Dec 31.

Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Electronic address:

Rationale: Adhesion molecules are key elements in stroke-induced brain injury by regulating the migration of effector immune cells from the circulation to the lesion site. Platelet endothelial cell adhesion molecule-1 (PECAM-1) is an adhesion molecule highly expressed on endothelial cells and leukocytes, which controls the final steps of trans-endothelial migration. A functional role for PECAM-1 in post-ischemic brain injury has not yet been demonstrated.

Objective: Using genetic Pecam-1 depletion and PECAM-1 blockade using a neutralizing anti-PECAM-1 antibody, we evaluated the role of PECAM-1 mediated trans-endothelial immune cell migration for ischemic injury, delayed brain atrophy, and brain immune cell infiltrates. Trans-endothelial immune cell migration was furthermore evaluated in cultured human cerebral microvascular endothelial cells.

Methods And Results: Transient middle cerebral artery occlusion (tMCAO) was induced in 10-12-week-old male Pecam-1 and Pecam-1 wildtype mice. PECAM-1 levels increased in the ischemic brain tissue due to the infiltration of PECAM-1 leukocytes. Using magnetic resonance imaging, we observed smaller infarct volume, less edema formation, and less brain atrophy in Pecam-1 compared with Pecam-1 wildtype mice. The transmigration of leukocytes, specifical neutrophils, was selectively reduced by Pecam-1, as shown by immune fluorescence and flow cytometry in vivo and transmigration assays in vitro. Importantly, inhibition with an anti-PECAM-1 antibody in wildtype mice decreased neutrophil brain influx and infarct.

Conclusion: PECAM-1 controls the trans-endothelial migration of neutrophils in a mouse model of ischemic stroke. Antibody blockade of PECAM-1 after stroke onset ameliorates stroke severity in mice, making PECAM-1 an interesting target to dampen post-stroke neuroinflammation, reduce ischemic brain injury, and enhance post-ischemic brain remodeling.
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http://dx.doi.org/10.1016/j.bbi.2020.12.026DOI Listing
March 2021

Neutrophil dynamics, plasticity and function in acute neurodegeneration following neonatal hypoxia-ischemia.

Brain Behav Immun 2021 Feb 14;92:234-244. Epub 2020 Dec 14.

Department of Pediatrics I, Neonatology & Experimental Perinatal Neurosciences, University Hospital Essen, University Duisburg-Essen, Essen, Germany. Electronic address:

Neonatal encephalopathy following hypoxia-ischemia (HI) is a major cause of long-term morbidity and mortality in children. Even though HI-induced neuroinflammation, involving infiltration of peripheral immune cells into the CNS has been associated with disease pathogenesis, the specific role of neutrophils is highly debated. Due to immaturity of the neonatal immune system, it has been assumed that neutrophils are less clinically relevant in neonatal HI-induced brain injury. In the present study, we demonstrate that neutrophils are rapidly activated in the neonatal brain after exposure to experimental HI, revealed by an enhanced proportion of CD86 cells and an increased expression of CD11b compared to splenic and blood neutrophils. Furthermore, production of reactive oxygen species and the proportion of hyperactivated/aged (CXCR4CD62L) cells was enhanced in brain compared to peripheral neutrophils. Delayed neutrophil depletion, initiated 12 h after HI resulted in reduced cellular neurodegeneration, associated with reduced micro- and astroglial activation. In the present study, we uncovered a new complex switch of the phenotype in brain neutrophils, which may offer new possibilities for the development of selective therapeutic approaches by modulation of neutrophils in the early post-hypoxic disease phase.
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http://dx.doi.org/10.1016/j.bbi.2020.12.012DOI Listing
February 2021

The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides.

Cell Microbiol 2021 Mar 5;23(3):e13301. Epub 2021 Jan 5.

Mikrobiologisches Institut - Klinische Mikrobiologie, Immunologie und Hygiene, Universitätsklinikum Erlangen and Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine-stabilised (CS)αβ defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore-associated defX gene products delay the growth of Gram-positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore-associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a 'virulence school' for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co-infecting microorganisms to break colonisation resistance at host surfaces.
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http://dx.doi.org/10.1111/cmi.13301DOI Listing
March 2021

Advances in the In Vivo Molecular Imaging of Invasive Aspergillosis.

J Fungi (Basel) 2020 Dec 4;6(4). Epub 2020 Dec 4.

Werner Siemens Imaging Center, Department of Preclinical Imaging and Radiopharmacy, Eberhard Karls University Tübingen, 72076 Tübingen, Germany.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection of immunocompromised patients with , a ubiquitous environmental mould. While there are numerous functioning antifungal therapies, their high cost, substantial side effects and fear of overt resistance development preclude permanent prophylactic medication of risk-patients. Hence, a fast and definitive diagnosis of IPA is desirable, to quickly identify those patients that really require aggressive antimycotic treatment and to follow the course of the therapeutic intervention. However, despite decades of research into this issue, such a diagnostic procedure is still not available. Here, we discuss the array of currently available methods for IPA detection and their limits. We then show that molecular imaging using positron emission tomography (PET) combined with morphological computed tomography or magnetic imaging is highly promising to become a future non-invasive approach for IPA diagnosis and therapy monitoring, albeit still requiring thorough validation and relying on further acceptance and dissemination of the approach. Thereby, our approach using the -specific humanized monoclonal antibody hJF5 labelled with Cu as PET-tracer has proven highly effective in pre-clinical models and hence bears high potential for human application.
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http://dx.doi.org/10.3390/jof6040338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761943PMC
December 2020

Intravital 2-Photon Microscopy of Diverse Cell Types in the Murine Tibia.

Methods Mol Biol 2021 ;2236:189-201

Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen, Essen, Germany.

Intravital imaging allows the visualization of fluorescently labeled structures like cells, blood flow, and pathogens in a living organism. Nowadays, numerous methods for imaging in several organs are available. In this chapter, we present a method for intravital 2-photon microscopy of the murine tibial bone marrow. It enables the observation of hematopoietic cells including cells of the innate and adaptive immune system under physiological conditions. Motility analyses within this complex environment led to insights into their migratory potential as well as their interactions with other cells or blood vessels.
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http://dx.doi.org/10.1007/978-1-0716-1060-2_15DOI Listing
March 2021

Ventricular assist device for a coronavirus disease 2019-affected heart.

ESC Heart Fail 2021 02 20;8(1):162-166. Epub 2020 Nov 20.

Department of Thoracic and Cardiovascular Surgery, West German Heart and Vascular Center Essen, University Hospital Essen, Essen, Germany.

Coronavirus disease 2019 (COVID-19) is challenging the care for cardiovascular patients, resulting in serious consequences with increasing mortality in pre-diseased heart failure patients. In the current state of the pandemic, the physiopathology of COVID-19 affecting pre-diseased hearts and the management of terminal heart failure in COVID-19 patients remain unclear. We outline the findings of a young COVID-19 patient suffering from idiopathic cardiomyopathy who was treated for acute multi-organ failure and required cardiac surgery with implantation of a temporary right ventricular and durable left ventricular assist device (LVAD). For deeper translational insights, we used in-depth tissue analysis by electron and light sheet fluorescence microscopy revealing evidence for spatial distribution of severe acute respiratory syndrome coronavirus 2 in the heart. This indicates that in-depth analysis may represent a valuable tool in understanding indistinct clinical cases. We conclude that COVID-19 directly affects pre-diseased hearts, but the consequences can be treated successfully with LVAD implantation.
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http://dx.doi.org/10.1002/ehf2.13120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753611PMC
February 2021

Co-option of Neutrophil Fates by Tissue Environments.

Cell 2020 Nov 23;183(5):1282-1297.e18. Epub 2020 Oct 23.

Area of Cell & Developmental Biology, Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid 28029, Spain; Institute for Cardiovascular Prevention (IPEK), Ludwig-Maximillians-Universitat, Munich 80802, Germany. Electronic address:

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.
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http://dx.doi.org/10.1016/j.cell.2020.10.003DOI Listing
November 2020

Homozygous Smpd1 deficiency aggravates brain ischemia/ reperfusion injury by mechanisms involving polymorphonuclear neutrophils, whereas heterozygous Smpd1 deficiency protects against mild focal cerebral ischemia.

Basic Res Cardiol 2020 10 14;115(6):64. Epub 2020 Oct 14.

Department of Neurology, University Hospital Essen, University of Duisburg-Essen, 45147, Essen, Germany.

By cleaving sphingomyelin into ceramide, which is an essential component of plasma membrane microdomains, acid sphingomyelinase (Asm) pivotally controls cell signaling. To define how the activation of the Asm/ceramide pathway, which occurs within seconds to minutes upon stress stimuli, influences brain ischemia/reperfusion (I/R) injury, we exposed male and female wildtype mice carrying both alleles of Asm's gene sphingomyelinase phosphodiesterase-1 (Smpd1), heterozygously Asm-deficient mice (Smpd1) and homozygously Asm-deficient mice (Smpd1) of different age (8, 12 or 16 weeks) to 30, 60 or 90 min intraluminal middle cerebral artery occlusion (MCAO). For studying the contribution of brain-invading polymorphonuclear neutrophils (PMN) to I/R injury, PMNs were depleted by delivery of a PMN-specific Ly6G antibody. In male and female mice exposed to 30 min, but not 60 or 90 min MCAO, homozygous Smpd1 consistently increased I/R injury, blood-brain barrier permeability and brain leukocyte and PMN infiltration, whereas heterozygous Smpd1 reduced I/R injury. Increased abundance of the intercellular leukocyte adhesion molecule ICAM-1 was noted on cerebral microvessels of Smpd1 mice. PMN depletion by anti-Ly6G delivery prevented the exacerbation of I/R injury in Smpd1 compared with wildtype mice and reduced brain leukocyte infiltrates. Our results show that Asm tempers leukocyte entry into the reperfused ischemic brain, thereby attenuating I/R injury.
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http://dx.doi.org/10.1007/s00395-020-00823-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7560939PMC
October 2020

Spatiotemporal restriction of endothelial cell calcium signaling is required during leukocyte transmigration.

J Exp Med 2021 Jan;218(1)

Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL.

Endothelial cell calcium flux is critical for leukocyte transendothelial migration (TEM), which in turn is essential for the inflammatory response. Intravital microscopy of endothelial cell calcium dynamics reveals that calcium increases locally and transiently around the transmigration pore during TEM. Endothelial calmodulin (CaM), a key calcium signaling protein, interacts with the IQ domain of IQGAP1, which is localized to endothelial junctions and is required for TEM. In the presence of calcium, CaM binds endothelial calcium/calmodulin kinase IIδ (CaMKIIδ). Disrupting the function of CaM or CaMKII with small-molecule inhibitors, expression of a CaMKII inhibitory peptide, or expression of dominant negative CaMKIIδ significantly reduces TEM by interfering with the delivery of the lateral border recycling compartment (LBRC) to the site of TEM. Endothelial CaMKII is also required for TEM in vivo as shown in two independent mouse models. These findings highlight novel roles for endothelial CaM and CaMKIIδ in transducing the spatiotemporally restricted calcium signaling required for TEM.
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http://dx.doi.org/10.1084/jem.20192378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953625PMC
January 2021

Lactate released by inflammatory bone marrow neutrophils induces their mobilization via endothelial GPR81 signaling.

Nat Commun 2020 07 15;11(1):3547. Epub 2020 Jul 15.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81 mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.
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http://dx.doi.org/10.1038/s41467-020-17402-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363928PMC
July 2020

Defective migration and dysmorphology of neutrophil granulocytes in atypical chronic myeloid leukemia treated with ruxolitinib.

BMC Cancer 2020 Jul 13;20(1):650. Epub 2020 Jul 13.

Department of Hematology, University Hospital, West German Cancer Center (WTZ), University Duisburg-Essen, Hufelandstrasse 55, 45147, Essen, Germany.

Background: The identification of pathologically altered neutrophil granulocyte migration patterns bears strong potential for surveillance and prognostic scoring of diseases. We recently identified a strong correlation between impaired neutrophil motility and the disease stage of myelodysplastic syndrome (MDS). Here, we apply this assay to study quantitively increased neutrophils of a patient suffering from a rare leukemia subtype, atypical chronic myeloid leukemia (aCML).

Methods: A 69-year-old male was analyzed in this study. Besides routine analyses, we purified the patient's neutrophils from peripheral whole blood and studied their migration behavior using time-lapse video microscopy in a standardized assay. These live cell migration analyses also allowed for the quantification of cell morphology. Furthermore, the cells were stained for the markers CD15, CD16, fMLPR, CXCR1 and CXCR2.

Results: Despite cytoreductive therapy with hydroxyurea, the patient's WBC and ANC were poorly controlled and severe dysgranulopoiesis with hypogranularity was observed. Neutrophils displayed strongly impaired migration when compared to healthy controls and migrating cells exhibited a more flattened-out morphology than control neutrophils. Because of a detected CSF3R (p.T618I) mutation and constitutional symptoms treatment with ruxolitinib was initiated. Within 1 week of ruxolitinib treatment, the cell shape normalized and remained indistinguishable from healthy control neutrophils. However, neutrophil migration did not improve over the course of ruxolitinib therapy but was strikingly altered shortly before a sinusitis with fever and bleeding from a gastric ulcer. Molecular work-up revealed that under ruxolitinib treatment, the CSF3R clone was depleted, yet the expansion of a NRAS mutated subclone was promoted.

Conclusion: These results demonstrate the usefulness of neutrophil migration analyses to uncover corresponding alterations of neutrophil migration in rare myeloid neoplasms. Furthermore, in addition to monitoring migration the determination of morphological features of live neutrophils might represent a useful tool to monitor the effectiveness of therapeutic approaches.
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http://dx.doi.org/10.1186/s12885-020-07130-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7359613PMC
July 2020

Durable and controlled depletion of neutrophils in mice.

Nat Commun 2020 06 2;11(1):2762. Epub 2020 Jun 2.

Swiss Institute for Experimental Cancer Research, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015, Lausanne, Switzerland.

Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.
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http://dx.doi.org/10.1038/s41467-020-16596-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265525PMC
June 2020

Systemic but not MDSC-specific IRF4 deficiency promotes an immunosuppressed tumor microenvironment in a murine pancreatic cancer model.

Cancer Immunol Immunother 2020 Oct 24;69(10):2101-2112. Epub 2020 May 24.

Center of Integrated Protein Science Munich (CIPSM) and Division of Clinical Pharmacology, University Hospital, LMU Munich, Munich, Germany.

Pancreatic ductal adenocarcinoma is characterized by a strong immunosuppressive network with a dense infiltration of myeloid cells including myeloid-derived suppressor cells (MDSC). Two distinct populations of MDSC have been defined: polymorphonuclear MDSC (PMN-MDSC) and monocytic MDSC (M-MDSC). Several factors influence the development and function of MDSC including the transcription factor interferon regulatory factor 4 (IRF4). Here, we show that IRF4 deficiency accelerates tumor growth and reduces survival, accompanied with a dense tumor infiltration with PMN-MDSC and reduced numbers of CD8 T cells. As IRF4 has been described to modulate myeloid cell development and function, particularly of PMN-MDSC, we analyzed its role using MDSC-specific IRF4 knockout mice with the Ly6G or LysM knock-in allele expressing Cre recombinase and Irf4. In GM-CSF-driven bone marrow cultures, IRF4 deficiency increased the frequency of MDSC-like cells with a strong T cell suppressive capacity. Myeloid (LysM)-specific depletion of IRF4 led to increased tumor weight and a moderate splenic M-MDSC expansion in tumor-bearing mice. PMN cell (Ly6G)-specific depletion of IRF4, however, did not influence tumor progression or MDSC accumulation in vivo in accordance with our finding that IRF4 is not expressed in PMN-MDSC. This study demonstrates a critical role of IRF4 in the generation of an immunosuppressive tumor microenvironment in pancreatic cancer, which is independent of IRF4 expression in PMN-MDSC.
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http://dx.doi.org/10.1007/s00262-020-02605-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511276PMC
October 2020

Community standards for open cell migration data.

Gigascience 2020 05;9(5)

VIB-UGent Center for Medical Biotechnology, VIB, A. Baertsoenkaai 3, B-9000, Ghent, Belgium.

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.
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http://dx.doi.org/10.1093/gigascience/giaa041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317087PMC
May 2020

Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles Induce Ischemic Neuroprotection by Modulating Leukocytes and Specifically Neutrophils.

Stroke 2020 06 21;51(6):1825-1834. Epub 2020 Apr 21.

From the Department of Neurology (C.W., M.S., J.S., R.P., N.H., E.D., J.G., C.K., D.M.H.), University Hospital Essen, Germany.

Background and Purpose- Small extracellular vesicles (sEVs) obtained from mesenchymal stromal cells (MSCs) were shown to induce neurological recovery after focal cerebral ischemia in rodents and to reverse postischemic lymphopenia in peripheral blood. Since peripheral blood cells, especially polymorphonuclear neutrophils (PMNs), contribute to ischemic brain injury, we analyzed brain leukocyte responses to sEVs and investigated the role of PMNs in sEV-induced neuroprotection. Methods- Male C57Bl6/j mice were exposed to transient intraluminal middle cerebral artery occlusion. After reperfusion, vehicle or sEVs prepared from conditioned media of MSCs raised from bone marrow samples of 3 randomly selected healthy human donors were intravenously administered. sEVs obtained from normoxic and hypoxic MSCs were applied. PMNs were depleted in vehicle and MSC-sEV-treated mice. Neurological deficits, ischemic injury, blood-brain barrier integrity, peripheral blood leukocyte responses, and brain leukocyte infiltration were evaluated over 72 hours. Results- sEV preparations of all 3 donors collected from normoxic MSCs significantly reduced neurological deficits. Preparations of 2 of these donors significantly decreased infarct volume and neuronal injury. sEV-induced neuroprotection was consistently associated with a decreased brain infiltration of leukocytes, namely of PMNs, monocytes/macrophages, and lymphocytes. sEVs obtained from hypoxic MSCs (1% O) had similar effects on neurological deficits and ischemic injury as MSC-sEVs obtained under regular conditions (21% O) but also reduced serum IgG extravasation-a marker of blood-brain barrier permeability. PMN depletion mimicked the effects of MSC-sEVs on neurological recovery, ischemic injury, and brain PMN, monocyte, and lymphocyte counts. Combined MSC-sEV administration and PMN depletion did not have any effects superior to PMN depletion in any of the readouts examined. Conclusions- Leukocytes and specifically PMNs contribute to MSC-sEV-induced ischemic neuroprotection. Individual MSC-sEV preparations may differ in their neuroprotective activities. Potency assays are urgently needed to identify their therapeutic efficacy before clinical application. Visual Overview- An online visual overview is available for this article.
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http://dx.doi.org/10.1161/STROKEAHA.119.028012DOI Listing
June 2020

Three-Dimensional Light Sheet Fluorescence Microscopy of Lungs To Dissect Local Host Immune-Aspergillus fumigatus Interactions.

mBio 2020 02 4;11(1). Epub 2020 Feb 4.

Department of Medicine II and IZKF Research Laboratory, Würzburg University Hospital, Würzburg, Germany

is an opportunistic fungal pathogen that can cause life-threatening invasive lung infections in immunodeficient patients. The cellular and molecular processes of infection during onset, establishment, and progression of infections are highly complex and depend on both fungal attributes and the immune status of the host. Therefore, preclinical animal models are of paramount importance to investigate and gain better insight into the infection process. Yet, despite their extensive use, commonly employed murine models of invasive pulmonary aspergillosis are not well understood due to analytical limitations. Here, we present quantitative light sheet fluorescence microscopy (LSFM) to describe fungal growth and the local immune response in whole lungs at cellular resolution within its anatomical context. We analyzed three very common murine models of pulmonary aspergillosis based on immunosuppression with corticosteroids, chemotherapy-induced leukopenia, or myeloablative irradiation. LSFM uncovered distinct architectures of fungal growth and degrees of tissue invasion in each model. Furthermore, LSFM revealed the spatial distribution, interaction, and activation of two key immune cell populations in antifungal defense: alveolar macrophages and polymorphonuclear neutrophils. Interestingly, the patterns of fungal growth correlated with the detected effects of the immunosuppressive regimens on the local immune cell populations. Moreover, LSFM demonstrates that the commonly used intranasal route of spore administration did not result in complete intra-alveolar deposition, as about 80% of fungal growth occurred outside the alveolar space. Hence, characterization by LSFM is more rigorous than by previously used methods employing murine models of invasive pulmonary aspergillosis and pinpoints their strengths and limitations. The use of animal models of infection is essential to advance our understanding of the complex host-pathogen interactions that take place during lung infections. As in the case of humans, mice need to suffer an immune imbalance in order to become susceptible to invasive pulmonary aspergillosis (IPA), the most serious infection caused by There are several immunosuppressive regimens that are routinely used to investigate fungal growth and/or immune responses in murine models of invasive pulmonary aspergillosis. However, the precise consequences of the use of each immunosuppressive model for the local immune populations and for fungal growth are not completely understood. Here, to pin down the scenarios involving commonly used IPA models, we employed light sheet fluorescence microscopy (LSFM) to analyze whole lungs at cellular resolution. Our results will be valuable to optimize and refine animal models to maximize their use in future research.
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http://dx.doi.org/10.1128/mBio.02752-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7002341PMC
February 2020

Multicolor two-photon imaging of in vivo cellular pathophysiology upon influenza virus infection using the two-photon IMPRESS.

Nat Protoc 2020 03 29;15(3):1041-1065. Epub 2020 Jan 29.

Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus-infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus-infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo.
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http://dx.doi.org/10.1038/s41596-019-0275-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086515PMC
March 2020

STAT3 activation through IL-6/IL-11 in cancer-associated fibroblasts promotes colorectal tumour development and correlates with poor prognosis.

Gut 2020 07 4;69(7):1269-1282. Epub 2019 Nov 4.

First Department of Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg, Universitätsklinikum Erlangen, Erlangen, Germany

Objective: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood.

Design: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11).

Results: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts.

Conclusion: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.
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http://dx.doi.org/10.1136/gutjnl-2019-319200DOI Listing
July 2020

Multidimensional imaging provides evidence for down-regulation of T cell effector function by MDSC in human cancer tissue.

Sci Immunol 2019 10;4(40)

Department of Otorhinolaryngology, University of Duisburg-Essen, University Hospital Essen, Essen, Germany.

A high intratumoral frequency of neutrophils is associated with poor clinical outcome in most cancer entities. It is hypothesized that immunosuppressive MDSC (myeloid-derived suppressor cell) activity of neutrophils against tumor-reactive T cells contributes to this effect. However, direct evidence for such activity in situ is lacking. Here, we used whole-mount labeling and clearing, three-dimensional (3D) light sheet microscopy and digital image reconstruction supplemented by 2D multiparameter immunofluorescence, for in situ analyses of potential MDSC-T cell interactions in primary human head and neck cancer tissue. We could identify intratumoral hotspots of high polymorphonuclear (PMN)-MDSC and T cell colocalization. In these areas, the expression of effector molecules Granzyme B and Ki67 in T cells was strongly reduced, in particular for T cells that were in close proximity or physically engaged with PMN-MDSC, which expressed LOX-1 and arginase I. Patients with cancer with evidence for strong down-regulation of T cell function by PMN-MDSC had significantly impaired survival. In summary, our approach identifies areas of clinically relevant functional interaction between MDSC and T cells in human cancer tissue and may help to inform patient selection in future combination immunotherapies.
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http://dx.doi.org/10.1126/sciimmunol.aaw9159DOI Listing
October 2019

A network of trans-cortical capillaries as mainstay for blood circulation in long bones.

Nat Metab 2019 02 21;1(2):236-250. Epub 2019 Jan 21.

Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen, Essen, Germany.

Closed circulatory systems (CCS) underlie the function of vertebrate organs, but in long bones their structure is unclear, although they constitute the exit route for bone marrow (BM) leukocytes. To understand neutrophil emigration from BM, we studied the vascular system of murine long bones. Here we show that hundreds of capillaries originate in BM, cross murine cortical bone perpendicularly along the shaft and connect to the periosteal circulation. Structures similar to these trans-cortical-vessels (TCVs) also exist in human limb bones. TCVs express arterial or venous markers and transport neutrophils. Furthermore, over 80% arterial and 59% venous blood passes through TCVs. Genetic and drug-mediated modulation of osteoclast count and activity leads to substantial changes in TCV numbers. In a murine model of chronic arthritic bone inflammation, new TCVs develop within weeks. Our data indicate that TCVs are a central component of the CCS in long bones and may represent an important route for immune cell export from the BM.
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http://dx.doi.org/10.1038/s42255-018-0016-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795552PMC
February 2019

Epithelial NOTCH Signaling Rewires the Tumor Microenvironment of Colorectal Cancer to Drive Poor-Prognosis Subtypes and Metastasis.

Cancer Cell 2019 09;36(3):319-336.e7

Cancer Research UK Beatson Institute, Glasgow, UK; Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK. Electronic address:

The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in Kras-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) β-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.
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http://dx.doi.org/10.1016/j.ccell.2019.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6853173PMC
September 2019

Next-generation imaging of the skeletal system and its blood supply.

Nat Rev Rheumatol 2019 09 8;15(9):533-549. Epub 2019 Aug 8.

Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen, Essen, Germany.

Bone is organized in a hierarchical 3D architecture. Traditionally, analysis of the skeletal system was based on bone mass assessment by radiographic methods or on the examination of bone structure by 2D histological sections. Advanced imaging technologies and big data analysis now enable the unprecedented examination of bone and provide new insights into its 3D macrostructure and microstructure. These technologies comprise ex vivo and in vivo methods including high-resolution computed tomography (CT), synchrotron-based imaging, X-ray microscopy, ultra-high-field magnetic resonance imaging (MRI), light-sheet fluorescence microscopy, confocal and intravital two-photon imaging. In concert, these techniques have been used to detect and quantify a novel vascular system of trans-cortical vessels in bone. Furthermore, structures such as the lacunar network, which harbours and connects osteocytes, become accessible for 3D imaging and quantification using these methods. Next-generation imaging of the skeletal system and its blood supply are anticipated to contribute to an entirely new understanding of bone tissue composition and function, from macroscale to nanoscale, in health and disease. These insights could provide the basis for early detection and precision-type intervention of bone disorders in the future.
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http://dx.doi.org/10.1038/s41584-019-0274-yDOI Listing
September 2019

Contribution of polymorphonuclear neutrophils in the blood periphery to ischemic brain injury.

Neurol Neuroimmunol Neuroinflamm 2019 07 15;6(4):e570. Epub 2019 May 15.

Department of Neurology (D.M.H.), University Hospital Essen, University of Duisburg-Essen; and Institute of Experimental Immunology and Imaging (M.G.), University Hospital Essen, University of Duisburg-Essen, Germany.

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http://dx.doi.org/10.1212/NXI.0000000000000570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624096PMC
July 2019

Distinct Spatio-Temporal Dynamics of Tumor-Associated Neutrophils in Small Tumor Lesions.

Front Immunol 2019 25;10:1419. Epub 2019 Jun 25.

Department of Otorhinolaryngology, University Hospital Essen, University Duisburg-Essen, Essen, Germany.

Across a majority of cancer types tumor-associated neutrophils (TAN) are linked with poor prognosis. However, the underlying mechanisms, especially the intratumoral behavior of TAN, are largely unknown. Using intravital multiphoton imaging on a mouse model with neutrophil-specific fluorescence, we measured the migration of TAN in distinct compartments of solid tumor cell lesions . By longitudinally quantifying the infiltration and persistence of TAN into growing tumors in the same animals, we observed cells that either populated the peripheral stromal zone of the tumor (peritumoral TAN) or infiltrated into the tumor core (intratumoral TAN). Intratumoral TAN showed prolonged tumor-associated persistence and reduced motility compared to peritumoral TAN, whose velocity increased with tumor progression. Selective pharmacological blockade of CXCR2 receptors using AZD5069 profoundly inhibited recruitment of TAN into peritumoral regions, while intratumoral infiltration was only transiently attenuated and rebounded at later time points. Our findings unravel distinct spatial dynamics of TAN that are partially and differentially regulated via the CXCR2 signaling pathway.
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http://dx.doi.org/10.3389/fimmu.2019.01419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603174PMC
October 2020