Escaping the traps of your own hunters Gunzer, Matthias
Science (American Association for the Advancement of Science),
12/2017, Letnik:
358, Številka:
6367
Journal Article
Recenzirano
Cleavage of extracellular DNA NETs is essential for inhibiting peripheral organ failure
During the first days of an infection, protection is almost entirely provided by innate immunity, a large part ...of which consists of one type of immune cell—the neutrophil. Neutrophils form the largest group of immune cells in the body humans produce ∼10
11
neutrophils daily (
1
), and they carry out a number of defense mechanisms, including pathogen ingestion (phagocytosis) and the production of toxic chemicals that kill intruders directly. They can also cause severe hyperinflammatory diseases, such as lethal influenza-associated lung destruction (
2
,
3
). In 2004, another neutrophil defense system was discovered. In a final act of protection, dying neutrophils mix their nuclear DNA with toxic components from their cytosolic granules and release it explosively to form a neutrophil extracellular trap (NET) in the surrounding environment. This process is called NETosis (
4
). In addition to carrying genetic information, DNA is a very long (each nucleus contains ∼2 m of DNA) and sticky polyanionic molecule. Hence, DNA binds to surfaces such as bacterial cell walls by means of charge interactions (
5
). Through this immobilization on NETs, pathogens are brought into direct contact with cytotoxic molecules on the NET-DNA complex. However, free extracellular DNA is also a potent trigger of severe side effects in the body, for example, by forming immune complexes with anti nuclear antibodies (antibodies that can bind to DNA) in systemic lupus erythematosus (SLE) (
6
) or by directly clogging blood vessels and establishing vessel-blocking thrombi (
7
). Consequently, finding mechanisms that mediate the degradation of NETs has been a matter of intense research. On page
1202
of this issue, Jiménez-Alcázar
et al.
(
8
) describe two deoxyribonucleases (DNases) DNase1 and DNase1-like 3 (DNase1L3), which cleave DNA and together degrade freshly formed NETs. By doing so, they inhibit otherwise lethal immunopathologies such as thrombus-mediated organ failure.
Neutrophils have been implicated as harmful cells in a variety of inappropriate inflammatory conditions where they injure the host, leading to the death of the neutrophils and their subsequent ...phagocytosis by monocytes and macrophages. Here we show that in a fully repairing sterile thermal hepatic injury, neutrophils also penetrate the injury site and perform the critical tasks of dismantling injured vessels and creating channels for new vascular regrowth. Upon completion of these tasks, they neither die at the injury site nor are phagocytosed. Instead, many of these neutrophils reenter the vasculature and have a preprogrammed journey that entails a sojourn in the lungs to up-regulate CXCR4 (C-X-C motif chemokine receptor 4) before entering the bone marrow, where they undergo apoptosis.
Abstract
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.
Neutrophil recruitment is an important early step in controlling tissue infections or injury. Here, we report that this influx depends on both tissue-resident mast cells and macrophages. Mice with ...mast cell deficiency recruit reduced numbers of neutrophils in the first few hours of intraperitoneal lipopolysaccharide (LPS) stimulation. Conversely, in mice with clodronate-ablated macrophages, neutrophils extravasate, but have limited ability to reach the peritoneal fluid. Tissue macrophages synthesize neutrophil chemoattractants CXCL1/CXCL2 (CXC chemokine ligands 1/2) in response to LPS. Mast cells also produce these chemokines of which a proportion are preformed in granules. Release of the granules and new CXCL1/CXCL2 synthesis is Toll-like receptor 4–dependent. Both in vivo studies with blocking monoclonal antibodies and in vitro chemotaxis experiments show the neutrophil response to mast cells and macrophages to be CXCL1/CXCL2-dependent. The data are in keeping with the model that mast cells, optimally positioned in close proximity to the vasculature, initiate an early phase of neutrophil recruitment by releasing the chemoattractants CXCL1/CXCL2. Having arrived within the stimulated tissue, neutrophils penetrate further in a macrophage-dependent manner. Therefore, we demonstrate a positive role for mast cells in tissue inflammation and define how this comes about with contribution from a second tissue cell, the macrophage.
• Mast cells contribute to early neutrophil recruitment.• Mast cells and macrophages both make CXCL1 and CXCL2.
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.
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.
Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) ...injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes.
The total number of glomeruli is a fundamental parameter of kidney function but very difficult to determine using standard methodology. Here, we counted all individual glomeruli in murine kidneys and ...sized the capillary tufts by combining in vivo fluorescence labeling of endothelial cells, a novel tissue-clearing technique, lightsheet microscopy, and automated registration by image analysis. Total hands-on time per organ was <1 hour, and automated counting/sizing was finished in <3 hours. We also investigated the novel use of ethyl-3-phenylprop-2-enoate (ethyl cinnamate) as a nontoxic solvent-based clearing reagent that can be handled without specific safety measures. Ethyl cinnamate rapidly cleared all tested organs, including calcified bone, but the fluorescence of proteins and immunohistochemical labels was maintained over weeks. Using ethyl cinnamate-cleared kidneys, we also quantified the average creatinine clearance rate per glomerulus. This parameter decreased in the first week of experimental nephrotoxic nephritis, whereas reduction in glomerular numbers occurred much later. Our approach delivers fundamental parameters of renal function, and because of its ease of use and speed, it is suitable for high-throughput analysis and could greatly facilitate studies of the effect of kidney diseases on whole-organ physiology.
Inflammation-related comorbidities contribute to stroke-induced immune responses and brain damage. We previously showed that hyperlipidemia exacerbates ischemic brain injury, which is associated with ...elevated peripheral and cerebral granulocyte numbers. Herein, we evaluate the contribution of neutrophils to the exacerbation of ischemic brain injury.
Wild-type mice fed with a normal chow and ApoE knockout mice fed with a high cholesterol diet were exposed to middle cerebral artery occlusion. CXCR2 was blocked using the selective antagonist SB225002 (2 mg/kg) or neutralizing CXCR2 antiserum. Neutrophils were depleted using an anti-Ly6G antibody. At 72 hours post ischemia, immunohistochemistry, flow cytometry, and real-time polymerase chain reaction were performed to determine cerebral tissue injury and immunologic changes in the blood, bone marrow, and brain. Functional outcome was assessed by accelerated rota rod and tight rope tests at 4, 7, and 14 days post ischemia.
CXCR2 antagonization reduced neurological deficits and infarct volumes that were exacerbated in hyperlipidemic ApoE-/- mice. This effect was mimicked by neutrophil depletion. Cerebral neutrophil infiltration and peripheral neutrophilia, which were increased on ischemia in hyperlipidemia, were attenuated by CXCR2 antagonization. This downscaling of neutrophil responses was associated with increased neutrophil apoptosis and reduced levels of CXCR2, inducible nitric oxide synthase, and NADPH oxidase 2 expression on bone marrow neutrophils.
Our data demonstrate a role of neutrophils in the exacerbation of ischemic brain injury induced by hyperlipidemia. Accordingly, CXCR2 blockade, which prevents neutrophil recruitment into the brain, might be an effective option for stroke treatment in patients with hyperlipidemia.
Polymorphonuclear neutrophil granulocytes (PMN) orchestrate the removal of cell debris in ischemic stroke and intracerebral hemorrhage. In both pathologies, high neutrophil to lymphocyte ratios in ...peripheral blood are predictive of poor outcome in human stroke patients. Following earlier studies indicating that the cerebral microvasculature forms an efficient barrier that impedes neutrophil brain entry, intravital microscopy and immunohistochemistry in the meantime unequivocally revealed the accumulation of PMN in the ischemic and hemorrhagic brain parenchyma. These studies provide definite evidence that PMN contribute to the degradation of the blood-brain barrier, predisposing the brain to secondary injury, edema, hemorrhage formation, hemorrhage growth and poor neurological recovery. Recent studies demonstrated the role of pro-inflammatory N1 neutrophils in brain edema and neurotoxicity, whereas anti-inflammatory N2 neutrophils were found to limit this excessive immune response, promoting neuronal survival and successful brain remodeling. In view of the recent failure of anti-inflammatory immunotherapies in clinical trials, strategies specifically modulating the brain accumulation, differentiation and action of PMN may open promising perspectives for stroke treatment.
•We here review the role of polymorphonuclear neutrophil granulocytes (PMN) in ischemic stroke and intracerebral hemorrhage.•We define how PMN may contribute to brain injury.•We discuss how PMN might be targeted to improve stroke outcome.
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