Chronic obstructive pulmonary disease (COPD) is associated with elevated ATP levels in the extracellular space. Once released, ATP serves as danger signal modulating immune responses by activating ...purinergic receptors. Accordingly, purinergic signalling has been implicated in respiratory inflammation associated with cigarette smoke exposure. However, the role of P2X4-signalling has not been fully elucidated yet.
Here, we analysed the P2X4 mRNA expression in COPD patients as well as cigarette smoke-exposed mice. Furthermore, P2X4-signalling was blocked by either using a specific antagonist or genetic depletion of P2rx4 in mice applied to an acute and prolonged model of cigarette smoke exposure. Finally, we inhibited P2X4-signalling in macrophages derived from THP-1 before stimulation with cigarette smoke extract.
COPD patients exhibited an increased P2X4 mRNA expression in cells isolated from the bronchoalveolar lavage fluid and peripheral mononuclear cells. Similarly, P2rx4 expression was elevated in lung tissue of mice exposed to cigarette smoke. Blocking P2X4-signalling in mice alleviated cigarette smoke induced airway inflammation as well as lung parenchyma destruction. Additionally, human macrophages derived from THP-1 cells released reduced concentrations of proinflammatory cytokines in response to cigarette smoke extract stimulation when P2X4 was inhibited.
Taken together, we provide evidence that P2X4-signalling promotes innate immunity in the immunopathologic responses induced by cigarette smoke exposure.
Blazars are very broadband cosmic sources with spectra spanning over twenty orders of magnitude in frequency, down to the 100 MHz regime in the radio range, up to VHE at several tens of TeV. The ...modelling of their spectral energy distribution at high energies currently considers two main classes of models, leptonic and lepto-hadronic, which both succeed fairly well in describing the observed spectra for the two populations of blazars, namely BL Lac objects (BL Lacs) and flat spectrum radio quasars (FSRQs). However they are both confronted with difficulties, in particular to reproduce flaring phenomena monitored with a good multi-spectral and temporal coverage, or to reproduce extreme sources which challenge the basic descriptions. Such a situation has led to a diversity of specific scenarios, the positioning of which in relation to the general context of the sources is generally not clearly fixed. The identification of the dominant particle acceleration mechanism at work and a better understanding of the location of the TeV emitting zone would make it possible to break the degeneracies between models. Multi-wavelength and multi-messenger studies should also help in this regard, with the perspective to elaborate a general reference scenario of blazars and AGNs.
Aims.
The multi-wavelength emission from a newly identified population of ‘extreme-TeV’ blazars, with Compton peak frequencies around 1 TeV, is difficult to interpret with standard one-zone emission ...models. Large values of the minimum electron Lorentz factor and quite low magnetisation values seem to be required.
Methods.
We propose a scenario where protons and electrons are co-accelerated on internal or recollimation shocks inside the relativistic jet. In this situation, energy is transferred from the protons to the electrons in the shock transition layer, leading naturally to a high minimum Lorentz factor for the latter. A low magnetisation favours the acceleration of particles in relativistic shocks.
Results.
The shock co-acceleration scenario provides additional constraints on the set of parameters of a standard one-zone lepto-hadronic emission model, reducing its degeneracy. Values of the magnetic field strength of a few mG and minimum electron Lorentz factors of 10
3
to 10
4
, required to provide a satisfactory description of the observed spectral energy distributions of extreme blazars, result here from first principles. While acceleration on a single standing shock is sufficient to reproduce the emission of most of the extreme-TeV sources we have examined, re-acceleration on a second shock appears needed for those objects with the hardest
γ
-ray spectra. Emission from the accelerated proton population, with the same number density as the electrons but in a lower range of Lorentz factors, is strongly suppressed. Satisfactory self-consistent representations were found for the most prominent representatives of this new blazar class.
ABSTRACT
Extracellular ATP binds to and signals through P2X7 receptors (P2X7Rs) to modulate immune function in both inflammasome‐dependent and ‐independent manners. In this study, P2X7‐/‐ mice, the ...pharmacological agonists ATP‐magnesium salt (Mg‐ATP; 100 mg/kg, EC50 ≈ 1.32 mM) and benzoylbenzoyl‐ATP (Bz‐ATP; 10 mg/kg, EC50 ≈ 285 μM), and antagonist oxidized ATP (oxi‐ATP; 40 mg/kg, IC50 ≈ 100 μM) were used to show that P2X7R activation is crucial for the control of mortality, bacterial dissemination, and inflammation in cecal ligation and puncture‐induced polymicrobial sepsis in mice. Our results with P2X7‐/‐ bone marrow chimeric mice, adoptive transfer of peritoneal macrophages, and myeloid‐specific P2X7‐/‐ mice indicate that P2X7R signaling on macrophages is essential for the protective effect of P2X7Rs. P2X7R signaling protects through enhancing bacterial killing by macrophages, which is independent of the inflammasome. By using the connexin (Cx) channel inhibitor Gap27 (0.1 mg/kg, IC50 ≈ 0.25 μM) and pannexin channel inhibitor probenecid (10 mg/kg, IC50 ≈ 11.7 μM), we showed that ATP release through Cx is important for inhibiting inflammation and bacterial burden. In summary, targeting P2X7Rs provides a new opportunity for harnessing an endogenous protective immune mechanism in the treatment of sepsis.—Csóka, B., Németh, Z. H., Törő, G., Idzko, M., Zech, A., Koscsó, B., Spolarics, Z., Antonioli, L., Cseri, K., Erdélyi, K., Pacher, P., Haskó, G. Extracellular ATP protects against sepsis through macrophage P2X7 purinergic receptors by enhancing intracellular bacterial killing. FASEB J. 29, 3626‐3637 (2015). www.fasebj.org
Several studies have shown that SARS-CoV-2 can induce a massive release of cytokines which contributes to disease severity and mortality. Therefore, cytokine levels in the serum may help to predict ...disease severity and survival in COVID-19 patients. In this prospective trial, 88 patients who were hospitalised for COVID-19 were enrolled. Blood samples for serum peptide measurements were taken at the time closest to hospitalisation, at day 5, 9 and 13 (±1). The concentrations of cytokines (IL-1alpha, IL-1beta, IL-1RA, IL-6, L-7, L-10, IFN-gamma and TNF-alpha), chemokines (CCL-3, CCL-4 and CCL-7) and growth factors (G-CSF, GM-CSF and VEGF) were assessed and correlated with the type of ventilation, occurrence of consolidations on imaging and the level of care. COVID-19 patients (median age 68 years, IQR 55-77) stayed in hospital between 5-171 days. Compared to patients in the general care unit, patients in the intermediate care unit (IMCU) and intensive care unit (ICU) presented significantly elevated serum IL-6 (p = 0.004) and lower IFN-gamma levels (p = 0.005), respectively. The peak inspiratory pressure in ventilated patients correlated positively with IL-1RA, G-CSF and inversely with IFN-gamma serum levels (all p<0.05). VEGF serum levels inversely correlated with the fraction of inspired oxygen in patients receiving high-flow nasal canula oxygen therapy (p = 0.047). No significant correlation between serum concentrations of the measured peptides and the type of ventilation, occurrence of radiological consolidations or in-hospital mortality has been observed. IL1-RA, IL-6, IFN-gamma, G-CSF, CCL-7 and VEGF serum levels could prove helpful as biomarkers to assess disease severity and the need for intensive care in COVID-19 patients.
Antagonists of the type 1 cysteinyl leukotriene receptor (CysLT1R) are widely used to treat asthma and allergic rhinitis, with variable response rates. Alveolar macrophages express UDP-specific P2Y6 ...receptors that can be blocked by off-target effects of CysLT1R antagonists. Sensitizing intranasal doses of an extract from the house dust mite Dermatophagoides farinae (Df) sharply increased the levels of UDP detected in bronchoalveolar lavage fluid of mice. Conditional deletion of P2Y6 receptors before sensitization exacerbated eosinophilic lung inflammation and type 2 cytokine production in response to subsequent Df challenge. P2Y6 receptor signaling was necessary for dectin-2-dependent production of protective IL-12p40 and Th1 chemokines by alveolar macrophages, leading to activation of NK cells to generate IFN-γ. Administration of CysLT1R antagonists during sensitization blocked UDP-elicited potentiation of IL-12p40 production by macrophages in vitro, suppressed the Df-induced production of IL-12p40 and IFN-γ in vivo, and suppressed type 2 inflammation only in P2Y6-deficient mice. Thus, P2Y6 receptor signaling drives an innate macrophage/IL-12/NK cell/IFN-γ axis that prevents inappropriate allergic type 2 immune responses on respiratory allergen exposure and counteracts the Th2 priming effect of CysLT1R signaling at sensitization. Targeting P2Y6 signaling might prove to be a potential additional treatment strategy for allergy.
Acute respiratory distress syndrome (ARDS) is associated with high morbidity and mortality resulting from a direct or indirect injury of the lung. It is characterized by a rapid alveolar injury, lung ...inflammation with neutrophil accumulation, elevated permeability of the microvascular-barrier leading to an aggregation of protein-rich fluid in the lungs, followed by impaired oxygenation in the arteries and eventual respiratory failure. Very recently, we have shown an involvement of the Gq-coupled P2Y2 purinergic receptor (P2RY2) in allergic airway inflammation (AAI). In the current study, we aimed to elucidate the contribution of the P2RY2 in lipopolysaccharide (LPS)-induced ARDS mouse model. We found that the expression of
in neutrophils, macrophages and lung tissue from animals with LPS-induced ARDS was strongly upregulated at mRNA level. In addition, ATP-neutralization by apyrase
markedly attenuated inflammation and blocking of P2RY2 by non-selective antagonist suramin partially decreased inflammation. This was indicated by a reduction in the number of neutrophils, concentration of proinflammatory cytokines in the BALF, microvascular plasma leakage and reduced features of inflammation in histological analysis of the lung. P2RY2 blocking has also attenuated polymorphonuclear neutrophil (PMN) migration into the interstitium of the lungs in ARDS mouse model. Consistently, treatment of
deficient mice with LPS lead to an amelioration of the inflammatory response showed by reduced number of neutrophils and concentrations of proinflammatory cytokines. In attempts to identify the cell type specific role of P2RY2, a series of experiments with conditional
knockout animals were performed. We observed that
expression in neutrophils, but not in the airway epithelial cells or CD4
cells, was associated with the inflammatory features caused by ARDS. Altogether, our findings imply for the first time that increased endogenous ATP concentration via activation of P2RY2 is related to the pathogenesis of LPS-induced lung inflammation and may represent a potential therapeutic target for the treatment of ARDS and predictably assess new treatments in ARDS.
Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and ...pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1 s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.
Mass cytometry (MC) is a powerful method for mapping complex cellular systems at single-cell levels, based on the detection of cellular proteins. Numerous studies have been performed using human ...blood, but there is a lack of protocols describing the processing and labeling of bronchoalveolar lavage fluid (BALF) and nasal polyps (NP) for acquisition by MC. These specimens are essential in the investigation of immune cell characteristics in airway diseases such as asthma and chronic rhinosinusitis with NP (CRSwNP). Here we optimized a workflow for processing, labeling, and acquisition of BALF and NP cells by MC. Among three methods tested for NP digestion, combined enzymatic/mechanical processing yielded maximum cell recovery, viability and labeling patterns compared to the other methods. Treatment with DNAse improved sample acquisition by MC. In a final step, we performed a comparison of blood, BALF and NP cell composition using a 31-marker MC antibody panel, revealing expected differences between the different tissue but also heterogeneity among the BALF and NP samples. We here introduce an optimized workflow for the MC analysis of human NP and BALF, which enables comparative analysis of different samples in larger cohorts. A deeper understanding of immune cell characteristics in these samples may guide future researchers and clinicians to a better disease management.
Eosinophils are major effector cells against parasites, fungi, bacteria, and viruses. However, these cells also take part in local and systemic inflammation, which are central to eczema, atopy, ...rhinitis, asthma, and autoimmune diseases. A role for eosinophils has been also shown in vascular thrombotic disorders and in cancer. Many, if not all, above-mentioned conditions involve the release of intracellular nucleotides (ATP, ADP, UTP, etc.) and nucleosides (adenosine) in the extracellular environment. Simultaneously, eosinophils further release ATP, which in autocrine and paracrine manners, stimulates P2 receptors. Purinergic signaling in eosinophils mediates a variety of responses including CD11b induction, ROI production, release of granule contents and enzymes, as well as cytokines. Exposure to extracellular ATP also modulates the expression of endothelial adhesion molecules, thereby favoring eosinophil extravasation and accumulation. In addition, eosinophils express the immunosuppressive adenosine P1 receptors, which regulate degranulation and migration. However, pro-inflammatory responses induced by extracellular ATP predominate. Due to their important role in innate immunity and tissue damage, pharmacological targeting of nucleotide- and nucleoside-mediated signaling in eosinophils could represent a novel approach to alleviate eosinophilic acute and chronic inflammatory diseases. These innovative approaches might also have salutary effects, particularly in host defense against parasites and in cancer.
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