Publications by authors named "Adriano G Rossi"

155 Publications

Isolated IgG2 Deficiency is an Independent Risk Factor for Exacerbations in Bronchiectasis.

QJM 2021 May 10. Epub 2021 May 10.

Centre for Inflammation Research at the University of Edinburgh, Queen's Medical Research Institute, Edinburgh BioQuarter, Edinburgh, EH16 4TJ, Scotland.

Background: Immunoglobulin G (IgG) subclass 2 deficiency is the most frequent IgG subclass deficiency identified in patients with bronchiectasis, but its clinical significance is not known.

Aim: To analyse if bronchiectasis patients with isolated IgG2 deficiency at risk of recurrent exacerbations and/or hospitalisation? Do patients with IgG2 deficiency have worse disease progression?

Design And Methods: This is a retrospective study (2015-2020) exploring independent risk factors for recurrent exacerbations (three or more per year) and/or hospitalisation with bronchiectasis exacerbations using multivariable models using binary logistic regression. There was no patient with IgG deficiency, IgG 1, 3 or 4 deficiency, or IgA or IgM deficiency included. In this model, the authors included: serum IgG2 level; lung function; body mass index; MRC breathlessness scale; age; sex; number of bronchiectatic lobes; bacterial colonisation; comorbidities; the use of long-term immunosuppressant drugs or antibiotics for more than 28 days. Analysing two-year longitudinal data, one-way ANOVA and Mann-Whitney U test were used to compare bronchiectasis severity between patients with different IgG2 levels.

Results: Serum IgG2 levels (<2.68 g/L, 2.68-3.53 g/L, 3.54-4.45 g/L); hospital admission in the preceding two years; bacterial colonisation with potentially pathogenic organisms and asthma were independent predictors for three or more bronchiectasis exacerbations. Those with low IgG2 levels (<2.68 g/L and 2.68-3.53 g/L), had worsening progression of their bronchiectasis, using the Bronchiectasis Severity Index, over one year compared with those who were IgG2 replete (>4.45 g/L))(p = 0.003, 0.013).

Conclusion: Reduced IgG2 levels was an independent predictor for bronchiectasis exacerbations and have increased disease progression.
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http://dx.doi.org/10.1093/qjmed/hcab129DOI Listing
May 2021

The Outcome of Neutrophil-T Cell Contact Differs Depending on Activation Status of Both Cell Types.

Front Immunol 2021 30;12:633486. Epub 2021 Mar 30.

Centre for Inflammation Research, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Neutrophils and T cells exist in close proximity in lymph nodes and inflamed tissues during health and disease. They are able to form stable interactions, with profound effects on the phenotype and function of the T cells. However, the outcome of these effects are frequently contradictory; in some systems neutrophils suppress T cell proliferation, in others they are activatory or present antigen directly. Published protocols modelling these interactions do not reflect the full range of interactions found ; they do not examine how activated and naïve T cells differentially respond to neutrophils, or whether de-granulating or resting neutrophils induce different outcomes. Here, we established a culture protocol to ask these questions with human T cells and autologous neutrophils. We find that resting neutrophils suppress T cell proliferation, activation and cytokine production but that de-granulating neutrophils do not, and neutrophil-released intracellular contents enhance proliferation. Strikingly, we also demonstrate that T cells early in the activation process are susceptible to suppression by neutrophils, while later-stage T cells are not, and naïve T cells do not respond at all. Our protocol therefore allows nuanced analysis of the outcome of interaction of these cells and may explain the contradictory results observed previously.
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http://dx.doi.org/10.3389/fimmu.2021.633486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042376PMC
March 2021

Why Is COVID-19 More Severe in Patients With Diabetes? The Role of Angiotensin-Converting Enzyme 2, Endothelial Dysfunction and the Immunoinflammatory System.

Front Cardiovasc Med 2020 3;7:629933. Epub 2021 Feb 3.

Institute for Health Research and Innovation, University of the Highlands and Islands, Inverness, United Kingdom.

Meta-analyses have indicated that individuals with type 1 or type 2 diabetes are at increased risk of suffering a severe form of COVID-19 and have a higher mortality rate than the non-diabetic population. Patients with diabetes have chronic, low-level systemic inflammation, which results in global cellular dysfunction underlying the wide variety of symptoms associated with the disease, including an increased risk of respiratory infection. While the increased severity of COVID-19 amongst patients with diabetes is not yet fully understood, the common features associated with both diseases are dysregulated immune and inflammatory responses. An additional key player in COVID-19 is the enzyme, angiotensin-converting enzyme 2 (ACE2), which is essential for adhesion and uptake of virus into cells prior to replication. Changes to the expression of ACE2 in diabetes have been documented, but they vary across different organs and the importance of such changes on COVID-19 severity are still under investigation. This review will examine and summarise existing data on how immune and inflammatory processes interplay with the pathogenesis of COVID-19, with a particular focus on the impacts that diabetes, endothelial dysfunction and the expression dynamics of ACE2 have on the disease severity.
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http://dx.doi.org/10.3389/fcvm.2020.629933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7886785PMC
February 2021

Prostaglandin E promotes intestinal inflammation via inhibiting microbiota-dependent regulatory T cells.

Sci Adv 2021 Feb 12;7(7). Epub 2021 Feb 12.

Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh EH16 4TJ, UK.

The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (T), yet how the microbiota-T cross-talk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E (PGE), a well-known mediator of inflammation, inhibits mucosal T in a manner depending on the gut microbiota. PGE through its receptor EP4 diminishes T-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE-EP4 signaling modulates mucosal T responses and exacerbates intestinal inflammation. Mechanistically, PGE-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor diminishes PGE-dependent T inhibition. Together, our findings provide emergent evidence that PGE-mediated disruption of microbiota-T communication fosters intestinal inflammation.
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http://dx.doi.org/10.1126/sciadv.abd7954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880593PMC
February 2021

Epithelial Cells and Inflammation in Pulmonary Wound Repair.

Cells 2021 Feb 5;10(2). Epub 2021 Feb 5.

University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh Bioquarter, Edinburgh EH16 4TJ, UK.

Respiratory diseases are frequently characterised by epithelial injury, airway inflammation, defective tissue repair, and airway remodelling. This may occur in a subacute or chronic context, such as asthma and chronic obstructive pulmonary disease, or occur acutely as in pathogen challenge and acute respiratory distress syndrome (ARDS). Despite the frequent challenge of lung homeostasis, not all pulmonary insults lead to disease. Traditionally thought of as a quiescent organ, emerging evidence highlights that the lung has significant capacity to respond to injury by repairing and replacing damaged cells. This occurs with the appropriate and timely resolution of inflammation and concurrent initiation of tissue repair programmes. Airway epithelial cells are key effectors in lung homeostasis and host defence; continual exposure to pathogens, toxins, and particulate matter challenge homeostasis, requiring robust defence and repair mechanisms. As such, the epithelium is critically involved in the return to homeostasis, orchestrating the resolution of inflammation and initiating tissue repair. This review examines the pivotal role of pulmonary airway epithelial cells in initiating and moderating tissue repair and restitution. We discuss emerging evidence of the interactions between airway epithelial cells and candidate stem or progenitor cells to initiate tissue repair as well as with cells of the innate and adaptive immune systems in driving successful tissue regeneration. Understanding the mechanisms of intercellular communication is rapidly increasing, and a major focus of this review includes the various mediators involved, including growth factors, extracellular vesicles, soluble lipid mediators, cytokines, and chemokines. Understanding these areas will ultimately identify potential cells, mediators, and interactions for therapeutic targeting.
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http://dx.doi.org/10.3390/cells10020339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7914803PMC
February 2021

Assays of Eosinophil Apoptosis and Phagocytic Uptake.

Methods Mol Biol 2021 ;2241:113-132

MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.

Eosinophil apoptosis (programmed cell death) plays an important role in several inflammatory and allergic conditions. Apoptosis triggers various mechanisms including activation of cysteine-aspartic proteases (caspases) and is characterized by morphological and biochemical changes. These include cellular condensation, nuclear fragmentation, increased mitochondrial permeability with loss of membrane potential, and exposure of phosphatidylserine on the cell membrane. A greater understanding of apoptotic mechanisms, subsequent phagocytosis (efferocytosis), and regulation of these processes is critical to understanding disease pathogenesis and development of potential novel therapeutic agents. Release of soluble factors and alterations to surface marker expression by eosinophils undergoing apoptosis aid them in signaling their presence to the immediate environment, and their subsequent recognition by phagocytic cells such as macrophages. Uptake of apoptotic cells usually suppresses inflammation by restricting proinflammatory responses and promoting anti-inflammatory and tissue repair responses. This, in turn, promotes resolution of inflammation. Defects in the apoptotic or efferocytosis mechanisms perpetuate inflammation, resulting in chronic inflammation and enhanced disease severity. This can be due to increased eosinophil life span or cell necrosis characterized by loss of cell membrane integrity and release of toxic intracellular mediators. In this chapter, we detail some of the key assays that are used to assess eosinophil apoptosis, as well as the intracellular signaling pathways involved and phagocytic clearance of these cells.
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http://dx.doi.org/10.1007/978-1-0716-1095-4_10DOI Listing
March 2021

A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation.

Chem Commun (Camb) 2021 Jan;57(1):97-100

EaStCHEM School of Chemistry, University of Edinburgh, David Brewster Road, EH9 3FJ Edinburgh, UK.

A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils.
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http://dx.doi.org/10.1039/d0cc07028aDOI Listing
January 2021

Src kinase inhibition with dasatinib impairs neutrophil function and clearance of Escherichia coli infection in a murine model of acute lung injury.

J Inflamm (Lond) 2020 Oct 30;17(1):34. Epub 2020 Oct 30.

Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

Background: Neutrophils rapidly respond to and clear infection from tissues, but can also induce tissue damage through excessive degranulation, when acute inflammation proceeds unchecked. A number of key neutrophil functions, including adhesion-dependent degranulation, are controlled by src family kinases. Dasatinib is a potent src inhibitor used in treating patients with chronic myeloid leukaemia and treatment-resistant acute lymphoblastic leukaemia. We hypothesized that dasatinib would attenuate acute inflammation by inhibiting neutrophil recruitment, degranulation and endothelial cell injury, without impairing bacterial clearance, in a murine model of bacteria-induced acute lung injury. C57BL/6 mice received intratracheal Escherichia coli, and were treated with intraperitoneal dasatinib or control. Bacterial clearance, lung injury, and markers of neutrophil recruitment and degranulation were measured. Separately, human blood neutrophils were exposed to dasatinib or control, and the effects on a range of neutrophil functions assessed.

Results: Dasatinib was associated with a dose-dependent significant increase in E. coli in the mouse lung, accompanied by impairment of organ function, reflected in significantly increased protein leak across the alveolar-capillary membrane. However, the number of neutrophils entering the lung was unaffected, suggesting that dasatinib impairs neutrophil function independent of migration. Dasatinib did not cause direct toxicity to human neutrophils, but led to significant reductions in phagocytosis of E. coli, adhesion, chemotaxis, generation of superoxide anion and degranulation of primary and secondary granules. However, no biologically important effect of dasatinib on neutrophil degranulation was observed in mice.

Conclusions: Contrary to our starting hypothesis, src kinase inhibition with dasatinib had a detrimental effect on bacterial clearance in the mouse lung and therefore does not represent an attractive therapeutic strategy to treat primary infective lung inflammation. Data from human neutrophils suggest that dasatanib has inhibitory effects on a range of neutrophil functions.
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http://dx.doi.org/10.1186/s12950-020-00261-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7597020PMC
October 2020

Neutrophil activation by nanomaterials : comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line.

Nanotoxicology 2021 02 4;15(1):1-20. Epub 2020 Dec 4.

Nano-Safety Research Group, Heriot-Watt University, Edinburgh, UK.

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses , a limited number of studies have investigated neutrophil responses to NMs . We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to models where necessary.
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http://dx.doi.org/10.1080/17435390.2020.1834635DOI Listing
February 2021

Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration.

Front Cell Dev Biol 2020 19;8:579943. Epub 2020 Oct 19.

Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.

Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our imaging led to the finding of a novel wound responsive + neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation.
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http://dx.doi.org/10.3389/fcell.2020.579943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604347PMC
October 2020

A fluorogenic cyclic peptide for imaging and quantification of drug-induced apoptosis.

Nat Commun 2020 08 12;11(1):4027. Epub 2020 Aug 12.

Centre for Inflammation Research, University of Edinburgh, EH16 4TJ, Edinburgh, UK.

Programmed cell death or apoptosis is a central biological process that is dysregulated in many diseases, including inflammatory conditions and cancer. The detection and quantification of apoptotic cells in vivo is hampered by the need for fixatives or washing steps for non-fluorogenic reagents, and by the low levels of free calcium in diseased tissues that restrict the use of annexins. In this manuscript, we report the rational design of a highly stable fluorogenic peptide (termed Apo-15) that selectively stains apoptotic cells in vitro and in vivo in a calcium-independent manner and under wash-free conditions. Furthermore, using a combination of chemical and biophysical methods, we identify phosphatidylserine as a molecular target of Apo-15. We demonstrate that Apo-15 can be used for the quantification and imaging of drug-induced apoptosis in preclinical mouse models, thus creating opportunities for assessing the in vivo efficacy of anti-inflammatory and anti-cancer therapeutics.
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http://dx.doi.org/10.1038/s41467-020-17772-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423924PMC
August 2020

Eosinophil Deficiency Promotes Aberrant Repair and Adverse Remodeling Following Acute Myocardial Infarction.

JACC Basic Transl Sci 2020 Jul 8;5(7):665-681. Epub 2020 Jul 8.

British Heart Foundation/University Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.

In ST-segment elevation myocardial infarction of both patients and mice, there was a decline in blood eosinophil count, with activated eosinophils recruited to the infarct zone. Eosinophil deficiency resulted in attenuated anti-inflammatory macrophage polarization, enhanced myocardial inflammation, increased scar size, and deterioration of myocardial structure and function. Adverse cardiac remodeling in the setting of eosinophil deficiency was prevented by interleukin-4 therapy.
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http://dx.doi.org/10.1016/j.jacbts.2020.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393409PMC
July 2020

Non-steroidal anti-inflammatory drugs, prostaglandins, and COVID-19.

Br J Pharmacol 2020 11 27;177(21):4899-4920. Epub 2020 Aug 27.

Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh, UK.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the novel coronavirus disease 2019 (COVID-19), a highly pathogenic and sometimes fatal respiratory disease responsible for the current 2020 global pandemic. Presently, there remains no effective vaccine or efficient treatment strategies against COVID-19. Non-steroidal anti-inflammatory drugs (NSAIDs) are medicines very widely used to alleviate fever, pain, and inflammation (common symptoms of COVID-19 patients) through effectively blocking production of prostaglandins (PGs) via inhibition of cyclooxyganase enzymes. PGs can exert either proinflammatory or anti-inflammatory effects depending on the inflammatory scenario. In this review, we survey the potential roles that NSAIDs and PGs may play during SARS-CoV-2 infection and the development and progression of COVID-19. LINKED ARTICLES: This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.
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http://dx.doi.org/10.1111/bph.15206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405053PMC
November 2020

Author Correction: Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart.

Nat Commun 2020 07 15;11(1):3648. Epub 2020 Jul 15.

British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17483-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363677PMC
July 2020

Mcl-1 protects eosinophils from apoptosis and exacerbates allergic airway inflammation.

Thorax 2020 07 17;75(7):600-605. Epub 2020 Apr 17.

University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh BioQuarter, UK.

Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.
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http://dx.doi.org/10.1136/thoraxjnl-2019-213204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7361019PMC
July 2020

Resolution of Inflammation and Gut Repair in IBD: Translational Steps Towards Complete Mucosal Healing.

Inflamm Bowel Dis 2020 07;26(8):1131-1143

Edinburgh IBD Science Unit, Centre for Inflammation Research, Queen's Medical Research Unit, University of Edinburgh, Scotland, United Kingdom.

Despite significant recent therapeutic advances, complete mucosal healing remains a difficult treatment target for many patients with inflammatory bowel diseases (IBD) to achieve. Our review focuses on the translational concept of promoting resolution of inflammation and repair as a necessary adjunctive step to reach this goal. We explore the roles of inflammatory cell apoptosis and efferocytosis to promote resolution, the new knowledge of gut monocyte-macrophage populations and their secreted prorepair mediators, and the processes of gut epithelial repair and regeneration to bridge this gap. We discuss the need and rationale for this vision and the tangible steps toward integrating proresolution therapies in IBD.
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http://dx.doi.org/10.1093/ibd/izaa045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365805PMC
July 2020

Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart.

Nat Commun 2019 11 15;10(1):5173. Epub 2019 Nov 15.

British Heart Foundation Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.

Three-dimensional fluorescence time-lapse imaging of the beating heart is extremely challenging, due to the heart's constant motion and a need to avoid pharmacological or phototoxic damage. Although real-time triggered imaging can computationally "freeze" the heart for 3D imaging, no previous algorithm has been able to maintain phase-lock across developmental timescales. We report a new algorithm capable of maintaining day-long phase-lock, permitting routine acquisition of synchronised 3D + time video time-lapse datasets of the beating zebrafish heart. This approach has enabled us for the first time to directly observe detailed developmental and cellular processes in the beating heart, revealing the dynamics of the immune response to injury and witnessing intriguing proliferative events that challenge the established literature on cardiac trabeculation. Our approach opens up exciting new opportunities for direct time-lapse imaging studies over a 24-hour time course, to understand the cellular mechanisms underlying cardiac development, repair and regeneration.
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http://dx.doi.org/10.1038/s41467-019-13112-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858381PMC
November 2019

Assessment of Neutrophil Apoptosis.

Methods Mol Biol 2020 ;2087:167-190

Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.

The process of neutrophil apoptosis has an important role in the resolution of acute inflammation. Apoptotic cell death is characterized by a coordinated sequence of cellular alterations that serve to uncouple neutrophil effector functions whilst maintaining plasma membrane integrity. In this way the release on neutrophil intracellular contents, including proteases, glycosidases, and reactive oxygen species, is limited during apoptosis. In addition, plasma membrane alterations associated with neutrophil apoptosis provide molecular cues that enable recognition by phagocytic cells, including macrophages. The recognition and uptake of apoptotic neutrophils by macrophages dampens proinflammatory responses to pathogen- or damage-associated molecular patterns and triggers release of proresolution mediators, that further promote resolution of inflammation. The key cellular and molecular events that act to control neutrophil apoptosis and subsequent macrophage phagocytosis have been characterized by in vitro studies, unveiling potential therapeutic targets for the manipulation of these regulatory pathways. In this chapter, we outline some of the key assays that are used to assess neutrophil apoptosis in vitro, together with methods to assess activation of the apoptotic machinery and phagocytic clearance of apoptotic neutrophils.
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http://dx.doi.org/10.1007/978-1-0716-0154-9_13DOI Listing
January 2021

Regulation of Apoptotic Cell Clearance During Resolution of Inflammation.

Front Pharmacol 2019 13;10:891. Epub 2019 Aug 13.

Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom.

Programmed cell death (apoptosis) has an important role in the maintenance of tissue homeostasis as well as the progression and ultimate resolution of inflammation. During apoptosis, the cell undergoes morphological and biochemical changes [e.g., phosphatidylserine (PtdSer) exposure, caspase activation, changes in mitochondrial membrane potential and DNA cleavage] that act to shut down cellular function and mark the cell for phagocytic clearance. Tissue phagocytes bind and internalize apoptotic cells, bodies, and vesicles, providing a mechanism for the safe disposal of apoptotic material. Phagocytic removal of apoptotic cells before they undergo secondary necrosis reduces the potential for bystander damage to adjacent tissue and importantly initiates signaling pathways within the phagocytic cell that act to dampen inflammation. In a pathological context, excessive apoptosis or failure to clear apoptotic material results in secondary necrosis with the release of pro-inflammatory intracellular contents. In this review, we consider some of the mechanisms by which phagocytosis of apoptotic cells can be controlled. We suggest that matching apoptotic cell load with the capacity for apoptotic cell clearance within tissues may be important for therapeutic strategies that target the apoptotic process for treatment of inflammatory disease.
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http://dx.doi.org/10.3389/fphar.2019.00891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701246PMC
August 2019

A Negative Feedback Loop Regulates Integrin Inactivation and Promotes Neutrophil Recruitment to Inflammatory Sites.

J Immunol 2019 09 19;203(6):1579-1588. Epub 2019 Aug 19.

Centre for Inflammation Research, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom;

Neutrophils are abundant circulating leukocytes that are rapidly recruited to sites of inflammation in an integrin-dependent fashion. Contrasting with the well-characterized regulation of integrin activation, mechanisms regulating integrin inactivation remain largely obscure. Using mouse neutrophils, we demonstrate in this study that the GTPase activating protein ARAP3 is a critical regulator of integrin inactivation; experiments with Chinese hamster ovary cells indicate that this is not restricted to neutrophils. Specifically, ARAP3 acts in a negative feedback loop downstream of PI3K to regulate integrin inactivation. Integrin ligand binding drives the activation of PI3K and of its effectors, including ARAP3, by outside-in signaling. ARAP3, in turn, promotes localized integrin inactivation by negative inside-out signaling. This negative feedback loop reduces integrin-mediated PI3K activity, with ARAP3 effectively switching off its own activator, while promoting turnover of substrate adhesions. In vitro, ARAP3-deficient neutrophils display defective PIP3 polarization, adhesion turnover, and transendothelial migration. In vivo, ARAP3-deficient neutrophils are characterized by a neutrophil-autonomous recruitment defect to sites of inflammation.
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http://dx.doi.org/10.4049/jimmunol.1900443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731454PMC
September 2019

Cathelicidin is a "fire alarm", generating protective NLRP3-dependent airway epithelial cell inflammatory responses during infection with Pseudomonas aeruginosa.

PLoS Pathog 2019 04 12;15(4):e1007694. Epub 2019 Apr 12.

Centre for Inflammation Research at the University of Edinburgh, Queens' Medical Research Institute, Edinburgh BioQuarter, Little France Crescent, Edinburgh, United Kingdom.

Pulmonary infections are a major global cause of morbidity, exacerbated by an increasing threat from antibiotic-resistant pathogens. In this context, therapeutic interventions aimed at protectively modulating host responses, to enhance defence against infection, take on ever greater significance. Pseudomonas aeruginosa is an important multidrug-resistant, opportunistic respiratory pathogen, the clearance of which can be enhanced in vivo by the innate immune modulatory properties of antimicrobial host defence peptides from the cathelicidin family, including human LL-37. Initially described primarily as bactericidal agents, cathelicidins are now recognised as multifunctional antimicrobial immunomodulators, modifying host responses to pathogens, but the key mechanisms involved in these protective functions are not yet defined. We demonstrate that P. aeruginosa infection of airway epithelial cells promotes extensive infected cell internalisation of LL-37, in a manner that is dependent upon epithelial cell interaction with live bacteria, but does not require bacterial Type 3 Secretion System (T3SS). Internalised LL-37 acts as a second signal to induce inflammasome activation in airway epithelial cells, which, in contrast to myeloid cells, are relatively unresponsive to P. aeruginosa. We demonstrate that this is mechanistically dependent upon cathepsin B release, and NLRP3-dependent activation of caspase 1. These result in LL-37-mediated release of IL-1β and IL-18 in a manner that is synergistic with P. aeruginosa infection, and can induce caspase 1-dependent death of infected epithelial cells, and promote neutrophil chemotaxis. We propose that cathelicidin can therefore act as a second signal, required by P. aeruginosa infected epithelial cells to promote an inflammasome-mediated altruistic cell death of infection-compromised epithelial cells and act as a "fire alarm" to enhance rapid escalation of protective inflammatory responses to an uncontrolled infection. Understanding this novel modulatory role for cathelicidins, has the potential to inform development of novel therapeutic strategies to antibiotic-resistant pathogens, harnessing innate immunity as a complementation or alternative to current interventions.
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http://dx.doi.org/10.1371/journal.ppat.1007694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481867PMC
April 2019

Inflammation Resolution and the Induction of Granulocyte Apoptosis by Cyclin-Dependent Kinase Inhibitor Drugs.

Front Pharmacol 2019 19;10:55. Epub 2019 Feb 19.

Queen's Medical Research Institute, University of Edinburgh Centre for Inflammation Research, Edinburgh BioQuarter, Edinburgh, United Kingdom.

Inflammation is a necessary dynamic tissue response to injury or infection and it's resolution is essential to return tissue homeostasis and function. Defective or dysregulated inflammation resolution contributes significantly to the pathogenesis of many, often common and challenging to treat human conditions. The transition of inflammation to resolution is an active process, involving the clearance of inflammatory cells (granulocytes), a change of mediators and their receptors, and prevention of further inflammatory cell infiltration. This review focuses on the use of cyclin dependent kinase inhibitor drugs to pharmacologically target this inflammatory resolution switch, specifically through inducing granulocyte apoptosis and phagocytic clearance of apoptotic cells (efferocytosis). The key processes and pathways required for granulocyte apoptosis, recruitment of phagocytes and mechanisms of engulfment are discussed along with the cumulating evidence for cyclin dependent kinase inhibitor drugs as pro-resolution therapeutics.
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http://dx.doi.org/10.3389/fphar.2019.00055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6389705PMC
February 2019

Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential.

J Pathol 2019 04 15;247(5):672-685. Epub 2019 Feb 15.

The University of Edinburgh Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, UK.

Acute respiratory distress syndrome (ARDS) is the often fatal sequelae of a broad range of precipitating conditions. Despite decades of intensive research and clinical trials there remain no therapies in routine clinical practice that target the dysregulated and overwhelming inflammatory response that characterises ARDS. Neutrophils play a central role in the initiation, propagation and resolution of this complex inflammatory environment by migrating into the lung and executing a variety of pro-inflammatory functions. These include degranulation with liberation of bactericidal proteins, release of cytokines and reactive oxygen species as well as production of neutrophil extracellular traps. Although these functions are advantageous in clearing bacterial infection, the consequence of associated tissue damage, the contribution to worsening acute inflammation and prolonged neutrophil lifespan at sites of inflammation are deleterious. In this review, the importance of the neutrophil will be considered, together with discussion of recent advances in understanding neutrophil function and the factors that influence them throughout the phases of inflammation in ARDS. From a better understanding of neutrophils in this context, potential therapeutic targets are identified and discussed. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6492013PMC
April 2019

Repurposing simvastatin as a therapy for preterm labor: evidence from preclinical models.

FASEB J 2019 02 12;33(2):2743-2758. Epub 2018 Oct 12.

Tommy's Centre for Maternal and Fetal Health, Medical Research Council (MRC) Centre for Reproductive Health, Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; and.

Preterm birth (PTB), the leading cause of neonatal morbidity and mortality, urgently requires novel therapeutic agents. Spontaneous PTB, resulting from preterm labor, is commonly caused by intrauterine infection/inflammation. Statins are well-established, cholesterol-lowering drugs that can reduce inflammation and inhibit vascular smooth muscle contraction. We show that simvastatin reduced the incidence of PTB in a validated intrauterine LPS-induced PTB mouse model, decreased uterine proinflammatory mRNA concentrations (IL-6, Cxcl1, and Ccl2), and reduced serum IL-6 concentration. In human myometrial cells, simvastatin reduced proinflammatory mediator mRNA and protein expression (IL-6 and IL-8) and increased anti-inflammatory cytokine mRNA expression (IL-10 and IL-13). Critically, simvastatin inhibited myometrial cell contraction, basally and during inflammation, and reduced phosphorylated myosin light chain concentration. Supplementation with mevalonate and geranylgeranyl pyrophosphate, but not farnesyl pyrophosphate, abolished these anticontractile effects, indicating that the Rho/Rho-associated protein kinase pathway is critically involved. Thus, simvastatin reduces PTB incidence in mice, inhibits myometrial contractions, and exhibits key anti-inflammatory effects, providing a rationale for investigation into the repurposing of statins to treat preterm labor in women.-Boyle, A. K., Rinaldi, S. F., Rossi, A. G., Saunders, P. T. K., Norman, J. E. Repurposing simvastatin as a therapy for preterm labor: evidence from preclinical models.
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http://dx.doi.org/10.1096/fj.201801104RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338657PMC
February 2019

Validation of the Incremental Shuttle Walk Test as a Clinical End Point in Bronchiectasis.

Chest 2018 12 6;154(6):1321-1329. Epub 2018 Oct 6.

MRC Centre for Inflammation Research, Queen's Medical Research Institute, Edinburgh, United Kingdom; Department of Respiratory Medicine, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom.

Background: A validated clinical end point is needed to assess response to therapies in bronchiectasis.

Objectives: The goal of this study was to assess the reliability, validity, and responsiveness of the incremental shuttle walk test (ISWT) as a clinical end point in bronchiectasis.

Methods: In clinically stable patients (n = 30), the ISWT was performed twice, 6 months apart. Correlation between the St. George's Respiratory Questionnaire (SGRQ) and the ISWT (n = 94) was performed. The 1-year gentamicin study was reanalyzed to assess the area under the curve (percent change of ISWT with a ≥ 4 unit improvement in total SGRQ). ISWT was performed prior to and following 14 days of antibiotics for an exacerbation (94 oral courses and 30 IV courses, n = 124) and reanalysis of the 1-year gentamicin study (n = 57).

Results: The ISWT did not significantly change over 6 months while clinically stable. The ISWT correlated inversely with the SGRQ (rs = -0.60; P < .0001), Bronchiectasis Severity Index score (rs = -0.44; P < .0001), and sedentary time (rs = -0.48; P = .0007) but correlated with physical activity (rs = 0.42; P = .004). The area under the curve for percent change in ISWT with ≥ 4 unit improvement in SGRQ was 0.79 (95% CI, 0.66-0.91; P = .001). A threshold of 5% improvement in the ISWT had a 92% sensitivity but 50% specificity, and from the responsiveness studies would capture 73% of all patients.

Conclusions: This study confirmed the ISWT to be reliable, valid, and responsive to change in patients with bronchiectasis. The authors propose that a minimum clinically important difference of 5% improvement in the ISWT would be a useful objective end point to assess therapies in bronchiectasis.
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http://dx.doi.org/10.1016/j.chest.2018.09.019DOI Listing
December 2018

Early PREdiction of sepsis using leukocyte surface biomarkers: the ExPRES-sepsis cohort study.

Intensive Care Med 2018 Nov 5;44(11):1836-1848. Epub 2018 Oct 5.

MRC Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, Edinburgh, UK.

Purpose: Reliable biomarkers for predicting subsequent sepsis among patients with suspected acute infection are lacking. In patients presenting to emergency departments (EDs) with suspected acute infection, we aimed to evaluate the reliability and discriminant ability of 47 leukocyte biomarkers as predictors of sepsis (Sequential Organ Failure Assessment score ≥ 2 at 24 h and/or 72 h following ED presentation).

Methods: In a multi-centre cohort study in four EDs and intensive care units (ICUs), we standardised flow-cytometric leukocyte biomarker measurement and compared patients with suspected acute infection (cohort-1) with two comparator cohorts: ICU patients with established sepsis (cohort-2), and ED patients without infection or systemic inflammation but requiring hospitalization (cohort-3).

Results: Between January 2014 and February 2016, we recruited 272, 59 and 75 patients to cohorts 1, 2, and 3, respectively. Of 47 leukocyte biomarkers, 14 were non-reliable, and 17 did not discriminate between the three cohorts. Discriminant analyses for predicting sepsis within cohort-1 were undertaken for eight neutrophil (cluster of differentiation antigens (CD) CD15; CD24; CD35; CD64; CD312; CD11b; CD274; CD279), seven monocyte (CD35; CD64; CD312; CD11b; HLA-DR; CD274; CD279) and a CD8 T-lymphocyte biomarker (CD279). Individually, only higher neutrophil CD279 [OR 1.78 (95% CI 1.23-2.57); P = 0.002], higher monocyte CD279 [1.32 (1.03-1.70); P = 0.03], and lower monocyte HLA-DR [0.73 (0.55-0.97); P = 0.03] expression were associated with subsequent sepsis. With logistic regression the optimum biomarker combination was increased neutrophil CD24 and neutrophil CD279, and reduced monocyte HLA-DR expression, but no combination had clinically relevant predictive validity.

Conclusions: From a large panel of leukocyte biomarkers, immunosuppression biomarkers were associated with subsequent sepsis in ED patients with suspected acute infection.

Clinical Trial Registration: NCT02188992.
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http://dx.doi.org/10.1007/s00134-018-5389-0DOI Listing
November 2018

Augmentation of Human Monocyte Responses to Lipopolysaccharide by the Protein S and Mer/Tyro3 Receptor Tyrosine Kinase Axis.

J Immunol 2018 11 24;201(9):2602-2611. Epub 2018 Sep 24.

Medical Research Council Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, United Kingdom; and

Resolution of the inflammatory response requires coordinated regulation of pro- and anti-inflammatory mediator production, together with clearance of recruited inflammatory cells. Many different receptors have been implicated in phagocytosis of apoptotic cells (efferocytosis), including Mer, a receptor tyrosine kinase that can mediate recognition and subsequent internalization of apoptotic cells. In this manuscript, we examine the expression and function of the Tyro3/Axl/Mer (TAM) family of receptors by human monocytes. We demonstrate that the Mer ligand, protein S, binds to the surface of viable monocytes via phosphatidylserine-dependent and -independent mechanisms. Importantly, we have identified a novel role for receptor tyrosine kinase signaling in the augmentation of monocyte cytokine release in response to LPS. We propose that low-level phosphatidylserine exposure on the plasma membrane of viable monocytes allows protein S binding that leads to TAM-dependent augmentation of proinflammatory cytokine production. Our findings identify a potentially important role for TAM-mediated signaling during the initiation phase of inflammation.
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http://dx.doi.org/10.4049/jimmunol.1800249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201838PMC
November 2018

Cerebral Concussion Primes the Lungs for Subsequent Neutrophil-Mediated Injury.

Crit Care Med 2018 09;46(9):e937-e944

MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, United Kingdom.

Objectives: Mild traumatic brain injury in the form of concussion is extremely common, and the potential effects on pulmonary priming have been underestimated. The aim of this study was to characterize the pulmonary response following mild traumatic brain injury and assess the pulmonary susceptibility to lung injury after a subsequent innocuous pulmonary insult.

Design: Experimental in vivo study.

Setting: University research laboratory.

Subjects: Male CD1 mice.

Interventions: We developed a model of concussive traumatic brain injury in mice followed by pulmonary acid microaspiration. To assess the dependent role of neutrophils in mediating pulmonary injury, we specifically depleted neutrophils.

Measurements And Main Results: Lateral fluid percussion to the brain resulted in neuronal damage and neutrophil infiltration as well as extensive pulmonary interstitial neutrophil accumulation but no alveolar injury. Following subsequent innocuous acid microaspiration, augmented alveolar neutrophil influx led to the development of pulmonary hemorrhage that was reduced following neutrophil depletion.

Conclusions: This model shows for the first time that innocuous acid microaspiration is sufficient to induce neutrophil-mediated lung injury following mild concussion and that the extracranial effects of mild traumatic brain injury have been underestimated.
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http://dx.doi.org/10.1097/CCM.0000000000003270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6110623PMC
September 2018

Cell-surface signatures of immune dysfunction risk-stratify critically ill patients: INFECT study.

Intensive Care Med 2018 05 7;44(5):627-635. Epub 2018 Jun 7.

MRC Centre for Inflammation Research, University of Edinburgh, 47 Little France Crescent, Edinburgh, Scotland, UK.

Purpose: Cellular immune dysfunctions, which are common in intensive care patients, predict a number of significant complications. In order to effectively target treatments, clinically applicable measures need to be developed to detect dysfunction. The objective was to confirm the ability of cellular markers associated with immune dysfunction to stratify risk of secondary infection in critically ill patients.

Methods: Multi-centre, prospective observational cohort study of critically ill patients in four UK intensive care units. Serial blood samples were taken, and three cell surface markers associated with immune cell dysfunction [neutrophil CD88, monocyte human leucocyte antigen-DR (HLA-DR) and percentage of regulatory T cells (T)] were assayed on-site using standardized flow cytometric measures. Patients were followed up for the development of secondary infections.

Results: A total of 148 patients were recruited, with data available from 138. Reduced neutrophil CD88, reduced monocyte HLA-DR and elevated proportions of T were all associated with subsequent development of infection with odds ratios (95% CI) of 2.18 (1.00-4.74), 3.44 (1.58-7.47) and 2.41 (1.14-5.11), respectively. Burden of immune dysfunction predicted a progressive increase in risk of infection, from 14% for patients with no dysfunction to 59% for patients with dysfunction of all three markers. The tests failed to risk stratify patients shortly after ICU admission but were effective between days 3 and 9.

Conclusions: This study confirms our previous findings that three cell surface markers can predict risk of subsequent secondary infection, demonstrates the feasibility of standardized multisite flow cytometry and presents a tool which can be used to target future immunomodulatory therapies.

Trial Registration: The study was registered with clinicaltrials.gov (NCT02186522).
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http://dx.doi.org/10.1007/s00134-018-5247-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006236PMC
May 2018

Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation.

Cell Death Dis 2018 06 4;9(6):665. Epub 2018 Jun 4.

The MRC Centre for Inflammation Research, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

Apoptotic cells modulate the function of macrophages to control and resolve inflammation. Here, we show that neutrophils induce a rapid and sustained suppression of NF-κB signalling in the macrophage through a unique regulatory relationship which is independent of apoptosis. The reduction of macrophage NF-κB activation occurs through a blockade in transforming growth factor β-activated kinase 1 (TAK1) and IKKβ activation. As a consequence, NF-κB (p65) phosphorylation is reduced, its translocation to the nucleus is inhibited and NF-κB-mediated inflammatory cytokine transcription is suppressed. Gene Set Enrichment Analysis reveals that this suppression of NF-κB activation is not restricted to post-translational modifications of the canonical NF-κB pathway, but is also imprinted at the transcriptional level. Thus neutrophils exert a sustained anti-inflammatory phenotypic reprogramming of the macrophage, which is reflected by the sustained reduction in the release of pro- but not anti- inflammatory cytokines from the macrophage. Together, our findings identify a novel apoptosis-independent mechanism by which neutrophils regulate the mediator profile and reprogramming of monocytes/macrophages, representing an important nodal point for inflammatory control.
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http://dx.doi.org/10.1038/s41419-018-0710-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986789PMC
June 2018