Calum T. Robb

Invertebrate extracellular phagocyte traps show that chromatin is an ancient defence weapon

Controlled release of chromatin from the nuclei of inflammatory cells is a process that entraps and kills microorganisms in the extracellular environment. Now termed ETosis, it is important for innate immunity in vertebrates. Paradoxically, however, in mammals, it can also contribute to certain pathologies. Here we show that ETosis occurs in several invertebrate species, including, remarkably, an acoelomate. Our findings reveal that the phenomenon is primordial and predates the evolution of the coelom. In invertebrates, the released chromatin participates in defence not only by ensnaring microorganisms and externalizing antibacterial histones together with other haemocyte-derived defence factors, but crucially, also provides the scaffold on which intact haemocytes assemble during encapsulation; a response that sequesters and kills potential pathogens infecting the body cavity. This insight into the early origin of ETosis identifies it as a very ancient process that helps explain some of its detrimental effects in mammals.

Prostaglandin E2 constrains systemic inflammation through an innate lymphoid cell–IL-22 axis

A prostaglandin barrier to inflammation Blood-borne bacterial infections and severe trauma can send the immune system into overdrive, causing it to pump out inflammatory mediators, sometimes at lethal doses. Duffin et al. now report on a role for prostaglandins in keeping systemic inflammation in check. Systemic inflammation correlates with decreased production of the prostaglandin E2 (PGE2). Blocking PGE2 signaling in mice led to severe inflammation associated with the translocation of gut bacteria. PGE2 acts on innate lymphoid cells, which produce interleukin-22, a secreted protein that helps promote intestinal integrity. Science, this issue p. 1333 Prostaglandin E2 prevents systemic inflammation by maintaining gut barrier integrity. Systemic inflammation, which results from the massive release of proinflammatory molecules into the circulatory system, is a major risk factor for severe illness, but the precise mechanisms underlying its control are not fully understood. We observed that prostaglandin E2 (PGE2), through its receptor EP4, is down-regulated in human systemic inflammatory disease. Mice with reduced PGE2 synthesis develop systemic inflammation, associated with translocation of gut bacteria, which can be prevented by treatment with EP4 agonists. Mechanistically, we demonstrate that PGE2-EP4 signaling acts directly on type 3 innate lymphoid cells (ILCs), promoting their homeostasis and driving them to produce interleukin-22 (IL-22). Disruption of the ILC–IL-22 axis impairs PGE2-mediated inhibition of systemic inflammation. Hence, the ILC–IL-22 axis is essential in protecting against gut barrier dysfunction, enabling PGE2-EP4 signaling to impede systemic inflammation.

Key mechanisms governing resolution of lung inflammation

Innate immunity normally provides excellent defence against invading microorganisms. Acute inflammation is a form of innate immune defence and represents one of the primary responses to injury, infection and irritation, largely mediated by granulocyte effector cells such as neutrophils and eosinophils. Failure to remove an inflammatory stimulus (often resulting in failed resolution of inflammation) can lead to chronic inflammation resulting in tissue injury caused by high numbers of infiltrating activated granulocytes. Successful resolution of inflammation is dependent upon the removal of these cells. Under normal physiological conditions, apoptosis (programmed cell death) precedes phagocytic recognition and clearance of these cells by, for example, macrophages, dendritic and epithelial cells (a process known as efferocytosis). Inflammation contributes to immune defence within the respiratory mucosa (responsible for gas exchange) because lung epithelia are continuously exposed to a multiplicity of airborne pathogens, allergens and foreign particles. Failure to resolve inflammation within the respiratory mucosa is a major contributor of numerous lung diseases. This review will summarise the major mechanisms regulating lung inflammation, including key cellular interplays such as apoptotic cell clearance by alveolar macrophages and macrophage/neutrophil/epithelial cell interactions. The different acute and chronic inflammatory disease states caused by dysregulated/impaired resolution of lung inflammation will be discussed. Furthermore, the resolution of lung inflammation during neutrophil/eosinophil-dominant lung injury or enhanced resolution driven via pharmacological manipulation will also be considered.

Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (Ri) from 19 to 7 h and improved organ dysfunction with enhanced alveolar–capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (Ri; 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation.

Novel role for endogenous mitochondrial formylated peptide-driven formyl peptide receptor 1 signalling in acute respiratory distress syndrome

Background Acute respiratory distress syndrome (ARDS) is an often fatal neutrophil-dominant lung disease. Although influenced by multiple proinflammatory mediators, identification of suitable therapeutic candidates remains elusive. We aimed to delineate the presence of mitochondrial formylated peptides in ARDS and characterise the functional importance of formyl peptide receptor 1 (FPR1) signalling in sterile lung inflammation. Methods Mitochondrial formylated peptides were identified in bronchoalveolar lavage fluid (BALF) and serum of patients with ARDS by liquid chromatography–tandem mass spectrometry. In vitro, human neutrophils were stimulated with mitochondrial formylated peptides and their effects assessed by flow cytometry and chemotaxis assay. Mouse lung injury was induced by mitochondrial formylated peptides or hydrochloric acid. Bone marrow chimeras determined the contribution of myeloid and parenchymal FPR1 to sterile lung inflammation. Results Mitochondrial formylated peptides were elevated in BALF and serum from patients with ARDS. These peptides drove neutrophil activation and chemotaxis through FPR1-dependent mechanisms in vitro and in vivo. In mouse lung injury, inflammation was attenuated in Fpr1−/− mice, effects recapitulated by a pharmacological FPR1 antagonist even when administered after the onset of injury. FPR1 expression was present in alveolar epithelium and chimeric mice demonstrated that both myeloid and parenchymal FPR1 contributed to lung inflammation. Conclusions We provide the first definitive evidence of mitochondrial formylated peptides in human disease and demonstrate them to be elevated in ARDS and important in a mouse model of lung injury. This work reveals mitochondrial formylated peptide FPR1 signalling as a key driver of sterile acute lung injury and a potential therapeutic target in ARDS.

Delayed neutrophil apoptosis enhances NET formation in cystic fibrosis

Background Cystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation. Methods Blood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR. Results CF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF. Conclusions CF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

The cyclin-dependent kinase inhibitor AT7519 accelerates neutrophil apoptosis in sepsis-related acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a neutrophil-dominant disorder with no effective pharmacological therapies. While the cyclin-dependent kinase inhibitor AT7519 induces neutrophil apoptosis to promote inflammation resolution in preclinical models of lung inflammation, its potential efficacy in ARDS has not been examined. Untreated peripheral blood sepsis-related ARDS neutrophils demonstrated prolonged survival after 20 hours in vitro culture. AT7519 was able to override this phenotype to induce apoptosis in ARDS neutrophils with reduced expression of the pro-survival protein Mcl-1. We demonstrate the first pharmacological compound to induce neutrophil apoptosis in sepsis-related ARDS, highlighting cyclin-dependent kinase inhibitors as potential novel therapeutic agents.

Mechanism of neutrophil activation and toxicity elicited by engineered nanomaterials

The effects of nanomaterials (NMs) on biological systems, especially their ability to stimulate inflammatory responses requires urgent investigation. We evaluated the response of the human differentiated HL60 neutrophil-like cell line to NMs. It was hypothesised that NM physico-chemical characteristics would influence cell responsiveness by altering intracellular Ca2+ concentration [Ca2+]i and reactive oxygen species production. Cells were exposed (1.95-125 μg/ml, 24 h) to silver (Ag), zinc oxide (ZnO), titanium dioxide (TiO2), multi-walled carbon nanotubes (MWCNTs) or ultrafine carbon black (ufCB) and cytotoxicity assessed (alamar blue assay). Relatively low (TiO2, MWCNTs, ufCB) or high (Ag, ZnO) cytotoxicity NMs were identified. Sub-lethal impacts of NMs on cell function were investigated for selected NMs only, namely TiO2, Ag and ufCB. Only Ag stimulated cell activation. Within minutes, Ag stimulated an increase in [Ca2+]i (in Fura-2 loaded cells), and a prominent inward ion current (assessed by electrophysiology). Within 2-4 h, Ag increased superoxide anion release and stimulated cytokine production (MCP-1, IL-8) that was diminished by Ca2+ inhibitors or trolox. Light microscopy demonstrated that cells had an activated phenotype. In conclusion NM toxicity was ranked; Ag>ufCB>TiO2, and the battery of tests used provided insight into the mechanism of action of NM toxicity to guide future testing strategies.

Prostaglandin E2 stimulates adaptive IL-22 production and promotes allergic contact dermatitis

Background: Atopic dermatitis (AD) and allergic contact dermatitis (ACD) are both forms of eczema and are common inflammatory skin diseases with a central role of T cell‐derived IL‐22 in their pathogenesis. Although prostaglandin (PG) E2 is known to promote inflammation, little is known about its role in processes related to AD and ACD development, including IL‐22 upregulation. Objectives: We sought to investigate whether PGE2 has a role in IL‐22 induction and development of ACD, which has increased prevalence in patients with AD. Methods: T‐cell cultures and in vivo sensitization of mice with haptens were used to assess the role of PGE2 in IL‐22 production. The involvement of PGE2 receptors and their downstream signals was also examined. The effects of PGE2 were evaluated by using the oxazolone‐induced ACD mouse model. The relationship of PGE2 and IL‐22 signaling pathways in skin inflammation were also investigated by using genomic profiling in human lesional AD skin. Results: PGE2 induces IL‐22 from T cells through its receptors, E prostanoid receptor (EP) 2 and EP4, and involves cyclic AMP signaling. Selective deletion of EP4 in T cells prevents hapten‐induced IL‐22 production in vivo, and limits atopic‐like skin inflammation in the oxazolone‐induced ACD model. Moreover, both PGE2 and IL‐22 pathway genes were coordinately upregulated in human AD lesional skin but were at less than significant detection levels after corticosteroid or UVB treatments. Conclusions: Our results define a crucial role for PGE2 in promoting ACD by facilitating IL‐22 production from T cells.

Models for the Study of the Cross Talk Between Inflammation and Cell Cycle

Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.