Airway neutrophilia is a common feature of many chronic inflammatory lung diseases and is associated with disease progression, often regardless of the initiating cause. Neutrophils and their products are thought to be key mediators of the inflammatory changes in the airways of patients with chronic obstructive pulmonary disease (COPD) and have been shown to cause many of the pathological features associated with disease, including emphysema and mucus hypersecretion. Patients with COPD also have high rates of bacterial colonisation and recurrent infective exacerbations, suggesting that neutrophil host defence mechanisms are impaired, a concept supported by studies showing alterations to neutrophil migration, degranulation and reactive oxygen species production in cells isolated from patients with COPD. Although the role of neutrophils is best described in COPD, many of the pathological features of this disease are not unique to COPD and also feature in other chronic inflammatory airway diseases, including asthma, cystic fibrosis, alpha-1 anti-trypsin deficiency, and bronchiectasis. There is increasing evidence for immune cell dysfunction contributing to inflammation in many of these diseases, focusing interest on the neutrophil as a key driver of pulmonary inflammation and a potential therapeutic target than spans diseases. This review discusses the evidence for neutrophilic involvement in COPD and also considers their roles in alpha-1 anti-trypsin deficiency, bronchiectasis, asthma, and cystic fibrosis. We provide an in-depth assessment of the role of the neutrophil in each of these conditions, exploring recent advances in understanding, and finally discussing the possibility of common mechanisms across diseases. Neutrophils are characterised by their multi-lobed nucleus and granular cytoplasm, the latter caused by azurophillic (primary), specific (secondary) and gelatinase (tertiary) granules, as well as secretory vesicles , having a basal production of 1 to 2 × 102) to the superoxide anion O2−2. Superoxide then can dismutate to form the highly oxidative hydrogen peroxide (H2O2), which can react further, forming the strongly bactericidal hypohalous acids 8. These products can also be released into the extracellular matrix by degranulation, but neutrophils require different levels of activation to release granules; secretory vesicles are released during minimal stimulation to facilitate migration and adhesion, and azurophil granules (the most cytotoxic) require the most stimulation.In response to infection, neutrophils leave the circulation and migrate to the affected sites, where they use a variety of mechanisms to contain and kill invading pathogens, preventing further dissemination. The phagocytosis of bacteria leads to intracellular pathogen killing within a contained structure (the phagolysosome) to protect the cell and surrounding tissue. The phagolysosome is formed when neutrophil granules  fuse with the lysosome containing the ingested bacterium. ROS production is a convoluted process, necessary to protect the host from the free radical-associated harm. NADPH oxidase is constructed from a series of subunits and then acts as a channel for electrons from the cytosol to enter the phagolysosome, stimulating reduction of oxygen  as they can ensnare and “trap” bacteria13. To protect the host from this immense arsenal, neutrophils are held in three states that they can fluctuate between: quiescent, primed and activated. The primed state provides both a mechanism to allow a rapid and pro-inflammatory response to infection but also a brake to prevent unwarranted degranulation14.Release of azurophil granules leads to areas of obligate tissue damage, as the proteinases contained therein readily digest components of the extracellular matrix until their inhibition by anti-proteinases can occur15, can release a wide range of context-specific products16 and have an adaptable lifespan depending on activation status and environment17. They also express more than 30 different receptors—including G protein–coupled receptors, Fc receptors, adhesion receptors, cytokine receptors and pattern recognition receptors—that can sense pro-inflammatory mediators and modulate neutrophil migration, function and behaviour18, suggesting plasticity in their responses. There have been descriptions of pro-angiogenic neutrophils19, characterised by increased matrix metalloproteinase 9 (MMP-9) release21 and anti-inflammatory neutrophils capable of suppressing other immune cells22. Research into the exact function of these phenotypes  remains unclear, but it might be that different subsets of cells have different functions; indeed, when neutrophils are viewed, it is clear that cells within a field behave heterogeneously23.There is increasing interest in neutrophil phenotypes or subtypes, and cells appear to have a broader repertoire of responses than the highly aggressive, cytotoxic response described above. The concept of cell phenotypes is well established with lymphocytes or macrophages but remains more controversial with neutrophils. However, despite previous theories, neutrophils are transcriptionally active26. However, recent research has shown that this is not the case in health, and there is no evidence of a retained neutrophil population in the lungs unless there is systemic priming of neutrophils and even this results in only a transitory retention of neutrophils27. It was then demonstrated that the process of manoeuvring through tight spaces promoted neutrophil de-priming28, leading to the hypothesis that the sinuous pulmonary vasculature might be a site where the primed neutrophil population (thought to be up to 40% of the whole population in some studies) can be “stood down” into a quiescent state29.What is evident is the immense potential that neutrophils have for host damage which requires constant check. Recently, it has been suggested that the lung may be involved in this process. Neutrophils are larger in diameter than some of the tortuous pulmonary vasculature that they must traverse. Initially, it was considered that the lungs hold a sequestered pool of neutrophils, slow in transit through the capillary bed and ready to respond to pulmonary infectionPerhaps then it is no surprise that there is evidence of neutrophil dysregulation during lung disease, that airway neutrophilia is a feature of multiple lung pathologies and that patients with airway disease often display heightened and more damaging neutrophilic inflammation. This may represent a physiological response to an infective or inflammatory trigger (such as neutrophil recruitment to the lung in response to a respiratory infection or cigarette smoke) or a physiological response to a pathological environment (the inflamed and damaged lung causing increased neutrophil recruitment and being less able to “de-prime” cells). However, there is amassing evidence that the neutrophil itself may inflict further harm to the host by intrinsic changes to key cellular functions. Understanding whether the neutrophil is a reactive responder or a creative actor in lung disease (or indeed both) is vital when considering the development of new therapeutics: would one target the environment or the neutrophil? The evidence for these processes in airway disease has been most thoroughly described in chronic obstructive pulmonary disease (COPD). This review will explore what is currently known about neutrophils in the pathogenesis of airway disease, focusing mainly on COPD. However, neutrophil function in alpha-1 anti-trypsin deficiency (AATD), bronchiectasis, cystic fibrosis (CF) and asthma will also be considered in order to assess the likelihood of common mechanisms and therefore potential therapies which could span diseases.32. In the UK, chronic cigarette smoking remains the largest cause of COPD, but after 25 years of smoking, only about 30 to 40% of adults will have developed COPD33, and COPD is diagnosed in never smokers 34, suggesting that smoking is neither necessary nor sufficient to cause COPD.COPD is a leading cause of morbidity and mortality worldwide and constitutes a significant healthcare burden35 (1), reduction in gas transfer, and development of emphysema40. Although patients with COPD demonstrate airway neutrophilia, they also experience airway colonisation and recurrent bacterial infections44. This raises the possibility that the function of neutrophils is impaired, leading to reduced anti-microbial function and at the same time contributing to lung damage and a number of observations support this concept.Airway inflammation is central to the pathophysiology of COPD and contributes to tissue damage and destruction and a wealth of data support a role for the neutrophil at the heart of this inflammatory process. All patients with COPD have airway neutrophilia, regardless of clinical phenotype , disease severity, and rate of decline or age of onset. COPD is very heterogeneous and although patients may share a cause (such as cigarette smoking), the disease presentation is variable, suggesting that COPD is more an umbrella term than a narrow clinical entity35 . Neutrop46; however, more recent studies have allowed the assessment of specific neutrophil migratory dynamics and have shown that neutrophils from patients with COPD migrate with increased speed but reduced directional accuracy towards a variety of chemoattractants compared with age-matched healthy control subjects40. This does not result from reduced chemoattractant receptor expression or impaired receptor localisation49 but could be improved by using a broad-spectrum PI3K inhibitor (LY294002), suggesting that the defective migration results from aberrant intracellular signalling processes40.Older studies of neutrophil migration in COPD yielded conflicting results as to whether there was any compromise in migratory function52. Substrates for these proteinases include elastin53, collagen54 and fibronectin55, which are major components of the extracellular matrix, and their degradation is linked to all clinical facets of COPD.As a neutrophil migrates, serine proteinases, including neutrophil elastase (NE), cathepsin G and proteinase 3 (PR3), are released from azurophil granules into the extracellular space, and some active enzyme is retained on the plasma membrane56 . NE is only partly inhibited until it diffuses away from the cell and an optimal NE-inhibitor ratio is reached. As this inhibition is not immediate, an obligate area of local, proteinase-mediated tissue damage occurs, a phenomenon known as “quantum proteolysis”. Furthermore, a proportion of NE remains bound to the neutrophil cell surface, making it less accessible to inhibitors and more resistant to inhibition, further increasing the local potential damage. There is emerging evidence that PR3 may be even more implicated in the pathology of COPD, especially the emphysema process59. PR3 is stored in higher concentrations than NE60 and has a lower association rate constant with AAT, suggesting that it is likely to have more prolonged activity than NE before inactivation, as demonstrated by mathematical modelling61 and in studies of airway secretions in both AAT-deficient (AATD) and non-deficient COPD62.Initially, NE was thought to be the most important proteinase in COPD. NE can be inhibited by a number of endogenous inhibitors, including alpha-1 anti-trypsin (AAT), secretory leucocyte proteinase inhibitor (SLPI) and α2-macroglobulin (α2M). However, at the time of NE release, its concentration (5.33 mM) is 15 to 1500 times greater than that of its inhibitors45;in vitro, there are increased levels of the NE footprint Aα-Val36063 and newly described PR3 footprint Aα-VAL54164 in plasma from patients with COPD.Reduced migratory accuracy of neutrophils in COPD may have implications for disease pathology because of the increased area of obligate tissue damage caused by proteinase release during poorly directed migration. In keeping with this, previous work has shown increased fibronectin degradation by migrating COPD neutrophils65, although similar studies using single chemokines as the chemoattractant do not replicate this finding66. As neutrophils and monocytes are derived from the same bone marrow precursor cells, this may reflect a common genetic/epigenetic or inflammatory cause. It remains unclear when neutrophil migratory dysfunction develops in COPD: during maturation in the bone marrow or following release into the circulation. If monocyte migration were found to be similarly impaired in COPD, this might suggest that the defect lies in bone marrow precursor cells.Of note, recent studies suggest that migration of monocytes from COPD patients to COPD sputum is also impaired67, and selective class I PI3K-δ or PI3K-γ inhibitors improve neutrophil migratory accuracy. It is possible that the poor migratory accuracy seen with age is exaggerated in patients with COPD, and inhibition of specific PI3K isoforms may offer a novel strategy to improve bacterial clearance and reduce tissue damage in COPD.COPD is more commonly seen in older patients and neutrophil migratory accuracy also declines with “healthy” ageing, a phenomenon also associated with constitutive PI3K activity69. Recently, the proteinase/anti-proteinase theory of COPD was revisited in a study which elegantly demonstrates a role for exosomes  in promoting NE activity, effectively tipping the local protease/anti-protease balance within the lung to favour tissue damage70. The authors describe a population of exosomes released from neutrophils which bind NE when it is released during degranulation, before its diffusion into the tissues. Unlike free enzyme, NE bound to these exosomes was found to be resistant to inhibition by AAT and to be able to bind to the extracellular matrix (via mac-1) whilst maintaining NE activity against the extracellular matrix proteins. These neutrophil-derived exosomes were found in clinical specimens from subjects with COPD but not healthy controls and importantly were capable of transferring a COPD-like phenotype from humans to mice70. Certainly, there is evidence of increased degranulation in COPD, and CD63 (a marker of primary granules) is found to be increased on the surface of unstimulated neutrophils from patients with COPD71.The proteinase/anti-proteinase theory of COPD suggests that damage to the lung tissue occurs when the levels of anti-proteinases in the lung are insufficient to effectively neutralise the proteinases present72, which, alongside the effects of cigarette smoke on mucosal cilia, reduces mucociliary clearance74. Mucus is able to build up in the airways, contributing to further obstruction, increasing the risk for bacterial colonisation and further inflammation75.Serine proteinases are potent stimulators of mucus secretion from submucosal and goblet cells of the airways77. Furthermore, the abundance of NETs within sputum has been shown to correlate with severity of airflow limitation assessed by FEV178 and overall severity of COPD using a composite scale including symptoms and exacerbation frequency alongside FEV178. Interestingly, the most recent study of NETs in COPD shows a correlation between NET complexes in sputum and microbial diversity, in particular a dominance of haemophilus species, whereby more than 40% haemophilus species within the lung microbiome were found to be associated with significantly greater DNA-elastase complexes78. Despite this, neutrophils isolated from the blood of patients with exacerbations of COPD have been shown to have a reduced ability to undergo NETosis compared with both stable patients and healthy controls, despite the increased presence of cell-free DNA in plasma79. This seems counterintuitive but it is possible that the clearance of NETs by DNases80 is impaired in COPD or that only a proportion of cells are able to produce NETs (a phenotype of cell) but this remains unknown.Recently, there has been significant interest in the role of NETs in COPD, but the current evidence is conflicting. Increased quantities of NET components have been described in the sputum of both stable and exacerbating COPD patients, alongside an increased proportion of “NET producing” neutrophilsHaemophilus influenzae andStreptococcus pneumoniae), fungi81 and apoptotic neutrophils via efferocytosis84. However, little research to date has focused on the phagocytic ability of neutrophils, and data so far provide conflicting results; some demonstrate no differences in phagocytosis87 and others suggest a reduction88. However, few studies have used phagocytic targets relevant to disease pathology, and studies in macrophages suggest that the use of non-physiological targets may provide misleading results. For example, phagocytosis of synthetic beads by macrophages was found to be unaltered in COPD, but bacterial studies showed a reduction in phagocytosis of the disease-relevant bacteriaH. influenzae andS. pneumoniae82, suggesting that the results of studies using non-physiological targets may be misleading88. This gap in knowledge clearly needs detailed exploration.Reduced phagocytic function of macrophages in COPD is well described, encompassing impaired phagocytosis of disease-relevant bacteria  or whether these changes are also seen in other diseases of the airways.96.AAT is a 52-kDa glycoprotein produced by hepatocytes but also macrophages and neutrophils and (as stated) functions as a serine protein inhibitor, providing essential protection of the lung tissue against the proteolytic actions of enzymes such as NE and PR3. In health, there is a constant diffusion of AAT into the lung, which is increased in the presence of inflammation (such as during respiratory infections). AATD is a genetic disorder in which the gene encoding AAT is mutated. There are many subtypes of AATD but in the most common severe form of deficiency (named PiZZ) this leads to mis-folding of the protein product, retention of AAT in AAT-producing cells and the formation of protein polymers in these cells, which causes damage and low circulating levels of AAT. AATD is the only robustly established genetic risk factor for the development of COPD and emphysema, and these disease processes can occur in patients with AATD, even in the absence of cigarette smoking97, and pulmonary disease is thought to develop, in part, from an imbalance of proteinases and AAT, although AAT has many non-proteolytic functions which protect against infection and inflammation, including immunomodulation and anti-microbial activity. It is well known that AAT deficiency is associated with a reduced ability to neutralise NE and PR3 adequately, leading to more tissue damage. In response to NE activity, epithelial cells and macrophages also release pro-inflammatory mediators such as CXCL898 and leukotriene B4 (LTB4)99, respectively. This chemoattractant production is perpetuated, further increasing neutrophil influx and increased NE activity within the lung, forming a vicious cycle of damage. In keeping with this, the inflammation present in AATD  is amplified when compared with non-AATD COPD with a similar burden of disease100 and this may influence immune function by cell priming or activation.Neutrophils play a central role in the pathophysiology of emphysema associated with AATD101 but these cells do not appear to be just “reactive responders”, and a number of studies have described abnormal neutrophil behaviour in AATD. A recent study described increased apoptosis of AATD neutrophils102. The authors proposed that this might reflect endoplasmic reticulum stress owing to the accumulation of mis-folded AAT within the neutrophil102. However, augmentation therapy, in which deficient AAT is “replaced” with purified plasma AAT from healthy individuals, was able to normalise cell apoptosis without altering endoplasmic reticulum stress markers, and apoptosis was a direct result of low circulating AAT. Internalised AAT is known to co-localise with and inhibit staurosporine-induced caspase-3 activation103, a potent signal for apoptosis recently described in neutrophils104.Lung neutrophilia has been much easier to demonstrate in AATD compared with non-AATD COPDper se102. AAT augmentation bothin vitro andin vivo could restore the bacterial killing capacity of ZZ-AATD neutrophils to that of non-deficient neutrophils but again this might reflect reduced apoptosis102. AAT is known to improve phagocytosis by both human alveolar macrophages (AMs) from patients with non-AATD COPD and AMs isolated from mice exposed to cigarette smoke105. This improvement included both efferocytosis (clearance of dead neutrophils) and phagocytosis and was associated with the upregulation of efferocytosis and scavenger receptors on the AM plasma membrane105. These receptors were also shown to be upregulated in patients with AATD following double-dose augmentation treatment with purified AAT compared with a single dose, suggesting that a similar mechanism to enhance efferocytosis may existin vivo.The same authors propose defective bacterial killing by AATD neutrophils but this appeared to result from accelerated neutrophil apoptosis rather than an intrinsic defect in neutrophil phagocytosis4 in sputum rather than the ability of neutrophils to migrateper se106. A further study demonstrated no difference in the ability of neutrophils from patients with AATD to migrate to a standard chemoattractant (CXCL8) compared with healthy controls despite finding reduced migratory accuracy of neutrophils from patients with COPD40. There is an increased burden of ROS in AATD, and AAT modulates neutrophil O2− production elicited byN-formylmethionine-leucyl-phenylalanine (fMLP) and CXCL8 in a dose-dependent manner107. However, the burden of ROS in AATD may be multi-faceted, and AAT is known to bind to a number of products with oxidative potential, including hemin108. There are few studies of NET formation in AATD. One study using the non-physiological stimulant phorbol myristate acetate (PMA) reported that AAT did not reduce NET formation from neutrophils isolated from patients with PiZZ AATD but this study has yet to be replicated using disease-relevant stimuli109.There are limited data regarding neutrophil migratory function in patients with AATD. One study has suggested that sputum from patients with AATD has greater chemotactic activity which likely relates to increased levels of neutrophil chemoattractants CXCL8 and LTB111, reflecting differences in pathophysiology between the two conditions. 18-fluorodeoxyglucose (18FDG) positron emission tomography–computed tomography (PET-CT) studies generate both quantitative and spacial data regarding pulmonary glucose uptake, which has been shown to relate to neutrophil activity in animal models113. When these studies were performed in patients with COPD,18FDG uptake was shown to be greater in emphysematous regions of the lung and correlated with physiological measures of disease severity114. Despite this, the increased pulmonary18FDG demonstrated in non-AATD COPD was not demonstrated in a small cohort of patients with AATD, in whom18FDG uptake was comparable to that of healthy controls114. This suggests important differences in the pathogenesis of emphysema in these two conditions, in particular with respect to the role of the neutrophil.To date, there are no studies of neutrophil phenotype in PiZZ AATD to determine whether distinct patterns are seen, but when the data are considered together, it appears that AATD is not merely an exaggerated form of non-AATD COPD and there appear to be differences in cellular function between the two groups. This is highlighted in clinical and imaging studies. The predominant phenotype of emphysema observed in non-AATD COPD is typically apical centrilobular but this differs in AATD, where emphysema is predominantly basal and panlobular115. Furthermore, patients with AATD experience clinical phenotypes similar to those of patients with non-AATD COPD115. Also, although AAT augmentation reduces the progression of lung disease in some patients, it has little impact on others, highlighting the fact that replenishing the deficient anti-proteinase is not enough to treat disease and more studies are needed to assess the utility of targeting the neutrophil in conjunction with augmentation strategies.Also, lung disease is heterogeneous in AATD, even in patients with PiZZ AATD who have never smoked. A proportion of never-smoking patients with similar deficiency levels do not develop lung disease although some do, and in those who do, rates of decline are variable and currently cannot be predicted at baseline screening118. Bacterial colonisation with potentially pathogenic micro-organisms is extremely common, and neutrophils are thought to play a fundamental role in bronchiectasis pathogenesis, partially in response to this infection. Impaired mucus clearance and recurrent infections cause rich sputum levels of potent neutrophil chemoattractants, including interleukin 1 beta (IL-1β), tumour necrosis factor alpha (TNFα), CXCL8, and LTB4119. Consequently, neutrophils dominate cell populations in both the sputum and bronchoalveolar lavage fluid of patients with bronchiectasis, and neutrophil counts positively correlate with bronchiectasis disease severity120. This heightened inflammation impacts on neutrophil function. Systemic neutrophils from individuals with bronchiectasis have a higher level of baseline activation compared with healthy individuals, as indicated by increased CD62L and CD11b120. Furthermore, blood neutrophil viability is significantly prolonged because of delayed apoptosis (a feature of inflammation) and these neutrophils release more myeloperoxidase (MPO) when unstimulated in more severe forms of the disease (suggesting constitutive priming and activation)120. Systemic neutrophils seem to retain their phagocytic and anti-microbial ability compared with airway counterparts120. Airway neutrophils in bronchiectasis exhibit impaired phagocytosis of pathogens, includingPseudomonas aeruginosa (PAO1), contributing to recurrent infections120. However, this appears to improve with antibiotic therapy120. In a study of 103 adults with bronchiectasis, the most frequent immune cell abnormality was reduced neutrophil oxidative burst121 but there was significant heterogeneity. A comprehensive screen of immune function confirmed that 13 subjects had low levels of IgG3, six had low levels of B-cell lymphocytes and seven had low T-helper cell lymphocytes when compared with controls. All subjects had a normal neutrophil phagocytic function, but 33 of the subjects had an oxidative burst that was below that seen in health121. In addition, airway neutrophils in bronchiectasis exhibit higher necrosis and impaired cell death as well as reduced clearance by macrophages, delaying inflammation resolution and causing persistent inflammation and further airway damage120. Furthermore, increased neutrophil degranulation causes further airway damage and correlates with worse clinical outcome122.Rather than being a pathological entity (such as AATD), bronchiectasis is a chronic lung condition caused by a number of pathological insults  characte123. Recently, neutrophils from patients with PCD have been shown to display reduced migration toward CXCR2 ligands (CXCL5 and CXCL8) but not to LTB4 and complement component 5a. The reduced response to CXCL8 was observed in all subgroups of patients with PCD and correlated with lung function, and CXCR2 expression was downregulated on the cell surface in about 65% of the patients with PCD124. However, in non-PCD bronchiectasis, neutrophil migration appears preserved125, and a trial of a CXCR2 antagonist given orally for 28 days resulted in about a 70% decrease in the percentage of sputum neutrophils, suggesting that CXCR2 ligands were strong drivers of neutrophil accumulation in the lung126.Although these studies highlight themes of neutrophil function and dysfunction across bronchiectasis, the diverse causes of disease may display different patterns. For example, primary ciliary dyskinesia (PCD) is a rare genetic disease caused by abnormal structure or function of motile cilia (or both) which leads to bronchiectasis127, a strategy that has not changed since bronchiectasis was first characterised in the 1950s. Despite advances in understanding the pathophysiology of bronchiectasis, the rates of exacerbation and mortality amongst patients with bronchiectasis have shown little improvement128. Therefore, further research is needed to understand how to prevent disease progression and to develop therapeutic targets accordingly but this may require better stratification of the cause of bronchiectasis and a diverse treatment strategy.The combination of infection and inflammation suggests that both anti-microbial and anti-inflammatory agents might help with disease management. Currently, the two main treatment options for bronchiectasis involve physiotherapy for clearance of mucus and antibiotics for treatment of infectionsCTFR) gene affects mucociliary clearance117. CF is the most common inherited disorder in the Caucasian population, affecting 1 in 2000 live births129, and a common cause of bronchiectasis, which is often more severe and progressive than non-CF bronchiectasis. With CF bronchiectasis, as with non-CF bronchiectasis, neutrophil dysfunction has been described. In particular, airway neutrophils in patients with CF exhibit a functional exhaustion and a pro-survival phenotype130, potentially reflecting the high levels of inflammation and structural damage present in the lung.CF is an autosomal recessive disease whereby a loss-of-function mutation in the CF transmembrane conductance regulator  is increased compared with both patients with non-neutrophilic asthma and healthy controls145. However, whether defects in neutrophil function are intrinsic or are a consequence of—and perhaps contribute to—heightened systemic and airway inflammation remains unclear.Asthma is a chronic inflammatory lung disease that affects 340 million people worldwide and accounts for 180,000 deaths worldwide every year asthma , and for136, highlighting the delicate balance between protective and destructive functions of neutrophils in airway disease, a common feature across all of the diseases we have considered.Of note, although neutrophils are associated with tissue damage in asthma, they have also been shown to have a role in controlling inflammation, restoring tissue homeostasis and promoting tissue repairThe data presented highlight many similarities in neutrophilic inflammation across airway diseases. First, an airway neutrophilia is common. Second, there are often markers of neutrophil degranulation and in particular ROS and proteinase activity which are associated with disease presentation and progression. Third, aspects of neutrophil function appear altered. Although this seems to most commonly affect migration and ROS production, studies also highlight aberrant NETosis and phagocytosis, although defects are variable. The commonality of neutrophil inflammation across different diseases might suggest common underlying mechanisms of effect, and studies have suggested potential themes as to how this might occur.146. Furthermore, TNFα can impact on cellular functions as it is a potent priming agent and able to increase ROS production by neutrophils, which will further contribute to tissue damage147. Second (and more speculatively), the inflammation present across diseases might impair the ability of the lungs to “de-prime” cells29, leading to a circulating population of primed cells, which might confer a more aggressive cellular phenotype. A third putative theme is that of altered cellular subtypes. This has only recently been described in COPD, but other studies suggest that neutrophils change in response to signals such as inflammation, hypoxia or physical pressure, resulting in different functional phenotypes. These changes are subtle and may relate to the immediate cellular environment, as described in mice models of cancer147 and pro-inflammatory culture conditions148.First, inflammation might impact on neutrophil functions irrespective of the initial insult . For example, TNFα—shown to be increased in airway secretions from patients with COPD, asthma, bronchiectasis and AATD—is able to increase the expression of capture receptors and adhesion molecules on the surface of blood vascular endothelial cells, enhancing neutrophil migration into the inflamed lungAlthough modifying neutrophilic inflammation is an attractive interventional strategy, neutrophils are a challenging target and one that comes with risks associated with neutropenia or excessive inhibition of neutrophil host defence mechanisms against infection. There are two potential therapeutic avenues to explore.4 are the dominant chemokines thought to be responsible for this149 and drugs targeting these mediators have been trialled in inflammatory airway disease. Clinical studies of a CXCR2 agonist (MK-7123) led to improvements in lung function and reduced exacerbations in active-smoking patients with COPD compared with placebo treatment150, but a large proportion of patients experienced neutropenia, raising concerns about immunosuppression. As discussed earlier, in patients with bronchiectasis, the CXCR2 antagonist AZD5069 resulted in a 70% decrease in the percentage of sputum neutrophils but this was not associated with improved clinical outcomes126. A small phase II trial investigated the effects of a leukotriene synthesis inhibitor, reducing LTB4 production, on bronchial inflammation in patients with stable COPD, showing some benefit151. However, a randomised placebo-controlled trial of an LTB4 receptor antagonist (BIIL 284 BS) in patients with CF was terminated early because of serious adverse effects, including increased respiratory symptoms requiring intravenous antibiotics and hospitalisation, reduced pulmonary function and increased circulating neutrophil numbers152, suggesting that LTB4 antagonism may result in acute pulmonary exacerbations and heightened inflammation152, although the mechanisms for this were poorly understood. An alternative strategy may be to target associated co-morbid conditions which contribute to the inflammatory load. For example, the treatment of periodontitis, a chronic inflammatory condition associated with neutrophilic inflammation and recruitment which shares many inflammatory features of COPD153, has been shown to improve changes in both lung function and exacerbation frequency in COPD154.The first is to target the chemoattractants responsible for neutrophil recruitment into the lung. CXCL8 and LTB155. A number ofin vitro studies have investigated strategies to improve the accuracy of neutrophil migration, thereby theoretically reducing the potential for migration-associated and protease-mediated tissue damage. Broad-range inhibition of PI3K signalling has been shown to restore migration of COPD neutrophils to levels similar to those of neutrophils from age-matched healthy controls40. However, broad-spectrum inhibition of PI3K therapeutically is likely to lead to significant side effects. Selective inhibition of class I PI3K-δ and PI3K-γ, which are enriched in leucocytes156, may offer a more acceptable therapeutic option; indeed, selective isoform inhibition of PI3K-δ and PI3K-γ has been shown to restore reduced migratory accuracy of neutrophils from healthy older adults67. Impaired migration in COPD is hypothesised to be a further exaggeration of age-related impairment in neutrophil migration, emphasising the potential of this strategy in COPD, but whether PI3K inhibition provides benefitin vivo or in other inflammatory airway diseases requires further investigation. Simvastatin is a safe and well-tolerated drug commonly used for its cholesterol-lowering abilities. Population studies and clinical trials suggested a survival benefit for patients taking statins during infection157, which prompted interest in the ability of these drugs to modulate immune function.In vitro simvastatin treatment has been shown to have beneficial effects on migration of neutrophils from patients with COPD158 and during pulmonary infection in otherwise healthy older adults but not during more severe infection or sepsis159. These beneficial effects on migration were replicated in a clinical trial of high-dose simvastatin in healthy older adults160. Similarin vivo studies are required to determine whether effects are maintained in a disease setting, but a clinical trial is currently under way161 and outputs are expected this year. Other commonly used drugs may also provide mechanistic insight into how neutrophil could be targeted. Aspirin induces resolvin-D signalling, which has been associated with improved pneumonia outcomes in murine models162, and aspirin is associated with improved survival in observational studies of pneumonia163. Metformin, commonly used for glycaemic control in diabetes, has also gained interest as a potential means to target neutrophil functions, potentially modifying chemotaxis and bacterial killing through 5′ adenosine monophosphate-activated protein kinase (AMPK) activation164.The second therapeutic option is to directly modulate neutrophil function166 but these models do not recapitulate all features of human disease. Effective clearance of apoptotic cells, via efferocytosis, is vital to prevent secondary necrosis and release of damaging pro-inflammatory cell contents which may heighten inflammation and contribute to further tissue damage167. However, in human studies, clearance of apoptotic cells has been shown to be reduced in many inflammatory airway diseases, including COPD84, asthma169, CF and bronchiectasis170. As such, induction of neutrophil apoptosisin vivo, without improvement of clearance mechanisms, needs to be approached with caution and may have the potential to cause more harm than good.Targeting local airway neutrophil apoptosis has also been suggested, and induction of airway neutrophil apoptosis reduced airway inflammation in mouse modelsThe final challenge is effective delivery of the desired drug to its target without impacting host defence against infection owing to neutropenia or excessive impairment of neutrophil function. Inhaled therapies may permit effective targeting of airway neutrophils whilst minimising systemic side effects; indeed, an inhaled PI3K-Δ inhibitor is in clinical trial for the treatment of COPD exacerbations . The key would be to ensure penetration of the inhaled compounds into the smaller airways and newer devices offer the promise of these effects.171 which are variable but impact on function and the resultant changes appear long-lived172. This might provide a mechanism for the self-perpetuating inflammation present across many airway diseases. It might be that chronic inflammation leads to epigenetic reprogramming of neutrophils, which alters their phenotype or responses. The physical damage to the lung infrastructure and especially the pulmonary vasculature might compound this by inhibiting de-priming. Unfortunately, the current evidence base for understanding neutrophil function across diseases is limited  but this is certainly worthy of more study. To ascertain whether there are shared mechanisms of neutrophil dysfunction across disease and more importantly how these might be targetable will require collaborative research across current disease silos.Neutrophilic inflammation is a common feature of many airway diseases and is associated with disease progression, often irrespective of the initiating cause or underlying diagnosis. This provides a potential therapeutic target, but the target is a challenging one. The crucial role of neutrophils in clearing bacteria means that merely inhibiting their responses in a blunt or indiscriminate fashion is likely to be detrimental to the host, as demonstrated by the manifestations of neutropenia. Targeting neutrophils requires a more subtle approach. Neutrophils appear to be susceptible to epigenetic changes


The two other insulin genes, insa and insb, have been retained but show differing levels of selective constraint suggesting that they might have diversified in function. Intriguingly, a duplicate copy of the insa gene, which I named insab, is found in many fish. The coding sequence encoded by insab genes is under weak selective constraint, with its predicted protein sequences losing their potential to be processed into a two-peptide hormone. However, these sequences have retained perfectly conserved cystine residues, suggesting that they maintain insulin’s three-dimensional structure and therefore might modulate the processing and secretion of insulin produced by the other genes.Insulin is a key hormone for the regulation of metabolism in vertebrates. Insulin is produced by pancreatic islet cells in response to elevated glucose levels and leads to the uptake of glucose by tissues such as liver and adipose tissue to store energy. Insulin also has additional functions in regulating development. Previous work has shown that the proglucagon gene, which encodes hormones counter regulating insulin, is duplicated in teleost fish, and that the peptide hormones encoded by these genes have diversified in function. I sought to determine whether similar processes have occurred to insulin genes in these species. Searches of fish genomes revealed an unexpected diversity of insulin genes. A triplication of the insulin gene occurred at the origin of teleost fish, however one of these three genes,  Oreochromis niloticus) compared to mammalian sources  and takifugu (Takifugu rubripes) only showed expression in the embryo of zebrafish  ins2 gene showed that it had negligible expression in the pancreas, thus likely is not a major contributor to the regulation of glucose metabolism and would not need to be silenced to allow the xenotransplantation of tilapia islets as a treatment for diabetes in humans  and Ensembl (http://www.ensembl.org) were searched in March/April 2018, for sequences that were predicted to encode proteins similar to proinsulin. Initial searches used the tBLASTn algorithm (Danio rerio) proinsulin protein sequences , 2 cartilaginous (Superclass Chondrichthyes), and one lobe-finned (Class Sarcopterygii)) were examined, with 52 of these contained in the NCBI Genome Data Viewer, and three  only in the Ensembl database (9 were in both NCBI and Ensembl). A list of species with genomes used here, and their phylogenetic relationships, is presented in igf1) or insulin-like growth factor-II (igf2) were used for the following analyses. Here I suggest a standardized set of names for fish ins genes, based on the analysis described below, with insa, insb, and insc representing the three ins paralogs that originated early in teleost fish evolution, and insaa and insab representing duplicates of the insa gene shared by multiple species. Arabic numerals are used for recent lineage-specific duplicates and do not indicate necessarily indicate orthology.Genome sequences databases maintained by the National Center for Biotechnology Information (NCBI: lgorithm  using prequences  as queriins) coding sequences were aligned using the MAFFT algorithm  and Rhincodon typus ) and lobe-finned fish ) were used as outgroups to root the trees for all insulin coding sequences. Subsequent analyses, using subsets of the sequence data, were conducted with similar approaches, and used sequences identified in our initial analyses as outgroups.Prior to phylogenetic analysis, proinsulin (lgorithm  as impleGenes adjacent to insulin genes in fish genomes were identified by examining genomic contigs in the NCBI and Ensembl databases using methods that I have previously used . BrieflyBLASTp searches of the zebrafish and human proteomes (from the genomic databases. The orthology of the genes adjacent to insulin genes was assessed (or confirmed if annotated) using roteomes .dN) and synonymous substitutions (dS). Sequences under stronger selective pressure for protein function will have lower nonsynonymous to synonymous (dN/dS) ratios. dN/dS rate ratios were obtained from analyses using RELAX  to 8 (Sinocyclocheilus anshuiensis). As expected, fish species that experienced recent genome duplications, i.e., carp , 92 were classified as insa (which includes insaa and insab genes), 57 as insb, and 13 as insc, with 11, 12, and 3 of the sequences found to be incomplete, respectively, for these three paralogs  genes used in our analysis described below are presented in Supplementary Figure S1.Searches of genome databases maintained by NCBI and Ensembl resulted in the identification of 168 insulin genes from the genomes of 55 species examined, with 141 of these predicted to be intact full-length coding sequences  genes found in the genomes of ray-finned fish, their phylogenetic relationships, rooted using ins gene sequences from cartilaginous and lobe-finned fish, were inferred using maximum likelihood  and teleost (infraorder Teleostei) fish lineages. While duplicated insa and insb genes were previously identified in teleost fish , and the genera Sinocyclocheilus)). In contrast, a large number (30 of 51) of teleosts have multiple insa genes  or single-gene duplications , many are due to a duplication event that occurred on an early Percomorpha lineage , some cichlids , and species of the genus Poecilia do not have an insab gene  originated on this lineage. If duplicate genes originated through a genome duplication, then one might expect similar genomic neighborhoods for these genes , as observed for the duplicated proglucagon (gcg) and glucagon receptor (gcgr) genes of teleost fish (tenm2 (teneurin transmembrane protein 2) was found adjacent to insa genes in Exox lucius, O. kisutch, O. mykiss, Paramormyrops kingsleyae, Salmo salar, and Sclerophages formosus, and insb genes in Pygocentrus nattereri, Salvelinus alpinus, and Sinocyclocheilus rhinocerous (Supplementary Table S2). This might suggest that the insa and insb genes originated via the teleost-fish specific genome duplication. However, as the linkage between the tenm2 and ins genes was only observed in a small number of species, this would require large number of independent losses (or genomic rearrangements) of tenm2-like genes to yield the absence of a linked tenm2-like with most ins genes, therefore the evidence for the origin of insa and insb by a genome duplication event is not strong. No genes near insc were similar to those adjacent to insa or insb, thus it more likely originated as a single gene duplication event rather than through a genome duplication. If the insa and insb genes originated via a genome duplication, an addition single-gene duplication would still be needed to explain the origin of insc. The gene neighborhood data does not help resolve the relationships among the three ins genes. Intriguingly, the genomic neighborhoods of none of the insulin gene paralogs in teleost fish is similar to the typical vertebrate insulin gene neighborhood, where insulin is flanked by insulin-like growth factor 2 (igf2) and tyrosine hydroxylase (th) genes  genes , althouginsb and insc genes were examined -like gene, while all insc genes are flanked by prr12 (proline rich 12) and scaf1 (SR-related CTD associated factor 1)-like genes -like gene adjacent to the insa gene (Supplementary Table S2) and is found in species representing Osteoglossomorpha , Paracanthopterygii , and Percomorpha . The only major group of teleost fish that does not have an insa gene adjacent to nipal are those of Otomorpha , the insaa genes are found adjacent to nipal-like genes -like genes  for the insab gene that was inserted into a new genomic locus.Orthologous genes often have similar genomic neighborhoods, thus can these neighborhoods can be used to assess orthology when gene sequence similarity is limited . When thinsa1 and insa2 genes in Parmormyrops kingsleyae and in Larmichthys crocea, the insaa1 and insaa2 genes in Hippocampus comes, and the insab1 and insab2 genes in Boleopththalmus pectinirostris. Both Cyprinus carpio and Sinocyclocheilus rhinocerous have tandemly arranged insb genes  that might have originated prior to the divergence of these two species . Sinocyclocheilus anshuiensis has two pairs of tandemly arranged genes, insa1/insa2 and insa3/insa4, which either originated prior to this species genome duplication (and then were lost in the closely related species that share this genome duplication), had parallel tandem duplications, or is an assembly error . The ins1 and ins2 genes of Lepisosteus oculatus might also have originated via a tandem duplication, which was then followed by rearrangements so that these lineage-specific gene duplicates are now found in opposite orientations on either side of a hmmr-like gene (see Supplementary Table S2). More complex duplication events, resulting in genes moving to new locations are need to explain the remaining genes, with some being extremely recent  or more ancient .The gene duplication events described above explain most, but not all, of the insulin genes identified (ins1 gene in mice was generated from an incompletely processed mRNA transcript (ankrd6 (ankyrin repeat domain 6) and lyrm2 (LYR motif containing 2)-like genes on the Lepisosteus oculatus lineage to generate ins; (2) within an apbb3 (amyloid beta precursor protein binding family 8 member 3)-like gene on the Monopterus albus lineage to generate insb; and (3) within a psd3 (PH and SEC7 domain containing protein 3)-like gene on the ancestral lineage for salmonids  and the Northern pike (Esox lucius) that generated insa in O. kisutch, S. salar, and S. alpinus (but was not found in O. mykiss) and an intact coding sequence (insa2) in Esox lucius  suggests that this retropositioned sequence, like the mouse ins1 gene (insb4 from Cyprinus carpio and insa3 from Paramormyrops kingsleyae) were identified (see Supplementary Table S1) that were located at unique location of the genome (see Supplementary Table S2) consistent with being inserted processed cDNAs, but the sequences were similar to either exon 1 or 2 (and not both), thus cannot be distinguished from a sequence generated by an DNA-mediated incomplete gene duplication event.While duplication of genomic DNA is a frequent mechanism for the origin of duplicate genes, they can also be generated by retroposition of cDNA generated from mRNA transcripts . Indeed,anscript . Typicalns1 gene , is stildN) to synonymous (dS) substitutions . If the protein encoded by these genes still had a function, then selection should act against a subset of nonsynonymous substitutions (the deleterious mutations) and be lower than the synonymous rate . Rarely, one might see dN/dS>1, which would indicate positive selection for change in amino acid sequence  for each of the three insulin gene paralogs, and to determine whether the selective pressure was intensified or relaxed compared to the other insulin genes, with sequences from a non-teleost ray-finned fish, Lepisosteus oculatus, used as outgroup. When all sequences were analyzed, insa genes showed the stronger levels of purifying selection  driven by positive selection, while insa and insb have been retained due to subfunctionalization of insulin functions between the two genes.While the protein products of the three insulin genes are being maintained for function, they might not be the same function. To examine whether any of the sequences might have gained new functions I tested for evidence of positive selection on lineages using aBSREL . Only oninsa, insb, and insc paralogs was that the calculated value for the selective constraint acting on insa varied considerably depending upon which sequences were used for the analysis  had a cystine replaced by an arginine residue. It is possible that the S. graham insc2 gene does not encode a functional insulin, but this is compensated by the presence of an insc1 gene that encodes an insulin that can be secreted processed and has all six cysteine residues. Surprisingly, when I examined A- and B-chain sequences homologous to those of other insulins in the protein encoded by insab genes, all of the cysteine residues were perfectly conserved, however much of the rest of the sequence showed greater variation than within other types of insulins  genome, unexpectedly revealed the existence of three insulin gene paralogs  genome (insc gene in both the Ensembl (gene ID: ENSDARG00000096862) and NCBI (GenBank accession No.: 100534937) zebrafish genomes fail to predict an intact open reading frame, with the intact insc coding sequence only coming from the linked mRNA sequence (XM_009299616.3) in the NCBI database (see Supplementary Table S1). I could find no raw sequence data  that supports the existence of an intact open reading frame for zebrafish insc, as all mRNA derived sequences  also contained the deletion found in the genomic sequence. This suggests that the zebrafish insc gene does not encode an intact protein product. However, our analysis of the selective forces acting upon the insc coding sequences indicate that the nonsynonymous rate of substitutions (dN) is much lower than the rate of synonymous substitutions (dS) consistent with selection to maintain a protein coding sequence  that was identified in zebrafish, is found in the “gut and internal organs” of adults, thus potentially it could be expressed in the pancreas and be replaced by insa. Further studies on the expression of insc in other fish are needed to identify its role in physiology.Unexpectedly I found an sequence  in most,Searches of ray-finned fish genomes have demonstrated that they contain more insulin genes than previously appreciated and suggest that the roles of these genes have diversified. The diversification of the functions of insulin parallels the diversification of the proglucagon-encoded peptides found in fish . Similar


Sleeping Beauty and piggyBac transposons for the integration of transgenes and genetic screens, and CRISPR-cas9 for generating genetic knockout and functional screens. Applications of these technologies are providing novel insights into the functional genetic drivers of gliomagenesis, how these genes cooperate with one another, and the potential cells-of-origin of gliomas, knowledge of which is critical to the development of targeted treatments for patients in the clinic. The most common brain tumours, gliomas, have significant morbidity. Detailed biological and genetic understanding of these tumours is needed in order to devise effective, rational therapies. In an era generating unprecedented quantities of genomic sequencing data from human cancers, complementary methods of deciphering the underlying functional cancer genes and mechanisms are becoming even more important. Genetically engineered mouse models of gliomas have provided a platform for investigating the molecular underpinning of this complex disease, and new tools for such models are emerging that are enabling us to answer the most important questions in the field. Here, I discuss improvements to genome engineering technologies that have led to more faithful mouse models resembling human gliomas, including new cre/LoxP transgenic lines that allow more accurate cell targeting of genetic recombination,  Gliomas are the most common intrinsic brain tumours, causing considerable morbidity and mortality. Given that the prognosis of malignant gliomas has not substantially improved in the last few decades, strong efforts are being made into understanding the genetics and biology of this disease in order to design targeted therapies against oncogenic pathways. Although whole-genome sequencing studies of human tumours have provided some insight into the genetic make-up of gliomas in the last few years, disentangling functional driver genes from other ‘passenger’ mutations in these tumours requires complementary studies. Genetically engineered mouse models (GEMMs) of gliomas are powerful experimental tools that can provide new insights into their genetic drivers and molecular mechanisms, in addition to providing resources for preclinical drug testing. Compared with in vitro systems and xenografts in immune-deficient mice, GEMMs in immune-competent mice have the major advantages of being able to model interactions between tumour cells and the microenvironment including the vasculature and immune cells. Such interactions are known to have a critical role in gliomagenesis ,2,3. A sDiffuse gliomas may be classified into low- and high-grade gliomas, and include oligodendrogliomas, astrocytomas, and glioblastomas. Low-grade gliomas  are a heterogeneous group of intrinsic brain tumours with a tendency for progression to higher-grade tumours. These tumours contain cells with similar histological appearances as glial cells, including astrocytes and oligodendrocytes. LGGs constitute 15% of all adult brain tumours, and they most commonly present with seizures (in 80% of cases) . A modelGlioblastoma (GBM), or high-grade glioma (HGG), is the most common malignant intrinsic brain tumour, and characteristically invades surrounding brain aggressively, making complete surgical resection of all cancerous cells impossible. The disease tends to affect middle-aged to elderly people and can either arise de novo (primary glioblastoma) or by transformation from an LGG (secondary glioblastoma). Its prognosis is therefore poor, with a median survival of only 14 months despite maximal therapy with surgery and chemo- or radiotherapy . These sMGMT, MSH2, and MSH6 [Temozolomide, a DNA alkylating agent, is the mainstay of chemotherapy for GBM and has been shown in randomised controlled trials to improve prognosis by around two months when given with radiotherapy ,11. Giveand MSH6 . However and phosphatase and tensin homolog  are the most commonly mutated in GBM, although mutations in NF1 and RAS have also been documented. Mutations within this pathway tend to enhance cellular proliferation. Of the p53 pathway, TP53 (25%) itself is most commonly mutated in GBM. The TP53 gene is normally activated following DNA damage to cells, inducing transcription of genes whose ultimate effects include apoptosis. Mutations in TP53 are thought to have effects such as inhibition of apoptosis, stimulation of cell proliferation and neovascularisation, which are hallmarks of cancer [RB gene itself is infrequently mutated, and instead, mutations in cyclin-dependent kinase inhibitor 2A  are particularly common. CDKN2A is the locus for two tumour suppressor genes—INK4A and P19-ARF. In vitro and in vivo models have validated a number of such mutations as driving tumour growth and invasion. Mouse models have been particularly helpful in demonstrating how mutations in multiple pathways can co-operate together to accelerate tumourigenesis [Recent genome-wide sequencing studies of GBMs have provided insight into common genetic drivers of this tumour and have highlighted genetic differences between primary and secondary GBMs. Primary GBMs usually have one or more mutations in three main molecular pathways: Ras/RTK (receptor tyrosine kinase) pathway, p53 pathway, and the retinoblastoma (Rb) pathway ,16. Withf cancer . Althougigenesis . IDH1 (isocitrate dehydrogenase 1) mutation is characteristically found more commonly in secondary GBMs and also in LGGs [IDH1 mutant status, which predicts a better prognosis, and the presence or absence of 1p/19q co-deletions, TERT promoter mutations, and ATRX mutations [The  in LGGs , and alt in LGGs . The recutations ,22. A reutations . Figure utations . The majority of glioma mouse models have employed cre/LoxP technology for specifically targeting cancer genes in certain neural tissues of interest. I will therefore describe this and related technologies before discussing examples of glioma mouse models in more detail. Site-specific recombination enables the creation of genetic alterations . The first system to allow site-specific recombination in multicellular organisms was the flippase/flippase recognition target (Flp/FRT) system, and this was originally used in Drosophila . Here, tAnother system for producing targeted mutations in a tissue-specific manner is the RCAS  vector system. Such vectors originate from the Rous sarcoma virus, which belongs to the avian sarcoma-leukosis virus (ASLV) family, and these vectors contain the src (oncogene) splice site and express an inserted gene (such as an oncogene) via a spliced message. Limitations of the RCAS system include the small size of the insert (2.5 kb), and the modest number of cells typically infected and that express the transgene , and fornestin-cre. Nestin is an intermediate filament protein expressed in neural stem cells and neural progenitors. Mice carrying the nestin-cre allele express cre from embryonic day 13, at which stage the embryonic neural progenitors are able to undergo differentiation into many cell types including astrocytes, neurons and oligodendrocytes. Therefore, in postnatal and adult mice carrying nestin-cre, cre is expressed throughout most of the central nervous system, eye, and also the kidneys—this was demonstrated by Dubois and colleagues who showed virtually complete cre-mediated recombination in these tissues by embryonic day 15.5 using LacZ based reporters [nestin-cre allele is insufficient for driving recombination in early embryonic ventricular zone neural progenitors and neural stem cells (before embryonic day 17.5), as determined using multiple cre-dependent reporters including LacZ [hGFAP-cre , which is also expressed from pre-natal stages and in the majority of cell types in the brain and spinal cord [Olig2-cre allows site-specific recombination in all oligodendrocyte lineages, Syn1-cre gives recombination specifically in neurons, and Glast-cre is a newer alternative line for recombination in neural stem cells in the subventricular zone (SVZ). A number of cre lines have been utilised for the generation of glioma GEMMs. One such line is eporters ,31. Howeing LacZ . An altenal cord . In ordenestin-creERT2 (cre fused to a mutant oestrogen ligand binding domain) and GFAP-creERT2 are inducible cre lines used for this purpose. These are particularly helpful for studying tumour origins from adult brain cells as opposed to embryonic cells . The cre expression onset is controlled by specifying the age of the mice at which tamoxifen is administered. However, an important drawback of all of these cre lines is the specificity of the regions and cell types in which recombination occurs, in that there is typically recombination in other cells than those of interest. Inducible cre lines are invaluable for studies where the timing of recombination needs to be controlled, requiring tamoxifen injections to induce cre expression. For example, EGFR) gene [EGFR is particularly common, which is a deletion of exons 2–7 of the gene (the extracellular ligand binding domain) that promotes constitutive signalling from the resulting receptor. An early study, aimed at determining whether excessive EGFR signalling can induce gliomas in vivo, employed the RCAS vector system to introduce an Egfr activating mutation (the EgfrvIII deletion and second deletion that removes the intracellular regulatory kinase domain) in mice expressing the avian tumour virus receptor A (TVA) under control of brain cell specific promoters ). The vector was introduced into the frontal lobes and hippocampus. After 15 weeks, none of the mice developed gliomas. In contrast, when an Egfr activating mutation was introduced in the presence of Cdkn2a loss, gliomas arose at a high frequency particularly on the nestin-TVA (Ntv) background. The authors concluded that Egfr activating mutations alone are insufficient to generate gliomas but can cooperate with predisposing mutations such as those of Cdkn2a to produce these tumours [One of the earliest oncogenes to be discovered in gliomas is the epidermal growth factor receptor (FR) gene , which i tumours . Given tG12DKras mutation and a constitutively active Akt mutant were virally transferred into the brain of mice using RCAS vectors. Each of these genes was insufficient to induce gliomas when expressed on their own. However, lesions histologically resembling human GBMs were observed when these mutations were expressed in combination with one other. Although it was initially believed that neither Kras or Akt mutations are present in human GBMs, more recent large-scale sequencing efforts have revealed Kras mutations of these cancers at a low frequency [Another early glioma mouse model utilising the RCAS vector system was that by Holland and colleagues in 2000 , whereinrequency . MoreoveTP53 and PTEN are mutated in ~30% and 40% of human GBMs, respectively, making these amongst the most frequently altered genes in this disease. Zheng et al. hypothesised that loss-of-function mutations in these two genes cooperate in driving gliomagenesis [hGFAP-cre mouse with Trp53 mutant and Pten knockout mice. The resulting mice developed grade III and grade IV gliomas at a median latency of approximately seven months [TP53 and PTEN mutations are commonly found in human low-grade gliomas as well as GBMs, all of the tumours generated in these mice were either grade III or IV. These findings were supported by data showing cooperation of Trp53 and Pten with Nf1 in another glioma GEMM derived from adult neural stem cells [The tumour suppressor genes agenesis . To expln months . Gliomasem cells . EGFRvIII mutation and other genes in gliomagenesis by also using transgenic mice [EGFRvIII transgenic mouse, in which this mutation was overexpressed at the Col1a locus, and an EGFR wild-type transgenic mouse with the human EGFR gene sequence inserted and over-expressed at the Col1a locus. These conditional transgenic mice required injections of cre into the brain for expression of the transgenes (cre was injected into the striatum of adult mice). Neither of these mutations was sufficient to initiate gliomas alone, but when each was expressed in combination with homozygous loss of Pten and Ink4a, both mutations were able to produce high-grade gliomas with a short latency. However, the EGFRwt allele produced tumours with a lower incidence and longer latency in comparison with a single EGFRvIII allele. Homozygous EGFRvIII was more tumorigenic than heterozygous EGFRvIII, although the difference was small. Zhu and colleagues explored the cooperation between the nic mice . They geIDH1 is frequently mutated in human LGGs, often manifesting as the R132HIDH1 mutation, it would be expected that these represent driver mutations. Conditional transgenic mice with R132HIdh1 expression in the central nervous system were not found to generate gliomas  in earlier studies, although there was clear evidence that the same allele induces acute myeloid leukaemia in conditional transgenic mice when expressed in myeloid cells [R132HIdh1 knock-in mouse. Crossing this with nestin-cre mice led to pups with perinatal brain haemorrhages, as shown previously. However, when R132HIdh1 mice were crossed with T2nestin-creER (an inducible cre) and tamoxifen administered at 5–6 weeks of age, these mice displayed increased neural stem and progenitor proliferation in the SVZ, as measured through increased GFAP, Ki67, and BrdU-labelled cells. At later stages, this expansion of SVZ neural progenitors led to microscopically detectable nodules, which were believed to represent possible glioma precursors [Idh1 mutation led to early gliomagenesis in the adult SVZ but could not be detected from perinatal neural precursor recombination. It also adds weight to the argument that an IDH1 mutation can be an initiating event in gliomagenesis, explaining the high frequency of clonal IDH1 mutations in human gliomas and their presence in both primary and recurrent gliomas [Given that id cells ,41,42. M gliomas . Table 1Trp53/Pten and Rb mutations in adult subventricular zone (SVZ) neural stem cells (NSCs) and in astrocytes of mice by injections of adenovirus expressing cre (with and without the control of the GFAP promoter) [Trp53 and Pten loss-of-function mutations together induced gliomas, whereas deletion of Rb in addition to Trp53/Pten loss led to primitive neuroectodermal tumours (PNETs) rather than gliomas. Importantly, despite containing the same mutations as those induced in the SVZ, mature astrocytes were unable to form tumours. A well-studied but still controversial topic in glioma biology is which cell type gives rise to the tumour. Technological developments in glioma GEMMs have enabled important insights to be gleaned regarding the cell-of-origin of gliomas. Although it remains unclear which is the key cell type of origin, evidence suggests that the combination of genetic alterations has a major influence on whether one particular cell type can give rise to a glioma. Jacques and colleagues introduced combinations of romoter) . Only SVEgfr (activation) and Cdkn2a (loss) mutations in various brain cell types [Cdkn2a loss led to the dedifferentiation of the mature astrocytes, enabling these cells to later be transformed if an activating Egfr mutation was introduced. The authors concluded that loss of Cdkn2a was a critical initial step in gliomagenesis that must precede Egfr activation if the latter is to trigger glioma formation, at least from mature astrocytes. A related study into the cellular origin of gliomas investigated the role of ll types . These mSyn1-cre and CamK2a-cre transgenic mouse lines enabled recombination specifically in neurons. They found that all of these cell types generated malignant gliomas in mice with these genetic alterations and concluded that the mature cell types undergo dedifferentiation in response to defined oncogenic mutations to neural stem cells or progenitors, enabling tumour initiation and maintenance [To expand on these observations, Friedmann-Morvinski and colleagues performed direct lentiviral vector injections to cause p53/Nf1 knockdown or H-ras overexpression with p53 knockdown in neurons, astrocytes and neural stem cells of mice. Their models used cre-inducible lentiviral vectors, and ntenance . AlthougEgfr activation [EGFRvIII mutation in absence of exogenous EGF would be on these cells. Another group investigated whether a particular cell type in the SVZ was particularly responsive to epidermal growth factor (EGF); they demonstrated in mice that infusion of EGF into the lateral ventricles caused increased proliferation of C-cells (transit amplifying progenitor cells that express nestin) in the SVZ, and these cells then invaded the brain parenchyma. Although no tumours occurred in this model, the study demonstrated that exogenous EGF can increase proliferation of neural stem cells through the wild-type tivation . It is up53/Nf1 inactivation in NSCs. MADM is a genetic mosaic system that uses cre/Loxp recombination to generate homozygous mutations in a small population of cells [hGFAP-cre and nestin-cre to label neural stem cells and all lineages derived from them. Prior to GBM establishment, MADM-based lineage tracing identified aberrant growth only in oligodendrocyte precursor cells (OPCs), but not in NSCs or other NSC-lineages. Moreover, induction of p53/Nf1 mutations directly in OPCs caused glioma formation, leading the authors to conclude that OPCs are the origin of glioma in this model, even if the initiating mutations occur in NSCs [A further study elegantly used mosaic analysis with double markers (MADM) in mice with of cells , labelli in NSCs . TMAscl1-creER transgenic mouse line to target Nf1, Trp53 and Pten loss-of -function mutations specifically in these cells to the exclusion of neural stem cells [Nf1−/−; Trp53−/− these type 1 tumours were seen in addition to more circumscribed GBMs with expression profiles reflecting OPCs were observed (type 2 tumours). To confirm therefore that type 2 tumours are derived from adult OPCs, the investigators leveraged the TMNG2-creER transgenic line to express the same mutations specifically in adult OPCs only. These mice developed type 2 GBMs only. These data together suggest that the cell-of-origin influences the phenotype of GBMs, with adult OPCs and adult neural progenitors being capable of malignant transformation with appropriate mutations [To test whether adult committed neural progenitors and adult OPCs can give rise to gliomas, a well-designed study leveraged the em cells . When muutations . Figure IDH-wild type GBM patients, including normal SVZ, tumour tissue and normal cerebral cortex. They found that normal SVZ displayed low level driver mutations  that were also present in the GBMs of the same patients in 56.3% of cases. The introduction of driver mutations in astrocyte-like NSCs in the SVZ in mice led to migration of these cells and formation of GBMs at distant brain regions. These data support cells in the SVZ as being potential origins of GBMs, although the precise cell type is unclear from this study alone, given that multiple neural progenitor populations reside in the SVZ. Recent work using sequencing data from human patients lends support to the subventricular zone (SVZ) being the origin of at least some GBMs —the inveSleeping Beauty (SB) transposon system. SB transposons are derived from the Tc1-Mariner family and are naturally found in salmonid fish. This transposon was reconstructed from phylogenetic data and was given the name ‘Sleeping Beauty’ to reflect that fact that it was ‘awakened from a long evolutionary sleep’. Ivics and colleagues discovered the sequence of the ancestral transposon from this class of fish, demonstrated to be two 250 bp terminal DNA sequences containing inverted repeats that flank an open reading frame that codes for the transposase enzyme [Sleeping Beauty is therefore a two-component system composed of the transposon vector and the transposase enzyme. When these are present in the same cell, the transposase recognises the inverted repeats of the transposon and excises it from the donor locus. The transposon can then insert itself at a TA dinucleotide region elsewhere in the genome. In this way, the transposase catalyses a ‘cut and paste’ reaction of the transposon. Modifications to the inverted repeats have been made in order to improve transposition efficiency [A powerful platform for functional cancer gene discovery and also transgene insertion is the e enzyme . Sleepinficiency , and sitficiency ,56. The P19Arf allele, a known tumour suppressor gene in gliomas, which slightly increased the incidence of brain tumours. A mixture of anaplastic astrocytomas (grade III) and glioblastomas (grade IV) were generated. Genetic sequencing of the resulting 21 gliomas from this screen yielded 887 common integration sites (CIS—sites in the genome that are significantly recurrently ‘hit’ by the transposon more frequently than the background rate of insertions) and identified Csf1 as a recurrently hit gene. Immunohistochemistry in human tumours demonstrated overexpression of CSF1 in high-grade astrocytomas, providing some support for a role of this gene in malignant glioma formation in humans as well as mice. However, the CIS genes did not include a number of well-established glioma genes such as Egfr, Pdgfr, and Tp53, and there were only single insertions found in other important tumour-specific genes including Pten and Akt. This may be explained by certain insertion site preference biases of SB or that SB-induced gliomas represent only a subset of gliomas that is not driven by the major cancer drivers in most human gliomas. It is therefore crucial that transposon-driven gliomas are studied in other genetically predisposed backgrounds in order to gain a more complete understanding of the cancer drivers, ideally with an alternative transposon system such as piggyBac as well. Several SB insertional mutagenesis screens have been conducted in mice, successfully contributing to driver gene discovery for many cancers ,66,67,68nestin-cre allele on a Trp53-mutant background to drive expression of the SB transposase in mouse neural stem cells, although these did not directly generate tumours in vivo. In vitro culturing of embryonic neural stem cells derived from the SVZ of these mice demonstrated that these cells can be immortalised by mobilisation of SB. Immortalisation occurred significantly more frequently in cell lines with both R172HTrp53 and mobilising SB than those with R172HTrp53 alone, but not in lines with neither R172HTrp53 nor mobilising SB. When these immortalised cells were subcutaneously transplanted into SCID mice, they generated tumours with a latency of 2–4 months. Through genetic sequencing for the SB insertions, the authors identified 106 CIS genes in the immortalised cell lines and 114 in the tumours, of which 34 CIS genes were present in both cohorts. Comparing the CIS genes from the immortalised cells with those of the tumours in mice showed that a further round of transposon mobilisation for in vivo tumour establishment was needed in addition to the insertions present in immortalised cells alone [Pten, and amongst the tumour CIS were genes such as Pdgfrb and Nf1. The study also identified putative cancer genes that were not previously linked with gliomas, such as Met and Klf3 which were amongst their top-ranking tumour CIS. The CIS genes clustered into biological pathways that are thought to underlie gliomagenesis, in particular the Ras-MAPK and PI3K-Akt pathways, confirming that these major pathways that are mutated in human tumours can promote neural stem cell immortalisation in vitro and subcutaneous glioma formation. To enable screens to be performed for tissue-specific cancers, a conditional SB transposase allele was also developed that is active in the presence of cre expression and has been used for screens of many cancer types in mice. One study which employed this conditional SB transposon system for investigating gliomas used a ls alone . The autSleeping Beauty transposons have been proven to be a useful tool for inserting and expressing transgenes in cells both in vitro and in vivo, with relevance for glioma GEMMs [ATRX (encoding a histone chaperone protein) is recurrently co-mutated with TP53 in human GBMs. Elegant mouse modelling efforts used an SB transposase vector in conjunction with an ATRX knockdown sequence (shATRX) in a plasmid with flanking sequences recognized by the SB transposase to generate mice with ATRX knockdown [Trp53 loss and Nras overexpression, confirming ATRX acts as a tumour suppressor in vivo by impairing non-homologous end joining. This has clinical implications, in that ATRX GBMs in mice were more sensitive to double-stranded DNA damaging treatment such as irradiation and doxorubicin. In addition to being used for in vivo genetic screens, ma GEMMs . ATRX  in the hindbrain and cerebral cortex of mouse embryos. When this was done in combination with CRISPR-cas9 induced Trp53 loss, mice developed gliomas in both hindbrain and cortex within 8 months suggesting that H3.3K27M mutation cooperates with Trp53 loss in gliomagenesis when they are present in embryonic neural precursor cells but not in adult neural precursors  [piggyBac genome-wide screens for cancer genes have been performed for haematological and other solid cancers in mice [capacity ,75. 90% capacity . A recenomoters) . Althoug in mice ,79, suchE.coli in 1987 and in other bacteria and archaea a decade later [cas-genes led to the hypothesis and subsequent demonstration that the CRISPR-Cas system had a role in microbial adaptive immunity [Clustered regularly interspaced short palindromic repeats (CRISPRs) were identified in de later ,81. The immunity . This isimmunity . In contrast to the zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) systems in which specificity is achieved by complex protein-nucleic acid interactions, the Cas-nuclease is directed to a genetic target by nucleic acid base pairing determined by a unique 20 nucleotide region of short guide RNA (sgRNA). Experimentally this sgRNA sequence can be adjusted to guide the nuclease to virtually any site in a complex genome . The effNf1, Pten, and Trp53 led to the confirmed loss of these genes, and subsequent development of tumours histologically resembling human GBMs [Glioma GEMMs based on CRISPR-Cas9 have demonstrated the versatility and efficiency of this technology for in vivo tumour models . For examan GBMs . A modifman GBMs , as illuB2m-Nf1 and Mll3-Nf1 as putative driver combinations. These data demonstrate the utility of CRISPR-Cas9 for in vivo glioma functional tumour suppressor screens. Challenges with this approach however include the difficulty in achieving genome-wide coverage given the large number of cells that need to be transduced in order to cover a genome-wide library with multiple sgRNAs. Over the last few years, a number of studies have demonstrated the usefulness of CRISPR-Cas9 in both positive and negative selection genome-wide screens, including in human cancer cells ,91,92,93Mouse models have yielded landmark insights into the biology of gliomas in recent decades, and they continue to be indispensable for preclinical therapeutic testing. In order to maximise their relevance to human disease, glioma GEMMs need to faithfully recapitulate the molecular processes taking place, including genetic, epigenetic, transcriptional, and microenvironmental ones, amongst others. As technologies are being refined for engineering mutations found in human gliomas into the precise cell types of interest in the mouse brain, including by cre/LoxP recombination and viral- or transposon-based integration, our tools for investigating the mechanisms and origins of gliomagenesis are reaching new heights. Myb-Qk) in mice [piggyBac transposons and different versions of Cas9. Excitingly, these promise to provide large functional datasets in the investigation of GBM driver events and also potentially of resistance to novel targeted and immune therapies that will inform the next generation of clinical therapeutics. Future efforts must focus on exploring how combinations of mutations seen in human tumours cooperate in driving glioma formation using these models. The generation and detailed characterisation of novel cre transgenic mouse lines will enable further targeting of mutations to increasingly specific brain cell types to investigate the glioma cell-of-origin. Oncogenic fusion events in GBM are beginning to receive increased appreciation as potential driver events with the advent of deeper genetic sequencing technologies, and appropriate genome engineering technologies are needed to validate these and explore their mechanisms with glioma GEMMs. Indeed, recent work has shown the potential of combining the RCAS-TVA system with CRISPR-Cas9 for somatic genome engineering of an oncogenic chromosomal translocation (such as  in mice . Perhaps


Cell death-Inducing DNA Fragmentation Factor Alpha (DFFA)-like Effector (CIDE) proteins have emerged as lipid droplet-associated proteins that regulate fat metabolism. There are three members in the CIDE protein family—CIDEA, CIDEB, and CIDEC ). CIDEA and FSP27 are primarily expressed in adipose tissue, while CIDEB is expressed in the liver. Originally, based upon their homology with DNA fragmentation factors, these proteins were identified as apoptotic proteins. However, recent studies have changed the perception of these proteins, redefining them as regulators of lipid droplet dynamics and fat metabolism, which contribute to a healthy metabolic phenotype in humans. Despite various studies in humans and gene-targeting studies in mice, the physiological roles of CIDE proteins remains elusive. This review will summarize the known physiological role and metabolic pathways regulated by the CIDE proteins in human health and disease. Maintenance of an energy exchange balance is important to all forms of life. CIDE proteins  are an important group of proteins that were originally identified as pro-apoptotic proteins ,2. SeverOriginally, the CIDE proteins were discovered as apoptotic proteins through a homology search targeting the CIDE-N domain of DNA fragmentation factor 45 (DFF45). The apoptotic pathway is highly regulated and the prevention of its normal activation is linked to several disease states, including cancer . Near thThe apoptotic role of CIDE proteins has been primarily demonstrated through ectopic overexpression in mammalian cells or through induced mutations to different amino acid residues of the individual CIDE proteins ,16,17. IFor the CIDE proteins to induce apoptosis and fragment DNA, they must localize to the mitochondria ,17. ThisA set of elegant studies were performed by Cynthia Smas’ group, in which they discovered a dual role for FSP27 in adipocyte metabolism and cell death. FSP27 is highly upregulated during adipogenesis and is dependent on the differentiation of preadipocytes; the FSP27 transcript is downregulated by the presence of tumor necrosis factor-α (TNF-α), but it is upregulated by the presence of insulin . AdditioOverall, the role of CIDE proteins in terms of apoptosis has not yet been fully elucidated, but several required factors have been identified. Taken together, these studies indicate that the CIDE proteins are involved in the DNA fragmentation step of the apoptotic cascade; however, there remains much to be discovered in this area of research. Since their discovery as lipid droplet-associated proteins, much of the work regarding the CIDE proteins has been shifted towards investigating their roles in lipid metabolism.FSP27 was discovered 25 years ago as an adipocyte-specific gene that is upregulated during adipogenesis ,25 and iIt appears that depending on the cell type and level of expression, the CIDE proteins localize to additional intracellular organelles. In the case of CIDEA, localization to the Golgi apparatus occurs, while CIDEB is targeted preferentially to the smooth ER. VLDL is transported from the ER in vesicles which contain CIDEB; the biogenesis of these vesicles is also regulated by CIDEB ,32,33. FEach of the three CIDE proteins localize to the surface of lipid droplets in their respective tissue types  ,37,38,39As regulators of lipid droplet dynamics, the CIDE proteins are also involved in the rate of intracellular lipolysis. Through the use of RNA interference, Nordström et al. disrupted CIDEA in human preadipocytes and observed an increase in lipolysis and secretion of (TNF-α) . However−/−) have a lean phenotype, higher metabolic rate and body temperature, enhanced lipolysis in brown adipose tissue, and are resistant to diet-induced obesity and diabetes . Th. Th68]. Another study conducted by Wu et al. studied the association of five polymorphisms in the CIDEA gene with obesity in the Chinese population . The polFunctionally, CIDEA was identified as a lipid droplet-associated protein, and its expression was in consonance with insulin sensitivity in obese humans . In thisSimilar to CIDEA, a specific SNP in FSP27 has also been shown to be associated with the causation of metabolic syndrome. The SNP (G→T) occurs at nucleotide position 556 within exon 6 of FSP27 and inserts a premature stop codon (TAA), resulting in a non-sense mutation (E186X) that disrupts the CIDE-C domain. This mutation has been reported to be associated with partial lipodystrophy and insulin resistance in a female patient see  12]..12].Homeostatic Model Assessment for Insulin Resistance  is positively correlated with FSP27 expression in BMI-matched obese individuals . ComplimFSP27 regulates lipid droplet dynamics and lipolysis in adipocytes through regulation of the catalytic capacity , as well273. PPARγ Ser273 phosphorylation has been linked with the development of insulin resistance [273 [Recently, the role of FSP27 in human growth hormone (GH)-induced diabetes was elucidated . GH-indusistance ,78,79. Ince [273 . Anothernce [273 . OverallTaken together, the various mouse models have shown contradictory results on the role of CIDE proteins in whole-body metabolic phenotype. On the contrary, all of the human studies published to date unequivocally show a negative association of CIDEA and FSP27 with the pathogenesis and pathophysiology of metabolic disease in humans , giving The studies reported here indicate that the CIDE proteins are important regulators of fat metabolism. The functionality of each CIDE protein appears to depend highly on intracellular localization and tissue expression. It is currently unclear as to why there is a discrepancy between the metabolic phenotypes from studies targeting CIDE proteins in mice versus humans. However, the studies agree that the CIDE proteins are involved in lipid storage. Furthermore, the metabolic consequences of tissue-specific CIDE protein knockout appears to be different than that of whole-body knockouts. The human studies published to-date unequivocally show a negative association of CIDEA and FSP27 with the pathogenesis and pathophysiology of metabolic disease in humans. Further work is required to elucidate the mechanism by which the expression of each CIDE protein contributes to the regulation of insulin sensitivity or resistance and to examine their use as a potential therapeutic.