Andrew E. Leitch

Neutrophil Apoptosis: Relevance to the Innate Immune Response and Inflammatory Disease

Neutrophils are the most abundant cell type involved in the innate immune response. They are rapidly recruited to sites of injury or infection where they engulf and kill invading microorganisms. Neutrophil apoptosis, the process of programmed cell death that prevents the release of neutrophil histotoxic contents, is tightly regulated and limits the destructive capacity of neutrophil products to surrounding tissue. The subsequent recognition and phagocytosis of apoptotic cells by phagocytic cells such as macrophages is central to the successful resolution of an inflammatory response and it is increasingly apparent that the dying neutrophil itself exerts an anti-inflammatory effect through modulation of surrounding cell responses, particularly macrophage inflammatory cytokine release. Apoptosis may be delayed, induced or enhanced by micro-organisms dependent on their immune evasion strategies and the health of the host they encounter. There is now an established field of research aimed at understanding the regulation of apoptosis and its potential as a target for therapeutic intervention in inflammatory and infective diseases. This review focuses on the physiological regulation of neutrophil apoptosis with respect to the innate immune system and highlights recent advances in mechanistic understanding of apoptotic pathways and their therapeutic manipulation in appropriate and excessive innate immune responses.

Novel pharmacological strategies for driving inflammatory cell apoptosis and enhancing the resolution of inflammation

Resolution of inflammation requires the effective downregulation of key inflammatory cells such as neutrophils and eosinophils, which normally undergo programmed cell death (apoptosis) to enable their detection and removal by phagocytes such as macrophages. Dysregulation of this process is thought to contribute to the pathogenesis and progression of chronic inflammatory disorders such as chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, allergic rhinitis and inflammatory bowel disease. Importantly, knowledge of the signalling pathways responsible for the induction and execution of granulocyte apoptosis and the phagocytic removal of apoptotic cells continues to increase and, with it, the potential for incisive pharmacological intervention. In this article, we highlight pharmacological strategies that could be used to drive the resolution of inflammation by augmenting apoptosis of inflammatory cells.

Targeting granulocyte apoptosis: mechanisms, models, and therapies

Summary:  The inflammatory process is a complex series of tightly controlled cellular and biochemical events initiated by the immune system, which has evolved to eliminate or contain infectious agents and to repair damaged tissue. Apoptosis is essential for the clearance of potentially injurious inflammatory cells, such as neutrophils, eosinophils, and basophils, and the subsequent efficient resolution of inflammation. In this review, we aim to cover key features of the granulocyte life‐cycle ranging from their differentiation within the bone marrow to their maturation and ultimate clearance, with a focus on granulocyte apoptosis and macrophage efferocytosis. We further aim to discuss current and emerging models of inflammation and suggest novel ways of terminating or resolving deleterious inflammatory responses with a specific view to the translation of these strategies into fully realized, pro‐resolution therapies.

The cyclin-dependent kinase inhibitor R-roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non‐inflammatory phagocytosis by professional phagocytes. Recently, cyclin‐dependent kinase (CDK) inhibitors (e.g. R‐roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF‐κB and MAPK pathways and key endogenous survival proteins (typified by Mcl‐1) are involved in the regulation of neutrophil apoptosis and, in cancer‐cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R‐roscovitine over‐rides TNF‐α and LPS‐induced survival (determined by morphological examination and binding of fluorescently labelled annexin‐V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF‐κB activation (assessed by cytoplasmic IκBα proteolysis and NF‐κB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down‐regulation (at protein and mRNA level) of the survival protein Mcl‐1 but not the pro‐apoptotic bcl‐2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.

Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation

Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation.

Cyclin‐dependent kinase inhibitor drugs as potential novel anti‐inflammatory and pro‐resolution agents

The cyclin‐dependent kinase inhibitor (CDKi) drugs such as R‐roscovitine have emerged as potential anti‐inflammatory, pharmacological agents that can influence the resolution of inflammation. Usually, once an inciting inflammatory stimulus has been eliminated, resolution proceeds by prompt, safe removal of dominant inflammatory cells. This is accomplished by programmed cell death (apoptosis) of prominent effector, inflammatory cells typified by the neutrophil. Apoptosis of neutrophils ensures that toxic neutrophil granule contents are securely packaged in apoptotic bodies and expedites phagocytosis by professional phagocytes such as macrophages. A panel of CDKi drugs have been shown to promote neutrophil apoptosis in a concentration‐ and time‐dependent manner and the archetypal CDKi drug, R‐roscovitine, overrides the anti‐apoptotic effects of powerful survival factors [including lipopolysaccharide (LPS) and granulocyte macrophage‐colony stimulating factor (GM‐CSF)]. Inflammatory cell longevity and survival signalling is integral to the inflammatory process and any putative anti‐inflammatory agent must unravel a complex web of redundancy in order to be effective. CDKi drugs have also been demonstrated to have significant effects on other cell types including lymphocytes and fibroblasts indicating that they may have pleiotropic anti‐inflammatory, pro‐resolution activity. In keeping with this, CDKi drugs like R‐roscovitine have been reported to be efficacious in resolving established animal models of neutrophil‐dominant and lymphocyte‐driven inflammation. However, the mechanism of action behind these powerful effects has not yet been fully elucidated. CDKs play an integral role in the regulation of the cell cycle but are also recognized as participants in processes such as apoptosis and transcriptional regulation. Neutrophils have functional CDKs, are transcriptionally active and demonstrate augmented apoptosis in response to CDKi drugs, while lymphocyte proliferation and secretory function are inhibited. This review will discuss current understanding of the processes of inflammation and resolution but will focus on CDKis and their potential mechanisms of action.

The CDK inhibitor, R‐roscovitine, promotes eosinophil apoptosis by down‐regulation of Mcl‐1

Eosinophils are major players in inflammatory allergic diseases such as asthma, hay fever and eczema. Here we show that the cyclin‐dependent kinase inhibitor (CDKi) R‐roscovitine efficiently and rapidly induces human eosinophil apoptosis using flow cytometric analysis of annexin‐V/propidium iodide staining, morphological analysis by light microscopy, transmission electron microscopy and Western immunoblotting for caspase‐3 cleavage. We further dissect these observations by demonstrating that eosinophils treated with R‐roscovitine lose mitochondrial membrane potential and the key survival protein Mcl‐1 is down‐regulated. This novel finding of efficacious induction of eosinophil apoptosis by CDKi drugs has potential as a strategy for driving resolution of eosinophilic inflammation.

Relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa

The respiratory mucosa is responsible for gas exchange and is therefore, of necessity, exposed to airborne pathogens, allergens, and foreign particles. It has evolved a multi-faceted, physical and immune defense system to ensure that in the majority of instances, potentially injurious invaders are repelled. Inflammation, predominantly mediated by effector cells of the granulocyte lineage including neutrophils and eosinophils, is a form of immune defense. Where inflammation proves unable to remove an inciting stimulus, chronic inflammatory disease may supervene because of the potential for tissue damage conferred by the presence of large numbers of frustrated, activated granulocytes. Successful recovery from inflammatory disease and resolution of inflammation rely on the clearance of these cells. Ideally, they should undergo apoptosis prior to phagocytosis by macrophage, dendritic, or epithelial cells. The outcome of inflammation can have serious sequelae for the integrity of the respiratory mucosa leading to disease. Therapeutic strategies to drive resolution of inflammation may be directed at the induction of granulocyte apoptosis and the enhancement of granulocyte clearance.

Induction of eosinophil apoptosis by the cyclin-dependent kinase inhibitor AT7519 promotes the resolution of eosinophil-dominant allergic inflammation.

Background Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo. Methodology/Principal Findings Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk). Conclusions Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.

Editorial: Neutrophil apoptosis: hot on the TRAIL of inflammatory resolution

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