Neutrophil apoptosis is an important process contributing to the resolution of inflammation. This is because it allows the neutrophil membrane to remain intact preventing it’s potentially histotoxic contents from being released into the extra-cellular milieu, a process that can contribute to the exacerbation of many inflammatory disorders such as rheumatoid arthritis. When considering the life-span of a neutrophil and how it can be augmented by various inflammatory mediators to allow it to carry out its essential protective role in the body’s innate immune defences it is also important to consider how to terminate this process when the inflammatory insult has been dealt with or when the system goes awry. It is this information that we believe may hold the key to developing novel anti-inflammatory therapies. Through exploitation of the mechanisms controlling neutrophil apoptosis, it may be possible to selectively target these cells to enter apoptosis, and therefore help aid the process of resolution, especially if used in conjunction with treatments that up-regulate phagocytosis of apoptotic cells. This is important given that the main treatment for disorders of the inflammatory response are glucocorticoids, which whilst proven to be a powerful treatment for eosinophil based diseases such as asthma where they increase eosinophil apoptosis in conjunction with enhancing phagocytic clearance of apoptotic cells, glucocorticoids have been found to have the converse affect on neutrophils, actually serving to prolong their life-span potentially exacerbating the condition. Furthermore, it has been previously shown that the transcription factor nuclear factor kappa B (NF-κB) plays a pivotal role in neutrophil apoptosis, becoming activated by inflammatory agents such as lipopolysaccharide (LPS) and tumour necrosis factor-alpha (TNF-α). NF-κB activation results in the transcription of many pro-inflammatory agents and anti-apoptotic proteins such as X-linked inhibitor of apoptosis (X-IAP) increasing the life-span of the neutrophil. Interestingly, it has also been demonstrated that key neutrophil survival proteins such as myeloid cell leukemia-1 (Mcl-1) are not directly regulated by NF-κB activation. Therefore it is because of the aforementioned reasons that I have chosen to investigate further neutrophil apoptosis including the role played by NF-κB. Thus, I have investigated the hypothesis that NF-κB-dependent and independent survival proteins critically regulate the rates of neutrophil apoptosis and that newly identified pro-apoptotic agents such as the cyclin-dependent kinase (CDK) inhibitor, R-roscovitine interferes with the expression of such survival proteins. It has been previously found by myself and others in our laboratory during the course of this thesis that cyclin dependent kinase inhibitor (CDKi) drugs such as R-roscovitine are powerful novel anti-inflammatory agents with the ability to up-regulate rates of neutrophil apoptosis in vitro and influence the resolution of neutrophilic inflammation in vivo. Whilst the exact mechanism of CDK inhibitor drugs on neutrophil apoptosis remains elusive, work shown in this thesis demonstrates that R-roscovitine has the ability to over-ride powerful anti-apoptotic signals from pro-inflammatory agents such as granulocyte-macrophage colony stimulating factor (GM-CSF) and LPS causing the neutrophils to enter apoptosis. Furthermore, it has been found that R-roscovitine causes a decrease in levels of the antiapoptotic protein Mcl-1 in as little as 2h and that it prevents the maintenance / protective effect that GM-CSF has on the Mcl-1 protein levels. In addition R-roscovitine may also reduce levels of the NF-κB regulated protein X-IAP. The effect of R-roscovitine on X-IAP was investigated further using an X-IAP HIV-tat construct, though results from this remain inconclusive. This is because although the X-IAP construct appeared to be extending neutrophil longevity, it was discovered that LPS contamination of the construct had occurred which could therefore pose an alternative explanation for the increase in neutrophil life-span. As X-IAP, TNF-α and LPS are all regulated by NF-κB and given that NF-κB is already known to be a key player in neutrophil biology, the effects of R-roscovitine on this important transcription factor were investigated. It was discovered that R-roscovitine does not directly activate NF-κB, since this CDK inhibitor drug does not cause degradation and loss of the cytoplasmic inhibitor of NF-κB, IκBα. This lack of NF-κB activation was confirmed since R-roscovitine did not mobilize the NF-κB subunit, p65, from the cytoplasm to the nucleus. Furthermore, R-roscovitine (unlike the NF-κB inhibitor gliotoxin) does not interfere with the ability of LPS or TNF-α to activate NF-κB. Therefore by R-roscovitine to induce apoptosis, although this does not rule out the involvement of NF-κB at a later stage. When considering a reagent for possible use as a novel anti-inflammatory agent I think it is important to assess what effects it has on the activation state of the neutrophil. Therefore the effects of R-roscovitine on the activation markers CD62L, CD11b and shape change were assessed. It was found that R-roscovitine alone did not cause any significant neutrophil activation as measured using the parameters stated above. Importantly, it was also found that R-roscovitine did not interfere with the activation states induced by the inflammatory mediators GM-CSF, LPS, TNF-α or leukotriene B4 (LTB4). Another important consideration is the effect of R-roscovitine on the removal of apoptotic cells by macrophage phagocytosis. Results demonstrated that pre-treatment of macrophages with R-roscovitine did not augment their uptake of apoptotic neutrophils. In addition Rroscovitine did not detrimentally affect the increase in phagocytosis that results from macrophage treatment with the synthetic glucocorticoid dexamethasone. The data presented in this thesis suggest that CDK inhibitor drugs such as R-roscovitine are novel powerful pro-apoptotic agents for neutrophils with the ability to over-ride antiapoptotic signals from multiple pro-inflammatory mediators. It has been discovered that Rroscovitine causes a reduction in one of the neutrophil’s most prominent anti-apoptotic proteins (Mcl-1) whilst not altering the activation state of the neutrophil and furthermore it does not interfere with the uptake of apoptotic neutrophils by macrophages or result in any alteration to the increase in phagocytosis caused by treatment with dexamethasone. In conclusion, CDK inhibitor drugs such as R-roscovitine have the potential to be promising candidates for novel anti-inflammatory agents with the ability to selectively target neutrophil apoptosis whilst not interfering with steroid induced up-regulation of phagocytosis, therefore allowing a two pronged attack to help treat neutrophil based inflammatory disorders.

No description available.

The neutrophil is the first haemopoetic cell to arrive at the site of infection. In acute respiratory distress syndrome (ARDS), dense neutrophilic infiltrates are found in the lung in response to bacterial infection as well as generalised inflammatory stimuli, such as pancreatitis. At sites of infection, phagocytosis of bacteria by neutrophils enhances their subsequent apoptosis and clearance by macrophages however at inflammatory sites, the lifespan of the neutrophil is influenced by both pro- and antiapoptotic factors in the inflammatory milieu. Furthermore subsequent macrophage phagocytosis of apoptotic neutrophils induces the macrophage to switch to an antiinflammatory phenotype thereby hastening resolution of inflammation. The Fas death receptor pathway is important in T lymphocyte apoptosis but its role in neutrophil apoptosis is controversial. We have shown that neutrophils express the Fas receptor (CD95) on their surface but there is no evidence of expression of its natural ligand (FasL). An agonistic anti-Fas monoclonal antibody (CH-11) accelerated neutrophil apoptosis under certain culture conditions. Lipopolysaccharide (LPS) originating from Gram-negative bacteria is often found at sites of inflammation. We have shown that LPS attenuated CH-11 - induced neutrophil apoptosis unless the Fas/FasL death receptor pathway was activated prior to the LPS signalling pathway. This LPS-mediated attenuation did not involve the p42/44 ERK, protein kinase C or phosphatidylinositol 3-kinase signalling pathway however the p38 MAPK and NF-κB pathway appeared to be partially involved. We have shown that neutrophils express the protein cFLIPs and that CH-11 and inflammatory mediators altered its expression. Although macrophages are principally phagocytes, they are also important in determining the composition of the milieu at an inflammatory site. Macrophages have been shown to express FasL which can be shed and may contribute to the pools of sFasL found in the bronchoalveolar lavage fluid (BALF) in ARDS patients. We have shown that the conditioned supernatants from LPS-activated macrophages induced neutrophil apoptosis at early time points. The pro-apoptotic activity was mediated by TNF-α and was found in the fraction containing proteins with molecular weights greater than 50kD. Macrophage phagocytosis of apoptotic neutrophils suppressed TNF-α production by LPS-activated macrophages and this was associated with loss of the pro-apoptotic activity. In summary, our data suggest that Fas/FasL fratricide does not appear to be involved in spontaneous neutrophil apoptosis. However LPS attenuates Fas-induced apoptosis unless the Fas/FasL death receptor pathway is activated prior to LPS signalling pathways. The signalling pathways involved in this attenuation are not clear but may involve cellular FLIP. Furthermore, activated macrophages secrete inflammatory mediators and at early time points, TNF-α appears to be the most important in inducing neutrophil apoptosis.

Neutrophils are a central component of the innate immune system, whose major role is to defend the host against invading microorganisms. As such they are integral players in the process of inflammation, the response of vascular tissues to injury. They are frequently the first immune cells recruited from the systemic circulation into a site of tissue injury or infection where they themselves play a key antimicrobial role. Direct killing of microbes can be accomplished by phagocytosis, degranulation, production of reactive oxygen species (ROS) or the release of DNA and antimicrobial peptides into the extracellular milieu (NETosis). In addition neutrophils orchestrate the recruitment and activation of other leucocytes, further contributing to host defence. The central importance of neutrophils in immunity is revealed by defects in either number or function leading to recurrent life threatening infection. However, as the toxic arsenal of neutrophil constituents lack specificity they can also be damaging to surrounding host tissues causing exacerbated inflammation. It is therefore essential that neutrophil function is tightly controlled to allow an appropriate response to be mounted against invading pathogens while simultaneously minimising host tissue injury. Therefore, once the inciting inflammatory insult has been successfully cleared or controlled it is imperative that these non-tissue resident specialised immune cells are rapidly ‘switched off’ or cleared to allow the return to homeostasis. This resolution phase of the inflammatory cascade is now recognised as an energy dependent, finely controlled endogenous process, the beginnings of which are activated at the onset of inflammation. One of the main aims of resolution is to ensure efficient clearance of leucocytes that are no longer necessary. It is likely that a major clearance route is by the highly regulated and energy dependent processes of neutrophil programmed cell death (apoptosis) with subsequent uptake and disposal of apoptotic neutrophils by tissue macrophages. This process of neutrophil apoptosis renders the neutrophils nonfunctional and preserves cell membrane integrity, thus preventing further release of histotoxic neutrophil-derived inflammatory mediators into the extracellular environment. Furthermore, the recognition, uptake and disposal of apoptotic neutrophils cause a dynamic change in the phagocytosing macrophage phenotype with alterations in inflammatory mediator production. The fundamental importance of neutrophil apoptosis and subsequent efferocytosis in inflammation resolution is highlighted by the pathological consequences of neutrophil necrosis or failed apoptotic cell clearance, which leads to enhanced tissue injury and autoimmunity. Acute lung infection (pneumonia) is a common and serious condition affecting both developed and developing countries; globally, childhood pneumonia is the leading cause of death in children aged less than 5 years and pneumonia is the most common fatal infection in the developed world. In over half of patients with community acquired pneumonia no causative organism is ever isolated suggesting that although the immune response has successfully controlled infection, continued uncontrolled neutrophilic inflammation in the lung continues to cause morbidity and mortality. Indeed, pneumonia frequently progresses to acute respiratory distress syndrome (ARDS), a devastating acute inflammatory condition of the lungs characterized by inflammatory cell recruitment and accumulation of protein rich oedema fluid leading to impaired lung function. ARDS affects 200,000 critically ill patients in the USA per year, and has a substantial mortality rate of up to 40%. Despite advances in intensive care treatment and antimicrobial therapy mortality from pneumonia has not fallen since the 1950s, and at present there are no specific therapies for infection-related lung inflammation or ARDS. Understanding the mechanism behind such uncontrolled, persisting inflammation, and the need for novel approaches to target infection related lung injury are therefore both urgent and essential. This thesis examines the potential of neutrophil apoptosis-inducing pharmacological agents as potential treatments for infection-associated lung inflammation. The primary agents used include a cyclin-dependent kinase inhibitor as well as plant-derived polyphenolic flavones. The ability of these compounds to induce human neutrophil apoptosis in vitro, the key importance of the intracellular neutrophil survival protein Mcl-1 in mediating this process, and the effect of targeting Mcl-1 in human macrophages is investigated. In addition, neutrophilic inflammation is modelled in zebrafish and mice with both sterile and bacterial-driven models of inflammation. A key role for Mcl-1 is delineated in vivo, with it acting as an endogenous controller of the innate immune response by influencing neutrophil apoptosis, but without effects on macrophage apoptosis or ability to phagocytose apoptotic cells. Driving neutrophil apoptosis by down-regulation of Mcl-1 accelerates resolution of inflammation in vivo. This therapeutic approach is also found to have indirect anti-bacterial effects in a model of E. Coli induced pneumonia, in stark contrast to established anti-inflammatory approaches which routinely cause immune paresis and life threatening infection. As such, targeting inflammatory cell apoptosis by changes in Mcl-1 offers a potential new therapeutic approach for the treatment of infection-associated inflammation.

The normal physiological response to bacterial infection or wounding with threat of infection, termed inflammation, has been shown to be dysregulated in certain human diseases including (but not limited to): idiopathic pulmonary fibrosis, acute lung injury, arthritis and glomerulonephritis. The earliest arriving and most abundant cell responding to an inflammatory stimulus is the neutrophil granulocyte. It has been shown that under inflammatory conditions neutrophil granulocytes have extended longevity, enhanced responsiveness and upregulated activation parameters. In the setting of non-infective, or prolonged, ineffectuallycleared infective disease where resolution of inflammation does not occur then neutrophil granulocytes may cause tissue damage which is mediated by excessive, misdirected exocytosis of toxic granule contents or by spillage of the same products from necrotic or netotic cell carcasses that have lost membrane integrity. A key process in the resolution of inflammation is the induction of apoptosis in recruited neutrophils following a successful response to an inflammatory stimulus. Cellular signalling from apoptotic cells and from professional phagocytes that have ingested apoptotic cells has been shown to favour resolution of inflammation and restoration of tissue homeostasis. Additionally, the removal of key inflammatory cells in a highly regulated, non-phlogistic fashion robustly assists the resolution process. Cyclin-dependent kinase (CDK) inhibitor drugs are being developed as anti-cancer agents as it is hypothesized that they should interfere with the enhanced cellcycling ability (increased proliferative capacity and extended longevity) which is such a key feature of cancer cell biology. The CDKs that drive the cell cycle are CDKs 1, 2, 4 and 6 and consequently agents were designed to have enhanced specificity for these targets. CDK inhibitor drugs target the ATP-binding domain of CDKs and as a result usually have activity against more than one CDK. The CDK inhibitor drug, R-roscovitine which targets CDKs 2, 5, 7 and 9 was shown to promote neutrophil apoptosis and consequently resolution of inflammation. This thesis aims to investigate the mechanism by which apoptosis is induced in neutrophil granulocytes by CDK inhibitor drugs. The first experimental chapter of this thesis explores in detail the time-course and active concentration range of CDK inhibitor drugs in comparison to known promoters and inhibitors of neutrophil apoptosis. It then dissects the apoptotic machinery which is responsible for the effects of CDK inhibitor drugs before investigating their capacity to promote apoptosis even in the presence of survival mediators relevant to the context of inflammatory disease. Flow-cytometry, light and confocal microscopy as well as western blotting for caspases, mitochondrial dissipation assay, fluorometric caspase assay and the detection of DNA laddering demonstrate that CDK inhibitor drugs promote classical neutrophil apoptosis by the intrinsic pathway and show similar kinetics of apoptosis induction to drugs that inhibit transcription. The second experimental chapter investigates the key neutrophil survival protein and bcl-2 homologue Mcl-1. By flow cytometry, western blotting and RT-PCR it is demonstrated that Mcl-1 is down-regulated at the level of transcription and that this occurs even in the presence of inflammatory mediators that would normally promote neutrophil survival. Additionally, it is shown that pro-apoptotic bcl-2 homologues are affected to a lesser degree suggesting an imbalance of bcl-2 proteins is caused by effects at a transcriptional level mediated by CDK inhibitor drugs. The third experimental chapter identifies CDKs and their binding partner cyclins in neutrophil granulocytes and investigates the impact of CDK inhibitor drugs on CDK protein levels and cellular distribution by differential lysis and western blotting as well as by confocal microscopy. The key transcriptional enzyme RNA polymerase II is also identified and the effect of CDK inhibitor drugs on phosphorylation of this enzyme is documented. Western blotting and confocal microscopy demonstrate the presence of key CDKs 2, 5, 7, 9 and cyclin binding partners of CDKs 7 and 9. It is shown that the phosphorylation of RNA polymerase II mediated by CDKs 7 and 9 is inhibited by CDK inhibitor drugs. This suggests that a key mechanism by which neutrophil apoptosis is induced by CDK inhibitor drugs is the inhibition of transcription of key proteins and suggests that neutrophils require survival proteins for functional longevity. The fourth experimental chapter addresses the production and use of HIV-tat dominant negative CDK 7 and 9 proteins to knockdown CDKs 7 and 9 in neutrophil granulocytes in vitro to provide a molecular biology surrogate for the pharmacological data already presented. The cloning, production, purification and use of HIV-tat dominant negative CDK proteins are described. The final chapter describes the use of a more specific pharmacological inhibitor of CDKs 7 and 9, DRB, in the mouse bleomycin lung injury model. Resolution of inflammation by a compound specifically targeting CDKs 7 and 9 is described. This thesis identifies CDKs 7 and 9 as key targets of CDK inhibitor drugs in neutrophilic inflammation. It shows these drugs acting at the level of transcription to drive neutrophil apoptosis by exploiting the unique dependency of neutrophils on the short-lived survival protein Mcl-1. In so doing the presence of functional and essential transcriptional machinery is identified in neutrophils and the transcriptional profile of resting, stimulated and inhibited neutrophils is delineated. These findings suggest novel approaches to the pharmacological promotion of resolution of inflammation and indicate key new targets for rational drug design. In future, it will be important to further characterize the effects of CDK inhibitor drugs on other cell-types including epithelial cells, fibroblasts and mononuclear cells. This information should prove important to the continued investigation of CDK inhibitor drugs in resolution of inflammation and also to the ongoing experimental trial of these drugs in idiopathic pulmonary fibrosis.

Horses are at high risk to develop systemic inflammation due to the release of bacterial endotoxin from an inflamed gastrointestinal tract. Neutrophils are critical for mounting an immune response to bacterial endotoxins. Neutrophil activation following engagement of bacterial endotoxin expands their lifespan through suppression of their constitutive apoptosis. The prolonged lifespan of neutrophils propagates acute inflammation and delays the resolution of inflammation. Since equine neutrophil lifespan has not been well-studied, I investigated the occurrence of equine neutrophil apoptosis in vitro and in vivo. First, I investigated the effect of Escherichia coli lipopolysaccharide (LPS) treatment on the occurrence of equine neutrophil apoptosis in vitro. LPS treatment delayed in vitro equine neutrophil apoptosis in a dose-dependent manner at concentrations of 0.1-10 μg/ml through toll-like receptor (TLR)-4 signaling and down-regulation of the intrinsic pathway of apoptosis, specifically through reduced caspase-9 activity. Next, I found that ex vivo neutrophil apoptosis was delayed in two models of intestinal inflammation, jejunal ischemia and reperfusion (IR) and oligofructose-induced colitis, through down-regulation of both the intrinsic and extrinsic apoptosis pathways via reduced caspase-3, -8, and -9 activities. Pulmonary intravascular macrophages (PIMs) depletion with systemic gadolinium chloride (GC) prevented the prolongation of ex vivo neutrophil lifespan in horses undergoing jejunal IR through modulation of caspase-3, -8 and -9 activities. PIM depletion in IR horses resulted in an earlier and greater increase in tumor necrosis factor-alpha and a concomitant decrease in interleukin-10 to suggest an enhanced systemic pro-inflammatory response. I examined the effect of neutrophil concentration and co-incubation with aged, apoptotic neutrophils on the occurrence of neutrophil apoptosis in vitro. Neutrophil apoptosis was delayed with increasing concentrations of neutrophils in vitro, which may contribute to delayed neutrophil apoptosis in systemic inflammation. However, co-incubation with aged, apoptotic neutrophils did not alter in vitro neutrophil lifespan. Taken together, the data show that LPS delays equine neutrophils apoptosis in vitro in a TLR4-dependent manner through inhibition of caspase-9. Ex vivo neutrophil apoptosis was also delayed with systemic inflammation via down-regulation of caspase activity. A novel finding of this work was the reversal of delayed neutrophil apoptosis by depletion of PIMs in horses experiencing intestinal IR.

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.

Efferocytosis, the clearance of apoptotic cells including apoptotic neutrophils by macrophage phagocytosis, is a key cellular mechanism for resolution of inflammation and tissue repair. Cyclin-dependent kinases (CDKs) 7 and 9 phosphorylate RNA polymerase II that is vital for neutrophil transcriptional capacity. CDK inhibitors such as R-roscovitine, and the more selective inhibitor AT7519, induce neutrophil apoptosis and promote resolution of several mouse models of inflammation including acute lung inflammation. The hypothesis investigated here was that AT7519 would promote neutrophil apoptosis (i) in the infarcted heart, leading to macrophage polarisation, angiogenesis, reduced infarct expansion and retention of cardiac function and (ii) in the peritoneum, enhancing resolution of sterile peritonitis. AT7519 (1μM) induced apoptosis of mouse unstimulated-bone marrow derived neutrophils and thioglycollate-stimulated neutrophils in vitro in a time- and caspase-dependent manner, but did not alter activation assessed by calcium flux in response to the synthetic formyl peptide (fMLF) or platelet-activating factor (PAF). Only high concentrations of AT7519 (10 μM) induced monocyte/macrophage apoptosis and this was likely due to saturated phagocytosis of apoptotic neutrophils induced by high concentration of AT7519. Myocardial infarction (MI) was induced by coronary artery ligation in adult male mice and infarct volume was assessed 7 or 21 days later by in vitro optical projection tomography (OPT). The novel use of OPT for this purpose was validated by demonstrating correlation with infarct volume obtained by late-gadolinium enhanced magnetic resonance imaging in vivo and with infarct area assessed by histological staining (Masson’s Trichrome) in tissue sections. AT7519 (30 mg/kg i.p.) increased the number of apoptotic neutrophils (cleaved caspase-3 and Ly6G +ve) in the heart when administered after MI, but this was not associated with any subsequent alteration in macrophage polarisation, vessel density, infarct expansion or structural and functional remodelling of the left ventricle. In contrast, induction of neutrophil apoptosis by AT7519 (30mg/kg i.p.) successfully promoted macrophage polarisation and the resolution of inflammation associated with peritonitis elicited by either 10% thioglycollate or by 1mg zymosan. AT7519 treatment also reduced the number of CD19+ B cells, Foxp3+CD4+ T cells and eosinophils in peritoneal lavage, and prolonged the phase of monocyte recruitment in zymosan-induced peritonitis. In conclusion, AT7519 successfully induced mouse neutrophil apoptosis in vitro, as well as in vivo in experimental MI and peritonitis. Subsequent promotion of inflammation resolution in peritonitis was not matched by improved outcome following MI. Unexpected effects of CDK inhibition on monocytes, T cells and eosinophils that are necessary for myocardial infarct repair may have compromised any beneficial effects resulting from promotion of in situ neutrophil apoptosis. CDK inhibition may therefore have therapeutic potential for the treatment of peritonitis, but not for prevention of infarct expansion and detrimental ventricular remodelling after MI.

Neutrophils are an important cell in host defence and migrate rapidly to sites of inflammation when the host is compromised (e.g., in infection or wounding). There, they produce and/or release inflammatory mediators (e.g., LTB4, TNF, IL-8) and ingest and degrade pathogens (e.g., by release of granule proteins and reactive oxygen species). Neutrophils then undergo apoptosis and are cleared by phagocytes such as macrophages, to allow efficient resolution of inflammation. Inducing neutrophil apoptosis by pharmacological means could be a therapeutic strategy to dampen inflammation in diseases where neutrophils are prevalent, e.g., acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA). Inhibition of cyclin-dependent kinases (CDKs) using CDK inhibitor (CDKi) compounds induces mammalian neutrophil apoptosis in vitro, and can drive resolution of inflammation in vivo in mouse models. Evidence indicated that this is due to inhibition of CDK9 and CDK7-mediated transcription of the anti-apoptotic protein Mcl-1. The hypothesis of this project was that CDK9, CDK7 and Mcl-1 are pivotal regulators of resolution of inflammation in vivo. The model selected to test this hypothesis was tailfin injury of embryonic zebrafish (Danio rerio). Zebrafish are optically transparent and reporter transgenic lines with neutrophils labelled by enhanced GFP (EGFP - Tg[mpx:EGFP]i114) and macrophages (Tg[MPEG1:mCherry]) have been created, permitting the imaging of the behaviour of these cells in vivo. The model of tailfin transection was chosen to cause an inflammatory response in these animals, with neutrophil and macrophage recruitment to the tailfin. This response was manipulated using CDKi compounds and specific gene knockdowns (using morpholino and CRISPR/cas9 technologies). It was shown that CDKi compounds could reduce neutrophil numbers at 24 h post-injury at the transected tailfin, but did not affect macrophage numbers. The CDKi AT7519 increased neutrophil apoptosis at 12 h post-injury. Specific CDK9 knockdown using morpholinos or CRISPR/cas9 also reduced neutrophilic inflammation at the tailfin 24 h after transection, accompanied by increased apoptosis levels at 8 h in the morpholino-treated group. Inhibition of an endogenous CDK9 inhibitor, LaRP7, had the opposite effect and increased neutrophil numbers; and could oppose the neutrophil- reducing effect of AT7519 and CDK9 morpholino knockdown. Preliminary genetic knockdown studies into the roles of CDK7 and Mcl-1 have been carried out. Taken together, the results demonstrate CDK9 is important in the resolution of neutrophilic inflammation, indicating that manipulation of CDK9 activity could be a good target for therapeutic intervention in inflammatory disease.

Abstract Introduction: Current evidence suggests that the lungs of infants with the debilitating disorder, bronchopulmonary dysplasia (BPD), react to the challenges of extra-uterine adaptation with inappropriately aggressive inflammation. The reasons for this are not entirely clear and this study hypothesizes that a deficiency of interleukin (IL)-10, a potent anti-inflammatory mediator, leads to the functional and architectural changes characteristic of BPD. Aim: To characterize the behaviour of IL-10 and neutrophil apoptosis in the tracheal fluids (TF) of infants at risk of developing BPD. Method: TF from intubated infants of varying gestations at the Royal Hospital for Women, Randwick was spun and ILs 8, 10 and 16 were measured in the supernatant. The residual pellets of white cells were used to determine differential white cell counts and neutrophil apoptosis. Results: None of the 20 TF specimens from the extremely premature infants with BPD (n=11) had detectable IL-10, compared to 14/20(70%) of the specimens from preterm infants without BPD (n=20) and to 5/19 (26%) of the specimens from term infants (n=19). BPD infants also had a significantly lower number of apoptotic neutrophils during the 1st week of life. Premature infants with TF IL-10 &gt5pg/ml did not develop BPD. Levels of IL-8, a neutrophil chemotaxin, and white cell counts, while not differing significantly between the groups, increased considerably towards the end of the first week of life in the BPD group. IL-16, a chemotaxin for inflammatory CD4+ cells, was also detected in more BPD than non-BPD specimens (BPD: 16/46 (35%) v 1/30 (0.3%) non-BPD preterm and 2/7 (28%) term TF specimens). Conclusions: Extremely premature infants prone to BPD have decreased pulmonary anti-inflammatory activity as demonstrated by decreased IL-10 and apoptotic neutrophils in tracheal fluids. The lack of a counter-regulatory response to the inflammatory processes that are an inevitable consequence of extra-uterine adaptation may therefore place the extremely premature newborn infant at a considerable risk of developing BPD.