Leishmania infantum chagasi (Lic) is a pathogenic protozoan parasite and one of the etiological agents of human visceral leishmaniasis (VL). VL is a potentially deadly disease characterized by variable fevers, cachexia, hepatosplenomegaly, and global immune suppression. Many questions regarding the pathogenesis of VL and the mechanisms of host defense during Lic infection remain to be elucidated. The primary focus of this thesis is the relationship between Lic and the mammalian immune system. We studied parasite-host interactions during Lic infection at the molecular, cellular, and organismal level. We generated transgenic parasites that expressed firefly luciferase and/or fluorescent proteins to expand our capacity to detect, observe, and quantify the parasites in a variety of experimental settings with modern analytical methodologies. Using luciferase-expressing Leishmania, we developed an experimental infection model in which parasites were detected and the relative parasite burden in specific anatomical locations could be quantified in a live animal host using bioluminescence imaging. This method allowed the parasite burden to be assessed in the same host throughout the course of infection. Utilizing this model we have made some intriguing observations relating to the kinetics and distribution of the parasite burden over time. The parasite burden was observed primarily in the liver and bone marrow over the first few weeks and then shifts to the spleen and bone marrow. To gain a better understanding of the initial parasite-host immune interactions in vivo, we studied the early inflammatory response after intradermal (i.d.) inoculation. We observed a rapid and abundant influx of neutrophils into the inoculated ears. The neutrophil influx was transient, dose dependent and specific for the local inoculation site. While there was not a significant neutrophil influx into the draining lymph nodes (dLN), there was an increase in the total cellularity and a striking increase in the relative proportion of B cells to T cells over the first week after intradermal parasite challenge. By inoculating transgenic mCherry-Lic we found that neutrophils were the primary parasite-laden host cell in the dermal tissue during the first day, but macrophages harbored most of the parasites by 2 days. Neutrophil depletion using low-dose antibody treatment resulted in a reduced rate of parasite uptake initially at the site of inoculation, but no significant change in the dLN dynamics. We further examined the parasite-host relationship by studying molecular signaling and cellular interactions between Leishmania and human neutrophils. We investigated the nature of the chemotactic activity of Leishmania-conditioned growth medium for human neutrophils by testing physical properties of the activity and ruled out some of the major Leishmania surface molecules as potential candidates. We aim to identify the agent(s) responsible for the activity in on-going studies. To this end, we are collaborating with a group at the NIH and testing biochemical purification/separation samples. We conclude that intradermal Lic challenge induces a rapid innate immune response at the local site of infection, that neutrophils sense Leishmania-derived factors leading to directed migration, and that neutrophils function as a primary site for Leishmania entry into the mammalian host.

Research Doctorate - Doctor of Philosophy (PhD) / Asthma involves chronic inflammation of the airways that is heterogeneous in nature. Eosinophilic airway responses are well described in asthma, however non-eosinophilic subtypes of asthma have been recently reported, and can involve the influx of neutrophils into the airways (neutrophilic asthma). Neutrophils are important effector cells of the innate immune system. These cells are the first to migrate to inflammatory sites, where they contain and eliminate pathogenic microorganisms. Neutrophils also release cytokines and chemokines that initiate and amplify inflammatory responses. The mechanisms of neutrophilic asthma remain largely unknown; however activation of the innate immune response is implicated, particularly increased levels of proinflammatory cytokines Interleukin (IL)-8 and IL-1beta and gene expression of Toll Like Receptor (TLR)-4 and TLR2 have been demonstrated in induced sputum samples. This thesis examines innate immune responses of airway and circulating neutrophils, with a focus on neutrophilic asthma. Innate immune neutrophil activation occurs in response to exposure to Lipopolysaccharide (LPS), which activates TLR4. The activation response consists of the release of preformed granule associated mediators such as Matrix Metalloproteinase (MMP)-9 and Oncostatin M (OSM), new gene transcription and release of inflammatory cytokines such as IL-8, IL-1beta and Tumor Necrosis Factor (TNF)-alpha, and new gene transcription of TLR2 & TLR4 which serve to amplify neutrophil responses. In addition, this thesis examines whole genome gene expression profiles of circulating neutrophils in neutrophilic and eosinophilic asthma. The aims of this thesis are based on the hypothesis that dysregulation of innate immune neutrophil responses occurs with ageing and airway disease, particularly neutrophilic asthma and chronic obstructive pulmonary disease (COPD). With advancing age, there were alterations in the innate immune responses of neutrophils, which were characterised by enhanced spontaneous activation of both airway and circulating neutrophils, and a decreased response of circulating neutrophils to LPS. There was a decreased activation of airway neutrophils in airway disease that was most pronounced in neutrophilic asthma and COPD, with decreased production and release of proinflammatory cytokines most likely due to a downregulation of TLR4. TLR2 was downregulated in resting and LPS stimulated circulating neutrophils in asthma, particularly neutrophilic asthma. Circulating neutrophils had a decreased spontaneous release of total MMP-9, and downregulation of OSM, TLR2 and TLR4 at rest in COPD. However when stimulated with LPS, subjects with COPD had an enhanced proinflammatory cytokine release, with increases in IL-8 and TNF-alpha compared to subjects with asthma or healthy controls. Analysis of whole genome gene expression of circulating neutrophils in asthma revealed distinct gene profiles relating to asthma subtype. There was upregulation of genes relating to cell motility, inhibition of apoptosis and the NF-kB in neutrophilic asthma, which would contribute to their accumulation in the airways. The innate immune response is critical in controlling infections by bacteria and viruses. The reduced innate immune response of airway neutrophils in airway disease could contribute to impaired local defense, which may lead to an increased susceptibility to infection by invading pathogens. Systemically, the molecular mechanisms of neutrophilic asthma are distinct from eosinophilic asthma and may involve the enhancement of neutrophil chemotaxis and survival, contributing to their accumulation in the airways.

No description available.

Sepsis is a systemic inflammatory response to infection, which may ultimately lead to multi-organ failure. Sepsis causes millions of deaths each year and creates tremendous financial burdens on the health care system, yet there is no effective cure for sepsis. Even years after the onset of sepsis, patients who have clinically recovered still die from sepsis-related complications due to chronic immune dysfunction. Neutrophils, the most dominant leukocytes in human circulatory systems, play a critical role in not only promoting inflammation to fight against microbe invasion but also facilitating inflammation resolution to restore immune homeostasis. While dysfunctional/exhausted neutrophils have been implicated in the long-term morbidity and mortality of sepsis, the cause of neutrophil exhaustion and the system to rejuvenate the dysregulated immunity are understudied. To fill in the missing piece here, we conducted our trilogy-like projects. First, we established an in vitro culture system to mimic sepsis-like conditions: murine neutrophils prolonged-stimulated with LPS exhibit exhaustion-related phenotype with the elevated expression of both proinflammatory and immunosuppressive makers on the cell surface as well as dysregulated swarming patterns. We found that by knocking out TRAM (TICAM2), an adaptive molecule regulating TLR4 downstream MyD88-independent signaling pathway, neutrophils exhibit attenuated exhaustion on both phenotypic and functional levels. Of note, TRAM contributes to the development of exhausted neutrophils through activating Src family kinases (SFKs)-STAT1 cascade, and deficiency in TRAM provides protective effects on systemic inflammation, reduces tissue injury, and improves survival in a murine colitis-induced sepsis model. Next, in my second project, we reported that neutrophils can be clustered into three subpopulations even at their naïve state based on the single-cell RNA sequencing (scRNAseq) analyses. Of note, neutrophils in one of the clusters are more mature but less apoptotic with the elevated expression of resolving-associated markers Cd86 and Cd200r, hence we termed these neutrophils as 'resolving neutrophils'. We found that the resolving neutrophil population can be expanded via pharmacologically reprogramming with sodium 4-phenylbutyrate (4-PBA) or genetic deletion of TRAM. Resolving neutrophils not only secrete more pro-resolving mediators, such as ResolvinD1 and SerpinB1, but also exert enhanced phagocytic and bactericidal capacities. Mechanistically, we discovered that the development of resolving phenotype in neutrophils is mediated by the PPARγ/LMO4/STAT3 signaling circuitry, which is constitutively suppressed by TRAM. To explore the translational applications of resolving neutrophils, in my third and final project, we conducted adoptive transfer experiments to examine the effects of TRAM-deficient resolving neutrophils in cecal slurry (CS)-induced septic mice. We found that TRAM-deficient mice are more resilient to severe sepsis with reduced tissue injury and less compromised lung integrity as compared to wild-type (WT) mice, and splenic neutrophils from TRAM deficient septic mice better preserve resolving-related features. Moreover, transfusing TRAM deficient neutrophils in WT septic mice renders therapeutic effects with alleviated lung and kidney damage. We also observed TRAM-deficient neutrophil-mediated resolving memory propagation in vitro to promote resolving features of neutrophils, monocytes, and T cells, as well as to strengthen endothelial cell barrier function. In terms of the mechanism, we reported that TRAM is critical for the secretion of neutrophil elastase, a potent protein to compromise endothelium; hence, endothelial cells cocultured with TRAM deficient neutrophils maintain higher levels of adhesion/tight junction markers than cocultured with WT neutrophils. Taken together, our trilogy projects better define exhausted and resolving neutrophils. And most importantly, our works demonstrate that TRAM, an underappreciated molecule, is responsible for inducing neutrophil exhaustion and suppressing resolving neutrophil generation. / Doctor of Philosophy / A 'good' inflammation upon the infection should include two steps: the initial proinflammatory response to combat invading pathogens followed by the later resolution process to repair damage and restore the balance of the host's immune system. The harmony of these reactions is essential to maintain immune homeostasis, and the disruption of immune homeostasis may lead to different pathogenic conditions, including sepsis. Neutrophils are the most dominant white blood cells in human circulation, and they play a critical role in both promoting proinflammatory response and facilitating inflammation resolution. While the dysfunction of neutrophils is associated with the pathogenesis of sepsis and implicated in long-term sepsis-related death, approaches to rejuvenate dysregulated/exhausted neutrophils to restore immune homeostasis in septic patients are still lacking. In our projects, we better defined the characteristics of exhausted neutrophils in a sepsis-mimicking condition and unveiled the underlying mechanisms of neutrophil exhaustion. In addition, we demonstrated that neutrophils with pro-resolving features can be expanded concurrently with the decrease of exhausted neutrophils by a genetic modification approach. Finally, we showed that neutrophils with pro-resolving features can offer therapeutic effects in sepsis mice to alleviate tissue injury and organ dysfunction.

Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils. To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils. Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells. In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS. In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05. The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05. All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide. Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours. Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001). Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC. The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%). These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies. / Master of Science

La nétose du polynucléaire neutrophile (PN) correspond à la libération de filaments d’ADN recouverts de protéines appelés Neutrophil Extracellular Trap (NETs). Outre leur rôle anti-infectieux, les NETs représentent des acteurs émergents de nombreuses pathologies inflammatoires et nous avons souhaité évaluer leur implication au cours de réactions allergiques.Au cours d’une étude clinique multicentrique notre équipe a mis en évidence un mécanisme alternatif de l’anaphylaxie aux curares, impliquant les PN. La phase aiguë de ces réactions s’accompagne d’une libération de NETs dont la concentration est corrélée avec la sévérité et avec une diminution de l’expression des récepteurs activateurs des IgG à la surface des PN (FcγRs) ; ceci suggère un rôle des complexes immuns (CI) IgG/curares dans la formation des NETs au cours de ces réactions anaphylactiques.Pour confirmer cette hypothèse, la capacité d’activation de la nétose par les CI IgG a été étudiée, via la mise au point d’un modèle de stimulation in vitro des PN humains purifiés.Ce travail montre que 2 récepteurs aux IgG du PN (FcγRIIa et FcγRIIIB) contribuent à la libération de NETs en réponse à différents types de CI.En parallèle, la formation des NETs a été explorée dans un modèle de réaction allergique chronique, l’asthme. Au niveau systémique, la concentration de NETs est associée à la présence d’un asthme sévère mal contrôlé et d’une obstruction bronchique peu réversible. Inversement, la concentration de NETs dans le lavage broncho-alvéolaire est plus élevée au cours de l’asthme modéré et semble traduire un recrutement pulmonaire et une activation des PN en réponse à une colonisation microbienne.Au total nous montrons que les NETs sont libérés au cours des deux modèles de réactions allergiques choisis, aiguë (anaphylaxie aux curares) et chronique (asthme) et qu’ils pourraient représenter des biomarqueurs de sévérité. Des travaux complémentaires sont nécessaires pour déterminer dans quelle mesure les NETs contribuent à la physiopathologie des allergies. / Neutrophil netosis consists in the release of extracellular DNA filaments bound to granular proteins, called Neutrophil extracellular traps (NETs). In addition to their anti-infectious role, NETs are emerging actors of many inflammatory diseases and we decided to investigate their involvement during allergy.In a multicenter clinical study, our team highlighted an alternative mechanism of anaphylaxis to neuromuscular blocking agents (NMBA) involving neutrophils (PN). The acute phase of these reactions is characterized by NETs release which level is correlated with severity and with a decrease in IgG activating receptors (FcγRs) expression on PN; this suggests a role of immune complexes (IC) IgG / NMBA in NETs formation during these anaphylactic reactionsTo confirm this hypothesis, the ability of IgG ICs to activate netosis was studied through the development of an in vitro stimulation model of purified human PNs.This work shows that two PN IgG receptors (FcγRIIa and FcγRIIIB) contribute to NET release upon cellular activation by different ICsIn parallel, NETs formation has been explored in a model of chronic allergic reactions, asthma. At systemic level, NETs levels are associated with severe and poorly controlled asthma as well with the presence of low reversible bronchial obstruction. Conversely, NETs levels in bronchoalveolar lavage are higher in moderate asthma and appear to reflect pulmonary recruitment and activation of PN in response to microbial colonization.Taking together these results show that NETs are released during the two selected models of allergic reactions : acute (NMBA anaphylaxis) and chronic (asthma) and could be used as biomarkers of severity. Furthers works are needed to determine to what extent NETs contribute to the pathophysiology of allergy.

Galectin-3, a unique member of the growing family of β-galactoside binding lectins, contains a single carbohydrate recognition domain and a glycine rich N-terminal domain through which it can form oligomers and functions to cross-link both carbohydrate and non-carbohydrate ligands. Galectin-3 is widely expressed in adult tissues, particularly on and secreted by activated macrophages and monocytes. Galectin-3 has been implicated in many facets of the inflammatory response including neutrophil and macrophage activation and function. In this thesis I have examined the role of galectin-3 during fibrosis, alternative activation of macrophages and pneumonia. Galectin-3 expression is upregulated in established human fibrotic liver disease and in a mouse model of liver fibrosis induced by carbon tetrachloride. Galectin-3 expression is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis in this model. In addition, disruption of the galectin-3 gene markedly attenuates liver and kidney fibrosis, induced by unilateral ureteric obstruction, with reduced collagen deposition and myofibroblast activation. Results suggest that galectin-3 may promote fibrosis by stimulating myofibroblast activation by a transforming growth factor-β (TGF-β)-independent mechanism. Recent reports suggest that alternative macrophage activation is one of the key steps toward the progression of fibrosis. Disruption of the galectin-3 gene specifically restrains interleukin-4 (IL-4)/IL-13-induced alternative macrophage activation in vitro. My results suggest that the key mechanism required for activation of an alternative macrophage phenotype is an IL-4-stimulated galectin-3 feed back loop which directly activates CD98 causing sustained phosphatidylinositol 3-kinase (PI3-K) activation. The gram-positive Streptococcus pneumoniae (S. pn) is the leading cause of community acquired pneumonia worldwide, resulting in high mortality. Galectin-3-/- mice demonstrate a clearance defect of S. pn with increased septicaemia and a greater extent of lung damage compared to wild type mice. This phenotype is markedly reduced in pneumonia induced by the gram-negative Escherichia coli (E.coli). I have shown that presence of galectin-3 reduces the severity of pneumonia induced by S. pn and this is achieved through a number of processes: 1) Galectin-3 has bactericidal properties towards S. pn in vitro. 2) Galectin-3-/- macrophages show reduced production of nitrite following incubation with both S. pn and E. coli and hence a reduction in bacterial killing. 3) Galectin-3 activates neutrophils to produce reactive oxygen species which enhances the bactericidal activity of neutrophils. 4) Activation of neutrophils by galectin-3 augments phagocytosis of bacteria. 5) Finally, initial data suggests that galectin-3-/- neutrophils apoptose more readily than wild type neutrophils in vitro and galectin-3-/- macrophages phagocytose apoptotic neutrophils less efficiently compared to wild type. In vivo this would result in an accumulation of dying cells in the lung. The damage these apoptotic cells would have on the lung tissue may enable the bacteria to enter the blood stream resulting in sepsis. In summary, in response to chronic tissue injury, persistant upregulation of galectin-3 causes myofibroblast and alternative macrophage activation, thus enhancing collagen deposition and scarring. However during an acute S. pn infection, galectin-3 plays a benefitial role to aid the clearance of bacteria through a variety of processes. Therefore, galectin-3 plays a critical role in a variety of inflammatory disorders.

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.

No description available.

Final publication is available at http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=26018088 / Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19635号 / 医科博第73号 / 32671 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妹尾 浩, 教授 渡邊 直樹, 教授 椛島 健治 / 学位規則第4条第1項該当