The phenomenon of directed cell motion in response to external directional cues has drawn significant interest for more than a century, with the first recorded observations of bacterial chemotaxis at the end of the 19th century. Furthermore, movies generated by David Rogers while at Vanderbilt University of a peripheral blood neutrophil tracking a bacterium are a staple of any college biology class to demonstrate the phenomenon of eukaryotic chemotaxis. In just the last decade, our understanding of the biochemical mechanisms underlying the process of directed eukaryotic cell migration. As a result, several generalized processes have been identified, connecting multiple phenomena from cancer metastasis to axon guidance. Making further sense of the complex biochemical pathways requires both quantitative mathematical models and fine control over the external cellular environment. To this end, microfluidics has proven extremely useful, allowing for precise quantification of both the external environment and the cellular response.

The mechanisms responsible for the enhanced inflammatory response in type 2 diabetes (T2DM) and its contribution to the severe ischemia/reperfusion (I/R) injury observed in the T2DM heart are unclear. I/R is associated with an acute inflammatory response recognized by reactive oxidant production, complement activation, and leukocyte-endothelial cell adhesion, among others. Complement activation plays an important role in the inflammatory response and is involved in the manifestation of I/R injury in the non-diabetic heart, and is a potent chemoattractant for circulating neutrophils (PMNs). The purpose of this dissertation research was to test the hypothesis that the complement system, predominantly the lectin pathway, is a significant contributor to the excessive response of the Zucker Diabetic Fatty (ZDF), a rat model of T2DM, to myocardial I/R injury. Following 30min of coronary artery occlusion and 120min of reperfusion we measured C3 deposition, PMN accumulation, PMN CD11b expression, and ICAM-1 expression. We found significantly more C3 deposition, PMN accumulation, ICAM-1 and PMN CD11b expression in diabetic samples compared to non-diabetic samples. To elucidate a role for complement system activation, we treated animals with FUT-175, a broad complement inhibitor. In vivo, FUT-175 treatment significantly decreased complement deposition (66%), PMN accumulation (59%), and infarct size (55%) compared to untreated animals in both non-diabetic Sprague-Dawley and diabetic ZDF rats. To specifically examine the role of the lectin pathway, we selectively inhibited rat MBL-A prior to myocardial I/R in ZDF rats. Anti-MBL treatment significantly decreased infarct size, C3 deposition and PMN accumulation in the ZDF post-ischemic left ventricle (LV). Genomic analysis revealed that gene expression of the pro-inflammatory cytokines IL-6 and IL-1α was enhanced in the ZDF heart following reperfusion, and quantitative RT-PCR results confirmed IL-6 upregulation. We found significantly increased complement C5a receptor (CD88) expression on diabetic neutrophils prior to ischemia, suggesting that diabetic PMNs are "primed" to respond to complement activation. Taken together, these results provide evidence that 1) the ZDF rat is a good model for chronic inflammation in the setting of T2DM, 2) lectin pathway activation plays a significant role in the inflammatory response to I/R injury in the ZDF heart, and 3) anti-complement therapy may be particularly cardio-protective in T2DM.

Rheumatoid arthritis is an autoimmune disease that can cause chronic inflammation of the joints and other areas of the body. Neutrophils contribute to the pathogenesis of arthritis, and are recruited to the site of inflammation by chemokines. CXCL8/IL-8 is a member of a sub-family of chemokines (ELR+CXC chemokines) that activate and attract neutrophils through the CXCR1 and CXCR2 receptors. Our lab developed a high affinity human CXCR1/CXCR2 antagonist, called human CXCL8(3-72)K11R/G31P (hG31P). This antagonist has been shown to be highly effective in blocking ELR+CXC chemokine-driven neutrophilic inflammation. In this study we looked at the therapeutic effect of blocking ELR+CXC chemokine receptors (CXCR1 and CXCR2) in an experimental model of arthritis. We induced type II collagen (CII)-induced arthritis (CIA) in mice and treated them with hG31P after the onset of disease. The parameters we looked at to assess disease severity were clinical scores (paws were graded on the severity of edema), clinical measurements (measuring inflammation by change in circumference of paw), serum levels of anti-CII antibodies, and inflammatory cytokines mRNA (IL-1β, TNF, KC, and MIP-2) and protein levels (IL-1β, IL-6, KC, and MIP-2) in paw tissue. Initially, when we analyzed all mice together, we were unable to see a change in clinical scores and measurement when CIA mice were treated with hG31P. All CIA mice did not develop arthritis simultaneously, but rather in a serendipitous fashion; therefore we subdivided our mice and analyzed data from mice that developed arthritis early versus those that developed it late. Treatment with hG31P in mice that developed arthritis early (within 5 weeks of initial CII injection) significantly reduced clinical scores (p=0.02) in one, but not both, of our experiments. When CIA mice were treated with hG31P we saw a significant reduction (p<0.05) in CII-specific IgG1 and MIP-2 protein levels in one of our experiments. Our results were variable and we did not see these changes in our other experiment. Treatment of CIA mice with G31P did not significantly affect inflammatory cytokine mRNA levels in the paws. During this study we found the production of anti-hG31P antibodies in our hG31P-treated mice. We used a Ca2+ influx assay to determine if these hG31P antibodies were neutralizing. When these antibodies were non-neutralizing we were able to see a significant reduction in the clinical scores (p=0.02) of our hG31P-treated CIA mice (that had developed early-onset arthritis) when compared to our saline-treated CIA mice. In the experiment in which we detected significant levels of neutralizing anti-hG31P antibodies, treatment with hG31P did not affect the clinical scores of our CIA mice. Although we cannot definitively say that hG31P has a therapeutic effect in CIA, we believe this line of research merits further investigation. Our research suggests to us that after some experimental refinement and reduction of the immune response mounted to hG31P, there could still be potential for hG31P to have a therapeutic effect in arthritis.

The contribution of single complement system (CS) activation pathways to cerebral IR injury has not yet been adequately studied after ischemic stroke and reperfusion. It remains unclear whether a specific activation pathway (alternative, classical or lectin), single complement factors within the CS, or anaphylatoxins are responsible for increased cerebral IR injury after ischemic stroke and reperfusion. Also poorly understood is the relationship between these elements (activation pathways, CS factors and anaphylatoxins) and neutrophil mediated cerebral IR injury. The objective of this dissertation was to test the hypothesis that mannose binding lectin (MBL) deficiency during cerebral ischemia and reperfusion will result in a significant reduction of systemic neutrophil activation and cerebral injury after ischemic stroke and reperfusion via decreased CS activation and subsequent decreased anaphylatoxin production. Using the intraluminal filament method, mice with targeted mutations to MBL A/C genes (MBL-/- ) or the C57Bl/6 strain (MBL +/+ ) were subjected to 60 minutes of cerebral ischemia and either 15 minutes or 24 hours of reperfusion. After reperfusion (15 min and 24 hour) blood was removed to assess systemic neutrophil CD11b expression via flow cytometry. After 24 hours of reperfusion, the brain was removed to assess cerebral injury. CS activation after ischemic stroke and reperfusion was assessed via immunofluorescent C3 staining and RT-rtPCR methods. Our primary findings are, after ischemic stroke and reperfusion: (1) hepatic MBLA gene expression is significantly increased, (2) systemic neutrophils significantly express CD11b, (3) MBL deficiency significantly decreased cerebral infarct volume in the striatum but not in the cortex or total hemisphere, and (4) systemic neutrophil activation is independent of MBL deficiency. This study is the first to examine the contribution of MBL-initiated lectin pathway activation to cerebral IR injury after ischemic stroke and reperfusion. These findings suggest that MBL deficiency does not significantly reduce neutrophil activation or protect brain tissue after ischemic stoke and reperfusion. A complete understanding of reperfusion events after ischemic stroke is necessary for successful development of future stroke therapies to prevent cerebral IR injury. In this way, the acquisition of knowledge from the bench serves the stroke population cared for by nurses at the bedside.

There is no doubt that eosinophils and neutrophils are important cells participating in asthma pathogenesis. The most prominent feature reflecting asthma pathogenesis is late-phase airway inflammation, which occurs a few hours after allergen inhalation. The worldwide published studies on asthma show that most attention is paid to individual, not complex, functions of neutrophils and eosinophils in the airways. Moreover, associations between peripheral blood neutrophil and eosinophil activity and infiltration of these cells in the airways during asthma have not been com¬pletely elucidated yet. There are no data about peripheral blood neutrophil and eosinophil activity during allergen-induced late-phase airway inflam¬mation in asthma patients. Therefore, the aim of this study was to evaluate peripheral blood neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma. We found that an inhaled allergen activates peripheral blood neutrophil and eosinophil chemotaxis, phagocytosis, generation of reactive oxygen species and also reduces apoptosis during late-phase airway inflammation in asthma. Furthermore, altered peripheral blood neutrophil and eosinophil functional activity is related with airway neutrophilia and eosinophilia. Our findings provide new evidence about neutrophil and eosinophil functional activity during allergen-induced late-phase airway inflammation in asthma patients. / Mokslininkai neabejoja, jog eozinofilai ir neutrofilai yra vienos svarbiausių ląstelių, dalyvaujančių astmos patogenezėje, kurią labiausiai atspindi vėlyva kvėpavimo takų uždegimo fazė, išsivystanti praėjus kelioms valandoms po alergeno įkvėpimo. Pasaulinėje literatūroje publikuojami darbai, nagrinėja atskirus kvė¬pavimo takų neutrofilų ir eozinofilų aktyvumo pokyčius. Ypač mažai darbų apie periferinio kraujo neutrofilų ir eozinofilų funkcijas bei jų ryšį su šių ląstelių pagausėjimu kvėpavimo takuose, sergant astma. Taip pat nėra tyrimų, vertinančių periferinio kraujo uždegimo ląstelių (neutrofilų ir eozi¬nofilų) funkcijų alergeno sukeltos vėlyvos fazės kvėpavimo takų uždegimo metu. Todėl šio tyrimo tikslas buvo įvertinti periferinio kraujo neutrofilų ir eozinofilų funkcinį aktyvumą alergeno sukeltos vėlyvos fazės kvėpavimo takų uždegimo metu sergant astma. Tyrimo metu nustatėme, kad įkvėptas alergenas aktyvina periferinio kraujo neutrofilų ir eozinofilų funkcijas - chemotaksį, fagocitozę, reaktyvių deguonies formų susidarymą, degranuliaciją bei silpnina apoptozę vėlyvos fazės kvėpavimo takų uždegimo metu. O šių ląstelių aktyvumo pokyčiai yra susiję su kvėpavimo takų neutrofilija ir eozinofilija. Moksliniame darbe pateikiami rezultatai suteikia naujų duomenų apie sergančiųjų alergine astma periferinio kraujo neutrofilų ir eozinofilų funkcinių savybių ypatumus ir parodo jų pokyčius alergeno sukeltos vėlyvos fazes kvėpavimo takų uždegimo metu.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Traumatic Brain Injury (TBI) accounts for 1,000,000 hospital admissions in the European Union every year and is the leading cause of death in individuals under 45 years of age in both Europe and the United States. This thesis examines the consequences to both the brain and lung following TBI using the lateral fluid percussion injury (FPI) in an in-vivo murine model. In the murine FPI model, alongside cerebral inflammation (associated with neuronal damage and the infiltration of inflammatory cells), there is significant neutrophil accumulation within the pulmonary interstitium 6 and 24 hours after TBI. This was associated with pulmonary haemorrhage and increased vascular permeability. In an attempt to reduce pulmonary injury, 17-DMAG, an HSP90 inhibitor, was applied but proved to be nonprotective. Since patients with TBI show increased susceptibility to bacterial infection, microaspiration and ventilator-induced lung injury, a double-hit model was established whereby mice first received the head injury and then received a lung injury. This demonstrated worse lung injury following intra-tracheal administration of hydrochloric acid after TBI. Depleting neutrophils with an anti-LY-6G depleting antibody improved outcome in this model, indicating increased susceptibility to damage was neutrophil dependent. To test whether neutrophil accumulation within the pulmonary interstitium was specifically related to brain injury, lung tissue following other distant organ injury such as renal ischemia-reperfusion injury (IRI) and renal transplantation were assessed. Significant pulmonary interstitial neutrophil accumulation was seen following both models and was associated with significant pulmonary haemorrhage. Inducing HSP70 activity with an HSP90 inhibitor was shown to be protective by reducing the degree of pulmonary haemorrhage in these models. In an attempt to identify the mechanisms behind neutrophil accumulation in TBI, renal IRI and renal transplantation, ICAM-1 (CD54), a marker of the reverse transmigration of neutrophils was investigated. No differences in ICAM-1 expression were seen following TBI, indicating that another mechanism must be responsible. This mechanism is the focus of on going work within the laboratory. Hypoxia is believed to contribute towards the development of secondary brain injury however little is known regarding its direct contribution. Working alongside chemists at the University of Edinburgh, a number of novel fluorescent hypoxia probes were designed and tested, but none proved to be able to detect hypoxia in-vitro. In conclusion, this thesis has demonstrated that following mild TBI, the lungs are “primed” with a massive interstitial neutrophil influx and that a subsequent micro aspiration of acid induces exaggerated lung injury. The mechanism by which this occurs is the focus of on-going investigation. Pulmonary sequestration of neutrophils is also a predominant feature of other distant organ injuries.

Neutrophils provide a rapid first response to invading pathogens and orchestrate the immune response. They are able to employ potent antipathogenic mechanisms such as phagocytosis, reactive oxygen species (ROS) generation, protease release from granules, and formation of neutrophil extracellular traps (NETs). Despite this, certain pathogens have evolved mechanisms to benefit from neutrophil effector functions. Human cytomegalovirus (HCMV) is a clinically important pathogen that infects the majority of the human population. Monocytes are considered the main vehicle of HCMV dissemination throughout the body, but little research has been done on its interaction with neutrophils. The virus encodes a range of immunomodulatory proteins including an IL-8 homologue that acts as a powerful neutrophil chemoattractant. Viral conservation of a protein that recruits neutrophils to the site of HCMV infection suggests that the interaction between neutrophils and HCMV provides an overall advantage to the virus, but little evidence exists so far to suggest this is the case. Here I report that human peripheral blood neutrophils exposed to a clinical strain of HCMV display a profound survival phenotype, as assessed by morphology, phosphatidylserine exposure, cell permeability, and caspase-3/7 activity. This occurs in the absence of viral gene production. Neutrophils also upregulated their release of inflammatory cytokines in response to HCMV, with higher concentrations of IL-6, IL-8, and MIP-1α detected in the secretomes of infected neutrophils. These secretomes induced monocyte chemotaxis and increased monocyte permissivity to HCMV infection, as well as augmented survival in healthy, uninfected neutrophils. These experiments were confirmed with clean HCMV after the discovery of contaminating Mycoplasma spp. in the viral inocula of the initial experiments. Mycoplasma-HCMV coinfection induced an autophagic phenotype in neutrophils, as assessed by Western blotting and qPCR of autophagy-related components. Inhibition of autophagy using 3-MA reversed a profound survival effect. The unintended inclusion of Mycoplasma spp. further led to the serendipitous discovery of yet another pathogenic ability to overcome neutrophil immune functions: contaminating Mycoplasma spp. as well as Mycoplasma pneumoniae profoundly degraded NETs. These extracellular chromatin structures were stimulated using PMA or pyocyanin, and their release was dependent on the generation of ROS: severely ROS-deficient murine bone marrow neutrophils were unable to generate NETs. However, small amounts of ROS were sufficient for NETs generation, as neutrophils from acute respiratory distress syndrome patients, including many that had attenuated ROS-responses, were still capable of NETs generation. The NETs-degradative properties of mycoplasma were confirmed by fluorescence confocal and scanning electron microscopy, as well as spectrophotometry and agarose gel electrophoresis. This study demonstrates that two pervasive pathogens, HCMV and M. pneumoniae, both frequently found in coinfections in clinical contexts, are able to overcome neutrophil antipathogenic mechanisms to potentially enhance pathogen dissemination. These data provide not only a novel example of manipulation of an anti-viral response in a cell not productively infected, but also a novel example of pathogenic NETs degradation. These findings may have implications on our understanding of mycoplasma and HCMV pathogenesis and provide new targets for the generation of therapies.

Binding of agonist of phagocytic receptors on surface of tumor cells caused significant reduction of tumor growth. This reduction was enhanced by stimulation of TLR receptors. This immunotherapy combines ligation of phagocytic receptors and signaling receptors to achieve anti-tumor effect. The next part of this thesis was focused on the study of mechanism of the anti-tumor activity of neutrophils.

A coleta de hemocomponentes por equipamentos de aférese tem aumentado muito nos últimos anos, sendo considerado um avanço na medicina transfusional, pois possibilita a retirada de um ou mais componentes de um doador único resultando em um hemocomponente padronizado e de alta qualidade. No entanto, os intervalos entre as doações de plaquetaférese em geral são curtos, podendo haver perda de células a cada doação e potencial desregulação do sistema hematopoiético. Pode ocorrer ainda um possível efeito patogênico após passagem das células pelo equipamento de aférese e ativação neutrofílica. Diante disso, há a preocupação se isso acarretaria riscos à saúde do doador em longo prazo. Objetivo: O objetivo deste estudo foi avaliar a perda da expressão de proteínas ancoradas ao glicosilfosfatidilinositol (GPI), presença de clone HPN (hemoglobinúria paroxística noturna) e ativação de neutrófilos em doadores de plaquetaférese de repetição. Métodos: Estudo de caso controle, sendo 44 amostras de doadores de plaquetaférese de repetição e 44 doadores de sangue total controle. Foram coletadas amostras de sangue periférico, marcadas com os anticorpos monoclonais CD157, CD45, CD64, CD10 e FLAER (do inglês, Fluorescent Aerolysin, aerolisina fluorescente) e analisadas por citometria de fluxo. Para análise de ativação de neutrófilos, foram analisadas 17 amostras de doadores de plaquetaférese de repetição e 17 amostras de doadores de sangue total marcadas com CD64. Conclusão: Não foram encontradas alterações significativas na expressão das proteínas ancoradas ao GPI e na expressão de CD64 entre os doadores de plaquetaférese de repetição e os controles. Sugere-se que a doação de plaquetaférese de repetição não altera a expressão de proteínas ancoradas ao GPI, não gera clone HPN tampouco altera a expressão de CD64. Palavras-chave: plaquetaférese, GPI, HPN, ativação de neutrófilos. / The collection of hemocomponents through apheresis equipment has increased much in recent years, which is considered an advance in transfusion medicine because it enables the withdrawal of one or more components from a single donor, resulting in a standardized and high-quality hemocomponent. Nonetheless, the intervals between the plateletpheresis donations are generally short, which can cause loss of cells in each donation and potential dysregulation of the hematopoietic system. What can also happen is a possible pathogenic effect after the transit of the cells through the apheresis equipment and neutrophilic activation. In light of this situation, there is the concern about whether that brings risks to the donor’s health, in the long term. Objective: the objective of this study was to evaluate the loss in the expression of some glycosylphosphatidylinositol-anchored (GPI-anchored) proteins, the presence of PNH clone and neutrophils activation in repeated plateletpheresis donors. Methods: Case-control study using 44 samples of donors of repeated plateletpheresis and 44 samples of donors of whole-blood donors as controls. Peripheral blood samples were collected into tubes containing EDTA, marked with CD157, CD45, CD64, CD10 and FLAER monoclonal antibodies, and analyzed by flow cytometry. For the analysis of neutrophil activation, 17 samples of repeated plateletpheresis donors and 17 samples of whole-blood donors, both marked with CD64 were analyzed. Conclusion: No alteration in the expression of glycosylphosphatidylinositol-anchored proteins and in the CD64 expression was found. It is suggested that repeated plateletpheresis donation does not alter the expression of GPI-anchored proteins, does not generate PNH clone and neither alters the expression of CD64.