No description available.

No description available.

The dairy cow experiences a period of immunosuppression around the time of calving that contributes to the increased incidence and severity of infectious diseases observed during this period. This reduction in immune capacity is due in part to the impairment of neutrophil function, a key component of the innate immune system. In fact, the success of the host defense mechanisms against infection depends on the ability of neutrophils to reach the site of the infection, recognize, engulf and ultimately destroy the pathogen using several mechanisms such as the generation and release of reactive oxygen species (ROS) and the recently described neutrophil extracellular traps or NETs. The alteration in some of these functions and the overall killing ability of neutrophils during the periparturient period has been widely described. However, the physiological mechanisms underlying the period of immunosuppression are not completely elucidated. Interestingly, the impairment of these immune defense mechanisms coincides with the profound metabolic changes associated with parturition and lactogenesis. Changes in several hormones and metabolites have been proposed to be the cause of the reduction in neutrophil function, but the effect of insulin on the functional capacity of these cells has not been investigated. Not only does the concentration of plasma insulin fall as parturition approaches, but also the animal experiences a period of impaired insulin action, termed insulin resistance, during this same time-frame. Therefore, we isolated circulating neutrophils from periparturient and midlactating cows and incubated them with insulin alone or in combination with the insulin-sensitizing agent 2,4- thiazolidinedione (TZD). Subsequently, we measured the total, extracellular, and intracellular generation of ROS, NETs release, phagocytic and killing ability. Insulin did not improve any of the parameters used to assess neutrophil function. In contrast, TZD had a potent inhibitory effect on the total ROS generation, despite an increase in extracellular superoxide anion production. Surprisingly, TZD did not alter the ability of neutrophils to phagocytose and/or kill Staphylococcus aureus during an in vitro coculture. Results suggest that TZD can reduce the oxidative stress that neutrophils experience during their respiratory burst and diminish the damage that ROS cause to the surrounding tissue without compromising the capacity of neutrophils to eliminate the invading pathogen.

Chronic, low-grade inflammation is an underlying condition across a globally increasing number of debilitating diseases. These diseases include obesity, atherosclerosis, and diabetes and their resultant low-grade inflammation can be effectivity modeled with low dose stimulants such as lipopolysaccharide (LPS). While the innate immunity plays a significant role in fighting infectious disease, an initial exposure to low dose LPS hinders secondary infection clearance and pre-disposes murine models for fatal sepsis. Neutrophils are the most prevalent circulating innate immune cell and their homotypic aggregation, or swarming, is a key mechanism in clearing pathogens greater than 20 μm in size. We hypothesize that neutrophil swarming ability is altered when in a low dose LPS primed state; potentially leading to an overall altered innate immune response in the face of infection. However, an in vitro model does not currently exist to reliably quantify and compare neutrophil swarms across treatment groups. Here we propose a novel model utilizing fungal zymosan coated beads as a uniform target to which neutrophils may swarm. / Master of Science / White blood cells are critical for our body’s ability to fight off infection. The pathogens that cause infections come in many forms including fungus, viruses, and bacteria. However, in many debilitating inflammatory diseases such as heart disease and obesity, chronic inflammation prevents one’s white blood cells from being able to properly fight off infection. In order to study white blood cell function without the variability that is analogous to living pathogens, we propose a model system that simulates an artificial pathogen target where both the target and the surrounding environment can be precisely controlled. This system can then be used to study white blood cell function, specifically how it may be impacted under inflammatory conditions.

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.

No description available.

Microparticles are small subcellular vesicles, which function in inter-cellular communication by transferring RNA, bioactive lipids, proteins and receptors to target cells. Neutrophil microparticles are abundant in rheumatoid arthritis synovial fluids. Current dogma dictates that cartilage is an impenetrable avascular matrix through which metabolites from the synovial fluid must diffuse; we present the first evidence that microparticles can access chondrocytes through the cartilage. Addition of neutrophil microparticles to chondrocytes in vitro and in vivo afforded protection from arthritogenesis, evidenced by reduced extracellular matrix degradation, increased expression of genes involved in cartilage matrix synthesis and reduced inflammatory mediator production. Adoptive transfer of fluorescently labelled neutrophils into mice with inflammatory arthritis migrated to the inflamed joints and released microparticles, which could be found abundantly within the cartilage. We propose a mechanism whereby microparticles deliver the pro-resolving protein Annexin A1, which engages the receptor FPR2/ALX on the chondrocyte, eliciting tissue protection. This protection could be blocked in part by TGF-β neutralising antibodies and by preventing microparticle phosphatidylserine interaction with chondrocytes by coating with Annexin V. Intriguingly, microparticle treatment directly inhibited the phosphorylation of Hsp27. Hsp27 exists as large oligomers within the resting cell, and upon phosphorylation are released as monomers which function to stabilise the mRNA of certain proinflammatory genes such as IL-8, IL-6 and COX-2; which were effects seen during microparticle/chondrocyte coculture. Thus, microparticles may directly reprogram chondrocytes to prevent the expression of pro-inflammatory cytokines and mediators, but also they exert anti-inflammatory effects via the exposure of phosphatidylserine and induce the production of protective TGF-β by chondrocytes. As cartilage has limited capacity for self-repair, there is an unmet need for therapies that actively repair or protect cartilage, especially in Rheumatoid Arthritis. We envisage these microparticles could offer therapeutic possibilities in the protection of cartilage in situ in these difficult-to-treat patients.

archives@tulane.edu / Neutrophils are the most abundant white blood cells in human blood and require continuous replenishment from bone marrow granulopoiesis throughout life. Their function as phagocytes in innate immunity has been well studied, but the dynamics and movement of neutrophils in vivo are less clearly understood. To quantitate the kinetics of neutrophil movement during homeostasis, acute inflammation, and aging, we applied 5-bromo-2'-deoxyuridine (BrdU) pulse-chase labeling followed by hematology and flow cytometry analyses in healthy, acutely-infected, and aged rhesus macaques (Macaca mulatta). We applied our kinetics results to a mathematical model and calculated that neutrophils have a half-life of 1.63 ± 0.16 days and a daily production of 1.42×109 cells / L / day in heathy young adult rhesus macaques. In rhesus macaques undergoing acute inflammation, we followed neutrophil kinetics during acute stages of a bacterial infection (Shigella) and viral infection (SIV). A massive replenishment of neutrophils from bone marrow to blood as indicated by BrdU kinetics occurred as early as 3 days post Shigella inoculation, the degree to which correlated with the Shigella inoculation doses. As animals aged, neutrophil production declined while plasma G-CSF levels increased, and there was an earlier release, as well as higher in-group variability in neutrophil kinetics, particularly upon reaching 20 years of age or older (equivalent to 70 years or more in humans). This suggested a dysregulated feedback mechanism whereby increased levels of G-CSF failed to restore neutrophil production in elderly rhesus macaques that was associated with induced production of pro-inflammatory cytokines and earlier release of less mature neutrophils and PMN-MDSCs. Taken together, we established a rhesus macaque model to study neutrophil kinetics and functions in vivo during homeostasis, acute inflammation, and aging. Our results emphasized the massive production of neutrophils during homeostasis and the continuous requirement but reduced replenishment capabilities during aging. The significance of the results also indicates an important role for these long-discovered leukocytes in maintaining homeostasis beyond phagocytic pathogen clearance. / 1 / Ziyuan He

Bisphosphonate-induced osteonecrosis of the jaw (BPONJ) has been identified as a severe complication of dental treatment in 1-10% of patients previously treated with intravenous bisphosphonates. The mechanism by which bisphosphonates induce BPONJ is uncertain. It has been noted that necrotic bone from BPONJ sites display signs of bacterial infection that suggests that an immune defect may play a role in the pathophysiology of BPONJ. The purpose of this thesis examined the effect of a potent bisphosphonate, zoledronate, on the innate immune system, specifically, neutrophil function, differentiation and survival with in vitro and in vivo murine models. Zoledronate exposure leads to decreased neutrophil migration, neutrophil NADPH oxidase activity, circulating neutrophil counts, as well as neutrophil survival, however does not appear to affect neutrophil differentiation. We present evidence that bisphosphonates have the potential to depress the immune system in mice and a subset of patients, possibly contributing to the pathogenesis of BPONJ.