Kevan Roberts - US grants

DESCRIPTION (provided by applicant): Allergic asthma is characterized by airway hyperreactivity and chronic mucosal inflammation mediated by CD4+ Th2 cells. There is increasing evidence to suggest that the chronic inflammation arises as a consequence of a defect in regulatory mechanisms. Our long-term goal is to elucidate which immunomodulatory events are normally operative to limit allergic airway inflammation mediated by CD4+ T cells. Our work has enabled us to formulate the central hypothesis that "allergic pulmonary inflammation is regulated by the action of PGI2 and CD4+CD25+ regulatory T cells, which cooperate in the suppression of lung mucosal Th2 responses". We base this hypothesis from studies demonstrating that selective inhibition of COX-2 in vivo specifically reduced PGI2 production and resulted in a concomitant increase in the level of allergic inflammation. The PGI2 receptor (IP-receptor) was induced by IL-4 and predominantly expressed by CD4+CD25+ T regulatory cells. CD4+CD25+ T cells were shown to play a crucial role in regulating Th2-mediated pulmonary inflammation. 3 aims pertaining to key regulatory mechanisms that control allergic inflammation will be investigated. 1. To elucidate the cellular and molecular requirements for the production of the anti-inflammatory prostanoid PGI2 and examine the induction and function of its receptor during lung inflammation. 2. To determine the mechanism by which CD4+CD25+ T regulatory cells suppress allergic pulmonary inflammation and the role of IL-10 and glucocorticoid-induced TNF receptor in modulating this regulation. The role of CD4+CD25+ T cells in suppressing (i) the development of CD4+ Th2 responses, and (ii) effector Th2-mediated inflammatory responses in vivo will be addressed. 3. To resolve the mechanism by which PGI2 and CD25+ T cells cooperate to limit allergic inflammation and the contribution of this form of immune modulation to NSAID-induced exacerbations of asthma.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The function of the Fluorescence Cytometry Core Facility is to provide state-of-the art technology for investigators to aid them in their quest for understanding cellular processes involved in environmental health challenges. Understanding the processes at this level enables researchers to formulate and test theories as to how environmental factors affect health. These discoveries are important in the quest for disease prevention and treatment. The Core is home to instruments that use fluorescent markers and/or monoclonal antibodies to provide data that illuminate cellular processes. The BD FACSAria" is a state-of-the art flow cytometer and high-speed cell sorter. The three laser, nine filter-set configuration allows the identification of multiple molecular signals to be analyzed simultaneously. Further, the instrument has capabilities of precise sorting of subpopulations of interest for further investigation. The BD FACSCalibur" is a 1-laser, 3-detector flow cytometer that provides basic cell analysis in 5 parameters. The CompuCyte Laser Scanning Cytometer (LSC) offers similar detection of molecular signals as in flow Cytometry, the difference being that the sample is fixed on a microscope slide or cell culture plates, rather than in a fluid suspension. This allows more types of tissues to be analyzed and visualization of individual cells. The Miltenyi Biotec autoMacs" cell sorter purifies subpopulations by magnetic bead separation for further investigation. The Luminex[unreadable] 100" analyzes addressable laser bead arrays for capture and detection of multiple analytes in small samples. The Zeiss" Fluorescence Microscope and Imaging System is used to visualize and capture images of fluorescently tagged cells.

DESCRIPTION (provided by applicant): Since the purchase of our laser scanning cytometer (LSC) in 2000, there have been major advances in technology, both in hardware and software for imaging cytometers. CompuCyte has initiated a program whereby LSC users can upgrade their instrument to the newer iCys imaging cytometer. The upgrade takes advantage of using some of the existing parts of the LSC, including the lasers and video camera with monitor, significantly lowering the cost of purchasing a new system. The microscope will be replaced with an inverted Olympus IX-71 microscope. This change allows high throughput scanning in many formats, including microscope slides, chamber slides, microtiter plates, and Petri dishes. In this way, live cells can be viewed in real time, and measurements can be taken at multiple time points in an assay. Some of the other technological advances of the iCys" include autofocus as the instrument scans various parts of a slide or plate, vastly improving scanning time and efficiency, and new automatic data analysis capabilities. CompuCyte is the only manufacturer of these unique analytical cytometers that allow not only for imaging of biological cell and tissue samples, but also sophisticated quantification of many fluorescence and chromatic parameters. Several investigators are interested in being able to analyze biochemical events in live cells, such as induction of apoptosis, changes in redox status, transcription factor translocation, and receptor- mediated endocytosis. Although the LSC allows end point analysis of these events, it is very difficult to capture the actual sequence and timing. The iCys" upgrade will therefore be a very important addition to our state-of-the-art biomedical research equipment on campus. It will complement the existing flow cytometric and confocal microscopy capabilities, allow for novel research applications, and will become the only quantitative imaging research cytometer in Montana. This puts our investigators at the cutting edge of research technologies and helps keep them competitive for major national grant funding on projects that have profound impacts on our understanding of toxicological and pharmacological events, all of which will affect our ability to address many public health issues. As outlined in the proposal, a number of investigators will benefit from this new system, not only in their existing research projects, but also in new questions that they are currently unable to address. In summary, our proposal covers not only an important upgrade of a valuable instrument that is already in use and maintained within an excellent instrument core facility, but adds significantly to its capabilities in addressing critical biomedical research questions.

PROJECT SUMMARY (See instructions): The mission of the Fluorescence Cytometry (FC) Core Facility is to provide investigators with state-of-the art technology in a cost-effective manner to aid in understanding cellular processes involved in environmental health challenges. Understanding the processes at this level enables researchers to formulate and test theories as to how environmental factors affect health. The Core is home to instruments that use fluorescent markers and/or monoclonal antibodies to investigate cellular processes. The BD FACSAria¿ is a state-of-the art flow cytometer and high-speed cell sorter. The three-laser, ten filter-set configuration allows the identification of multiple molecular signals to be analyzed simultaneously. Further, the instrument has capabilities of precise sorting of subpopulations of interest for further investigation. The BD FACSCalibur¿ is a 1-laser, 3-detector flow cytometer that provides basic cell analysis in 5 parameters. The CompuCyte iCys¿ Laser Scanning Cytometer (LSC) offers similar detection of molecular signals as in flow Cytometry, the difference being that the sample is fixed on a microscope slide or cell culture plates, rather than in a fluid suspension. This allows more types of tissues to be analyzed and permits visualization of tissues and individual cells. The Miltenyi Biotec autoMacs¿ cell sorter purifies subpopulations by magnetic bead separation for further investigation. The Luminex¿ 100¿ analyzes addressable laser bead arrays for capture and detection of multiple analytes in small samples. The Zeiss¿ Fluorescence Microscope and Imaging System is used to visualize and capture images of fluorescently tagged cells. In addition to providing reliable, well-maintained instrumentation, the Core offers scientific expertise in experiment design and analysis. The complexity of the technology of the instruments, reagents and protocols require a thorough understanding and training. Having a Core Director and Staff Scientist available to oversee projects ensures reliable and reproducible results using accepted methodologies. The combination of available instrumentation and expertise puts the researchers at CEHS as well as other investigators at the University of Montana at a competitive advantage in publishing and obtaining extramural funding.

PROJECT SUMMARY Asthma is a chronic inflammatory disease that is increasing in severity and prevalence, particularly in developed countries, and currently affects 11% of the US population. Airborne allergens, including those of the house dust mite (HDM) Dermatophagoides pteronyssinus, are the most common trigger of allergic asthma. The disease is characterized by pulmonary eosinophilic inflammation, type 2 cytokine production, goblet cell hyperplasia, and airway hyperreactivity and remodeling. Recent findings have showed that the innate immune system plays a critical role in the inflammatory response in asthma. It is believed that intrinsic properties of allergens contribute to promoting type 2 innate lymphoid cells that secrete IL-4, IL-5 and IL-13 and amplify CD4+ Th2 differentiation and inflammation. In addition, a series factors idiosyncratic to the airway and host genetics likely facilitate allergic sensitization. However, the immunological events that contribute to this outcome remain largely unclear. IL-15, a cytokine that induces expansion and maturation of natural killer (NK) cells, has been shown to be critically involved in chronic inflammatory processes in the intestinal mucosa. However, very little is known about the contribution of this cytokine to lung mucosal inflammatory responses elicited by inhalation of environmental allergens. Our preliminary findings revealed that intranasal administration of IL-15 complex reduced HDM allergen-induced pulmonary eosinophilic inflammation. Moreover, we have shown that allergic inflammatory responses elicited by allergen entering the airway was compromised in the absence of PGI2-IP receptor signaling because of the increased infiltration of NK cells into the lungs. These studies are based on our observations demonstrating a role for endogenous eicosanoids in regulating the mucosal expression of IL-15 and, de facto, the number and properties of pulmonary NK cells. We will test the overall hypothesis that: IL-15 expression in the lung is regulated by endogenous eicosanoids and this cytokine promotes the accumulation of immunoregulatory NK cells that prevent airway inflammation elicited by airborne allergens by suppressing type 2 innate lymphoid cells. To this end, the aim is to investigate the anti-inflammatory effects of IL-15 on HDM allergen- induced pulmonary inflammation and whether this effect is NK cell-dependent. Moreover, we will examine the role of eicosanoids in regulating endogenous IL-15 production and determine the mechanism of NK cell- mediated suppression of allergic airway inflammation. These studies will advance our knowledge of how IL- 15 and lung resident NK cells prevent sensitization of the airways and inflammatory responses to environmental allergens.

Project Summary/Abstract The Fluorescence Cytometry Core (FCC) at the University of Montana is requesting funding to purchase a state- of-the-art Flow Cytometer and High-speed Sorter. Specifically, we seek to acquire the BD FACSAria Fusion to replace our aging BD FACSAria IIu. This core facility has a successful track record and prides itself on maintaining the highest standards of instrumentation to assist our NIH-funded investigators in their current funded research as well as in their competitive applications for further funding. The University of Montana has made concentrated efforts in the last decade to bring successful biomedical research to the campus and depends on solid and updated infrastructure to recruit and retain high-impact investigators. Our core remains committed to emerging technologies and reliable access to sorting technologies, this necessitates replacement of an aging, but highly utilized BD FACSAria IIu. There are several limitations with the current Aria that the newer version will address. The newer FACSAria Fusion will offer much needed biosafety protection, an additional laser which will allow users to use popular fluorescent proteins used in transgenic applications, updated computer/software package, and critical service and maintenance support by the manufacture. Each of these benefits will be further outlined in this application. We have identified eight major users and two minor users across several departments, colleges, and disciplines at the University of Montana. Disciplines include: immunology, toxicology, developmental biology, cancer, genomic studies, pathogenic entities, and neurobiology. The upgrade of this essential tool will benefit the investigators outlined in this application, as well as new users, future hires, and the entire research community of the University of Montana.