Neutrophils are essential to innate host defense such that a decrease in the number of these cells in the peripheral blood presents heightened susceptibility to life threatening infections. In addition to being produced in adequate number, host defense also depends on the ability of neutrophils to function effectively. Patients with Leukocyte Adhesion Deficiency have normal numbers of neutrophils but they are genetically deficient in expression of integrins. Neutrophils in patients with Chronic Granulomatous Disease fail to produce adequate levels of microbiocidal oxidative species. In both cases, these functional deficits predispose patients to infections with opportunistic pathogens, recurrent infectious episodes and impairment of wound healing. Alternatively, an excessive neutrophil response can result in disease states characterized by collateral tissue damage such as respiratory distress syndrome, asthma, inflammatory bowel disease, ischemia/reperfusion injury, vasculitis and rheumatoid arthritis. Therefore, neutrophils must execute a sufficient but finely regulated response to infection or injury to promote a return to homeostasis. Neutrophils infiltrate any compromised tissue in the body in order to initiate an inflammatory response regardless of the fact that different bodily tissues offer substantial variations in composition an structure. Like all cells, neutrophils express a set of specific cell surface receptors that elicita functional response to biological elements within a microenvironment. It is clear that the physical nature of a microenvironment, such as its relative stiffness, is also an important regulator of function. The mechanisms that neutrophils use to respond to physical cues are not as clear as they are for receptor-ligand induced responses. Prior work from our laboratory hypothesized that soft, elastic tissues such as brain, may affect neutrophil function differently than stiffer tissues such as skin or muscle. Indeed, neutrophil adhesion, production of traction forces and migration were significantly different on fibronectin-coated matricies that varied only in stiffnes. The stiffnesses were all within the physiological range of tissue stiffnesses found in the body. Although this model provided a more relevant physiologic substrate than rigid materials such as plastic and glass, it is limited in that inflammatory neutrophils function within a 3-dimensional tissue environment. This is a proposal to engineer a tunable 3D in vitro system that will permit neutrophils to be studied under conditions that more closely model a tissue microenvironment. Moreover, the novelty of our system lies in that a given cell can be tracked in 2-dimensions and then in 3-dimensions thereby isolating dimension as a single experimental variable. Experiments will also determine whether integrins mediate the generation of traction forces and control migration of neutrophils in 3D as compared to 2D systems. A 3D in vitro system that accounts for the salient mechanical features of organs and tissues will provide a better means to predict effectiveness of therapeutics indicated for control of inflammation.
Pathology results when an inflammatory response is either insufficient or excessive. One of the reasons new therapeutics designed to optimize inflammation are slow to develop is that research on inflammatory white blood cells such as neutrophils is usually conducted on laboratory dishes and slides composed of glass and plastic that are much stiffer than our bodily tissues. In addition, these flat surfaces only allow for a tw dimensional experiments to be performed. Our previous work showed that neutrophils function differently on surfaces of different stiffnesses. There are also new indications that cells respond differently on 2D surfaces than on 3D surfaces, and that 3D experiments better reflect cell behavior in living tissues. This is a proposal to engineer a system in which cell function can be studied in real time on surfaces that are the same stiffness as in bodily tissues. Furthermore, by adding a top layer of matrix we will be able to directly compare the function of a single cell on a 2D system and a 3D system. This will reveal the importance of studying cells under a microscope under conditions that are close to that encountered in real life.
|Stout, David A; Bar-Kochba, Eyal; Estrada, Jonathan B et al. (2016) Mean deformation metrics for quantifying 3D cell-matrix interactions without requiring information about matrix material properties. Proc Natl Acad Sci U S A 113:2898-903|
|Toyjanova, Jennet; Flores-Cortez, Estefany; Reichner, Jonathan S et al. (2015) Matrix confinement plays a pivotal role in regulating neutrophil-generated tractions, speed, and integrin utilization. J Biol Chem 290:3752-63|