UC Davis

Neutrophils are vital for elimination of foreign particulates and pathogens, but their inappropriate activation in circulation can lead to tissue damage upon recruitment to sites of inflammation. Thus, neutrophil activation and recruitment is tightly regulated by the synergy between mechanical forces acting on neutrophil adhesion receptors and chemotactic stimulation mediated by inflammatory mediators. However, the synergy between outside-in signaling through selectins and integrins and inside-out signaling via G-protein coupled and Tumor necrosis factor receptors can be altered by foreign materials and chronic exposure to endogenous molecules that signal inflammatory responses such as Toll-like receptors during disease. In this thesis we explored how iron oxide nanoparticles and damage associated molecular patterns DAMPs alter the neutrophil's inflammatory response to chemokines and cytokines in freshly obtained blood samples from healthy individuals and patients with psoriasis. Intravenous delivery of nanoparticles is increasingly being employed for diagnostics and therapeutics. Feraheme is an FDA approved intravenous delivered iron oxide nanoparticle for the treatment of iron deficiency anemia (IDA) in adult patients with chronic kidney failure, but research on its effect on the innate immune response of neutrophils during inflammation has not been reported. Increasing interest in use of Feraheme for novel imaging techniques and therapeutics motivates the first objective of this dissertation; to study the immunomodulatory effects of Feraheme on neutrophil adhesive capacity and responses to GPCR agonists and function. In contrast to foreign nanoparticles, DAMPS and cytokines are produced endogenously to regulate inflammatory responses of immune cells, however during chronic inflammation continuous exposure to inflammatory stimuli can lead to aberrant neutrophil function. Psoriasis is a systemic inflammatory disease that is characterized by exuberant neutrophil recruitment into skin plaques where neutrophils are primed for production of ROS and release of extracellular traps that exacerbate inflammation. Here we explored the role of neutrophil activating cytokines IL-8 and TNF-α as well as DAMPs S100A8/A9 and LL37, known to be upregulated in psoriasis, to prime circulating neutrophils for enhanced β2-integrin-mediated recruitment and effector functions. Exposure to the foreign iron oxide nanoparticles Feraheme had no effect on baseline levels of neutrophil adhesion marker expression, indicating no priming or activation. However, Feraheme treatment reduced neutrophil response to IL-8 mediated upregulation of membrane CD11b/CD18 receptors, activation of high affinity CD18, and shedding of CD62L. Feraheme uptake by neutrophils also inhibited N-formyl-Met-Leu-Phe (fMLP) induced reactive oxygen species (ROS) production. Preincubation of neutrophils with Feraheme greatly reduced capacity capture efficiency on a substrate presenting E-selectin and ICAM-1 in vascular mimetic flow channels. Lastly, Feraheme modulated calcium flux dynamics in neutrophils with accelerated clearance of cytosolic calcium following IL-8 induced flux. Ca2+ is a vital signaling molecule downstream of chemotactic signaling and integrin mechanosignaling. Thus, we conclude that inducing rapid sequestration of calcium is a likely mechanism by which Feraheme uptake inhibits neutrophil chemotactic activation and recruitment efficiency. Psoriatic neutrophils in blood display a primed phenotype characterized by elevated β2-integrin receptor number and binding affinity that correlated with plasma calprotectin levels. Psoriatic neutrophils were exquisitely sensitive to ex vivo stimulation with TNF-α that elicited secretion of calprotectin and synergistically amplified CD18 activation. In accordance with increased β2-integrin activation, psoriatic neutrophils arrest on E-selectin and ICAM-1 twice as efficiently and had greater capacity for calcium flux and production of ROS than healthy neutrophils. These studies highlight the contrasting effects that foreign and innate stimuli can have on modulating neutrophil responsiveness. We report that the Feraheme iron oxide nanoparticles limit neutrophil recruitment and inhibit inflammatory responses to GPCR simulation by accelerating clearance of calcium after stimuli induced flux. In contrast, psoriatic neutrophils were primed for increased adhesion and effector function that correlated with circulating levels of DAMPs and cytokines, particularly S100A8/A9 which is secreted more readily in response to TNF-α by psoriasis neutrophils compared to healthy control.