Post-translational modification is a fundamental biological regulatory mechanism that controls the location and activity of a wide range of proteins. The goal of this proposed study is to elucidate the role of reactive oxygen species (ROS)-induced actin glutathionylation in controlling actin dynamics in neutrophils. In response to inflammatory stimuli, neutrophils migrate from the circulating blood to infected tissues, where they protect their host by phagocytosing, killing, and digesting bacterial and fungal pathogens. The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators is still poorly understood. We recently reported that NADPH oxidase-dependent physiologically generated ROS negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Protein glutathionylation is a dynamic process and the deglutathionylation is tightly regulated by glutaredoxin 1 (Grx1). Reducing actin glutathionylation by over-expressing Grx1 increased F-actin level, while elevating actin glutathionylation by disrupting Grx1 decreased F-actin level. Consistently, disruption of Grx1 impaired neutrophil chemotaxis. Moreover, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a novel physiological role for glutaredoxi and ROS-induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils, leading us to hypothesize that ROS-induced actin glutathionylation is a key regulatory mechanism that controls neutrophil function in innate immunity. Our previous study demonstrates that actin glutathionylation plays a critical role and needs to be well regulated in neutrophil chemotaxis. To further understand the role of ROS-induced actin glutathionylation in neutrophil recruitment and function during infection and inflammation, we will continue to elucidate the contribution of Grx1 and actin glutathionylation in regulating actin dynamics in cellular processes other than chemotaxis, including adhesion, phagocytosis, bacterial killing, turning, and signal transduction (Aim I). Moreover, the role of Grx1 in regulating neutrophil trafficking in vivo will be investigated in a murine cremaster muscle model using intravital microscopy (Aim II). Finally. we will directly elucidate the role of Grx1 in innate immunity and host defense in a murine Escherichia coli pneumonia model (Aim III).
These studies will provide a better understanding of the role of ROS-induced actin glutathionylation in neutrophil recruitment and function in innate immunity and host defense, with the ultimate goal of establishing ROS, glutaredoxin 1, and related pathways as novel therapeutic targets for modulating neutrophil functions. Accordingly, more efficient and effective therapies can be developed to treat a variety of infectious and inflammatory diseases such as acute lung injury, pneumonia, asthma, multiple sclerosis, and rheumatoid arthritis.
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|Loison, Fabien; Zhu, Haiyan; Karatepe, Kutay et al. (2014) Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation. J Clin Invest 124:4445-58|