NOX2-mediated regulation of NLRP3 inflammasome activation in traumatic brain injury

Abstract

Traumatic brain injury (TBI) is described as damage to the brain due to a mechanical impact to the head and is the leading cause of death and disability in developed countries. Following the primary mechanical injury, secondary injury evolves and activates molecular, biochemical, and cellular events such as oxidative stress, cell death and glial cell activation contributing to increased neuroinflammation and neurological impairments. NADPH oxidase 2 (NOX2) is an enzyme complex responsible for reactive oxygen species (ROS) production in phagocytes, including microglia. Chronic NOX2 expression induces oxidative stress, drives neuroinflammation, and leads to progressive cortical and hippocampal degeneration. NOX2 also acts as a priming signal for NLRP3 inflammasome activation, which has been implicated in many neurodegenerative diseases and plays a pivotal role in secondary TBI. GSK2795039 is a small molecule brain penetrable drug that inhibits NOX2 in an NADPH competitive manner. The hypothesis of this study states that pharmacological inhibition of NOX2 by GSK2795039 can attenuate pro-inflammatory mediators, including NLRP3 inflammasome activation, in brain-resident microglia and infiltrating myeloid cells, and improve neurological outcomes following TBI. In vitro, Immortalised Microglial (IMG) cells or primary microglia from p1 Wistar rat pups were used to assess microglial activation. Cells were pre-treated with GSK2795039, diphenyleneiodonium (antioxidant) or MCC950 (NLRP3 inhibitor) and stimulated with lipopolysaccharide (LPS) and ATP/nigericin to induce NOX2/ROS and NLRP3 inflammasome activation. In vivo studies, using controlled cortical impact (CCI), multi-dimensional flow cytometry, histology and neuro-behavioural tasks aimed to translate in vitro findings to an experimental TBI model in C57BL6/J mice. Results from the in vitro studies revealed that GSK2795039 attenuated LPS/nigericin-induced microglial NOX2 activity, ROS production in the cells and reduced nitrite, TNF⍺, LDH, IL-1β and IL-18 release, as well as the major components of the NLRP3 inflammasome; ASC, cleaved caspase-1 and cleaved IL-1β in the conditioned media. In vivo studies demonstrated increased infiltration of NOX2/ROS/Caspase-1/IL-1β+ inflammatory neutrophils and monocytes over a time-course analysis with peak microglial and monocytic NOX2/ROS production at 3 days post-injury (DPI). Pharmacokinetic results indicated that GSK2795039 entered the brain following CCI. Systemic administration of GSK2795039 (100mg/kg; IP) starting 2h post-injury attenuated NOX2+/IL-1β+ microglia/macrophage activation at 3 DPI. In addition, GSK2795039 reduced numbers of IL-1R+CD4+ and IL-1R+CD8+ T cells in the brains of CCI mice, indicating that microglial-T cell crosstalk was altered by treatment. These neuroimmune changes were associated with minor improvements in motor function post-TBI. At 28 DPI, GSK2795039 treatment showed modest improvements in neuro-behavioural and neuropathological deficits. In conclusion, TBI is a highly complex disease resulting from a multitude of secondary injury cascades. Addressing the many research gaps of the innate and adaptive immune responses is essential for future TBI therapies. The findings from this thesis imply the NOX2-NLRP3 inflammatory axis along with microglial-T cell crosstalk as effective targets in the neuroinflammatory response following TBI. Our translational studies indicate that GSK2795039 may be a promising therapeutic drug for mitigating NOX2-mediated neuroinflammation in microglia, and peripheral immune cells, following experimental TBI in mice.