Dynamics of neutrophilia at the neurovascular unit arising from repeated pulmonary inflammation

The role of neutrophils in mediating neurovascular vulnerability has been increasingly implicated in various acute inflammatory models of neuroimmune crosstalk between the periphery and the brain. This study aimed at understanding the early phases of crosstalk following repetitive inflammation to the lung and ensuing neuropathology. Such a model of frequent inflammatory injury to the lung is pertinent to understanding the focal neurologic risk of constant exposure to aerosolized environmental hazards leading to progressive pulmonary disease. To model repeated pulmonary inflammation, we applied a three-dose regimen of intranasal (i.n.) lipopolysaccharide (LPS) in C57BL/6J mice and studied the impact on the inflammatory environment of the brain, with a specific focus on neutrophil dynamics at the neurovascular unit (NVU). Tissue and circulatory inflammatory profiles were screened via bronchoalveolar lavage (BAL) protein content and cellularity, transcript analysis of brain tissue, and flow cytometry of peripheral blood. Intravital two-photon microscopy (2PM) of the brain vasculature identified neutrophil dynamics at the NVU. Immunofluorescence validated neutrophil dynamics and identified neuroinflammatory hallmarks and peripheral immune factor interactions at the NVU. In vivo findings were corroborated and replicated in murine and human microphysiological systems (MPS) modeling the blood-brain barrier as a proxy demonstration of the translational relevance of our findings. 2PM of tdTomato-Ly6G+ neutrophils demonstrated increased levels of circulating neutrophils and corresponding engagement with cortical brain vasculature after the three-dose repeated i.n. exposure regimen. Neutrophilia at the NVU was corroborated with increased transcript levels of Ly6G and other pro-inflammatory markers. This coordination between endothelial pathophysiology and neutrophil phenotypes was recapitulated in separate murine and human MPS models. Systemic neutrophilia in the lung and circulation was found to be cotemporaneous to neutrophilia at the NVU based on the cellularity of BAL and peripheral blood samples collected at the same endpoints. Immunohistochemical analysis of brain tissue implicates temporal coordination between vascular surface adhesion molecules with changes in neutrophil dynamics from adhesion, crawling, stalling, and transmigration. Extravasation of neutrophils was accompanied by sustained paravascular deposition of fibrinogen and microgliosis up to 72 h after the final i.n. dosing. Microglia-associated effector functions for synaptic pruning and regulation of neutrophil activity demonstrated distinct temporal profiles in the hippocampus independent from transduction along the primary olfactory cortex. Our results identify systemic levels of neutrophilia accompanied by ingress and extravascular accumulation in brain parenchyma that correlated with sustained microglial activation. Pathology to the brain parenchyma is further complemented by the observation of paravascular fibrinogen deposition that alters synaptic metabolism. Thus, we highlight a key role for neutrophil signaling and ensuing neuroimmune interactions from the lung to the brain as a generalizable model of repeated respiratory exposure to inflammatory agents.