Dynamics of Neutrophilia at the Neurovascular Unit Arising from Repeated Pulmonary Inflammation

BackgroundThe 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. Whether neurovascular vulnerability is similarly modulated in the context of frequent, but not acute, inflammatory activation in the periphery is the aim of our study. Such a model of frequent inflammatory irritation is pertinent to understanding the neurologic risk of constant exposure to aerosolized environmental hazards leading to progressive pulmonary disease. MethodsTo 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. Results2PM of tdTomato-Ly6G+ neutrophils demonstrated increased levels of circulating neutrophils and corresponding engagement with the 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 physiology and neutrophil phenotypes was recapitulated in murine and human MPS models. System-wide 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 complemented by sustained paravascular deposition of fibrinogen and microgliosis up to 72 hours after the final i.n. dosing. Microglia-associated effector functions for synaptic pruning and regulation of neutrophil activity demonstrated distinct temporal profiles. ConclusionsOur results identify systemic levels of neutrophilia accompanied by ingress and extravascular accumulation in brain parenchyma that correlated with sustained microglial activation. This neutrophil-centric lung-brain interaction is complemented by the observation of paravascular fibrinogen deposition that alters synaptic metabolism. Thus, we propose a mechanistic role for neutrophilia and associated inflammatory dysregulation as essential mediators along the lung-brain neuroimmune axis in a generalizable model of repeated respiratory exposure to inflammatory agents. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/562508v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@113d747org.highwire.dtl.DTLVardef@19a8a5corg.highwire.dtl.DTLVardef@f1a1eaorg.highwire.dtl.DTLVardef@1bd23c7_HPS_FORMAT_FIGEXP M_FIG C_FIG