Investigations of altered neutrophil apoptosis in Alzheimer’s disease

Abstract

AbstractBackgroundThere is strong evidence that neutrophils are dysregulated in Alzheimer’s disease (AD) and exacerbate disease pathogenesis. Under homeostatic conditions, neutrophils are short‐lived, and the least inflammatory mechanism of neutrophil clearance from tissues is caspase‐3 mediated apoptosis followed by engulfment by macrophages. However, in pathological conditions, neutrophil apoptosis is delayed, resulting in cell death mechanisms that exacerbate inflammation, including necrosis, pyroptosis, and the release of neutrophil extracellular traps (NETosis). Here we address the hypothesis that neutrophil apoptosis is delayed in AD, contributing to increased NET release.MethodWe assessed neutrophil apoptosis, frequency, and activation in blood from 4‐5 month old mice with 5 familial mutations for AD (5xFAD; n = 8) compared with C57BL6/J (B6; n = 8) as controls. Active caspase‐3 was stained in neutrophils using a fluorescent probe and distinguished from secondary necrosis via a membrane impermeable viability stain. Activation was assessed as the percentage of neutrophils expressing low levels of CD62L. Neutrophils were assessed fresh and after 4 hours of rest in complete culture medium. In addition, we investigated the ability of the osmolyte ectoine to rescue neutrophil apoptosis by incubating blood with 1mM ectoine for 4 hours. Finally, plasma was assessed for neutrophil elastase (NE) via ELISA as a marker of NET release.ResultIn 5xFAD blood, we found significantly decreased neutrophil apoptosis and increased total neutrophils compared to B6 blood. Upon resting for 4 hours in complete culture media (R10), neutrophil apoptosis nearly normalized in 5xFAD mice compared to controls, suggesting a continual in vivo stimulus is required. We observed significantly increased NE in plasma of the 5xFAD mice compared with controls that correlated with neutrophil apoptosis. Using a level of ectoine previously demonstrated to promote neutrophil apoptosis, we observed increased neutrophil apoptosis and reduced neutrophil frequencies in blood from B6 mice but not 5xFAD mice after 4 hours.ConclusionThese data provide evidence of dysregulated neutrophil apoptosis in AD that associates with increased NET release. These data also suggest that AD neutrophils may be resistant to some apoptosis promoting agents, prompting further research into the mechanisms involved in delayed neutrophil apoptosis in AD.