Comparison of in vivo bioluminescence imaging and lavage biomarkers to assess pulmonary inflammation

Abstract

Gram-negative bacterial endotoxin triggers innate immunity via TLR-4 and NF-kB signal activation. The aim of this study was to evaluate the use of transgenic mice expressing luciferase as a marker of NF-kB activation for exploring innate immune responses to pulmonary endotoxin exposure over time thus obviating the need for serial necropsies. Transgenic rNF-kB-Luc BALB/c mice were exposed to two different types of endotoxin (Neisseria meningitidis lipooligosaccharide, and Escherichia coli lipopolysaccharide) at multiple doses by nasal instillation. Bioluminescence was quantified in vivo at five time points in three separate experiments. In the fourth experiment lungs were imaged ex vivo 8h post exposure and tissue was analyzed for luciferase activity. Non-transgenic BALB/c mice were similarly exposed to lipooligosaccharide and bronchoalveolar lavage was assessed for neutrophil recruitment and IL-6. Non-transgenic BALB/c mice exhibited highly significant increases of IL-6 and neutrophils in bronchoalveolar lavage 4h after the exposure to instilled doses as low as 30EU/mouse. In contrast, luciferase imaging of NF-kB signal activation in vivo in transgenic rNF-kB-Luc mice did not show significant changes over time or over doses from 30EU to 300,000EU/mouse of nasally-instilled endotoxin. Ex vivo lung imaging 8h after endotoxin exposure to 3000EU demonstrated a strong signal. An intravenous LPS dose of 300,000EU/mouse produced a measurable luminescence signal in vivo. This non-terminal assessment method is useful only with extremely high doses of endotoxin that induce systemic injury and cannot be applied to research of occupational and environmental exposures at relevant levels of endotoxin.