Bioluminescent Detection of Endotoxin Effects on HIV-1 LTR-driven Transcription in Vivo

Abstract

S U M M A R YWe investigated the effects of Gram-negative bacterial lipopolysaccharide (LPS) on luciferase expression in transgenic reporter mice in which luciferase expression is driven by the nuclear factor B (NF-B)-dependent portion of the human immunodeficiency virus-1 (HIV-1) long terminal repeat (HIV-1 LTR). Using these mice, we dissected the sources of luciferase activity at the organ level by (a) assessing luciferase activity in organ homogenates, (b) bioluminescence imaging in vivo, and (c) bioluminescence imaging of individual organs ex vivo. Luciferin dosage was a critical determinant of the magnitude of photon emission from these reporter mice. Photon emission increased at doses from 0.5-6 mg of luciferin given by intraperitoneal (IP) injection. The differential between basal and LPS-induced bioluminescence was maximal at 3-6 mg of luciferin. Luciferase expression was highly inducible in lungs, liver, spleen, and kidneys after a single IP injection of LPS, as assessed by luciferase activity measurements in organ homogenates. Luciferase activity was also induced in the forebrain by treatment with IP LPS. In contrast, aerosolized LPS produced a response localized to the lungs as assessed by both bioluminescence and ex vivo luciferase assay measurements. These studies demonstrate the utility of luciferase reporter mice for determining organ-specific gene expression in response to local and systemic stimuli.

Airway Epithelium Controls Lung Inflammation and Injury through the NF-κB Pathway

“…Escherichia coli LPS (serotype 055:B5) was obtained from Sigma-Aldrich. For studies involving aerosolized LPS, 8 ml of a 1 g/ l LPS solution in PBS was delivered by ultrasonic nebulization in a closed chamber for 30 min using a previously published methodology (13). To deliver systemic LPS, an osmotic pump (2001D Alzet pump; ALZA) filled with LPS solution (1 g/ l in PBS) was implanted surgically in the peritoneal cavity using sterile technique (9).…”

“…Initially, WT and DNTA mice were treated with dox for 1 wk to induce transgene expression and then were administered aerosolized E. coli LPS (8 ml of a 0.1 g/ml solution) as reported previously (13). At 4 h after LPS treatment, lungs were lavaged and harvested for determination of neutrophilic alveolitis and NF-B activation by EMSA.…”

Bioluminescence imaging reveals a significant role for complement in liver transduction following intravenous delivery of adenovirus

“…This relationship was validated by comparing luciferase measurements from the live animals with independent measurements obtained from tissue homogenates as described. 30 These comparisons were accomplished at termination by removal of liver and spleen (mice injected with 2.3 Â 10 9 v.p. ), followed by independent in vitro luciferase analyses as described.…”

TLR4 Mediates Early Graft Failure After Intraportal Islet Transplantation

“…BLI was performed as previously described (38,39) (30,39). Because bioluminescence is dependent on tissue penetration and luciferin dosage, the intensity or light emission may vary.…”

“…Figure 1D), which is consistent with our previous report (19 Figure 2B and C Figure 2D). Reduced proinflammatory cytokines, (34,45,46 (39). Specific TLR expression was analyzed by flow cytometry using anti-mouse …”