Aerosol-mediated delivery of AAV2/6-IκBα attenuates lipopolysaccharide-induced acute lung injury in rats

Abstract

Inhibition of the proinflammatory transcription factor NF-κB has previously been shown to attenuate the inflammatory response in tissue after injury. However, the feasibility and efficacy of aerosolized adeno-associated viral (AAV) vector-delivered transgenes to inhibit the NF-κB pathway are less clear. Initial studies optimized the AAV vector for delivery of transgenes to the pulmonary epithelium. The effect of repeated nebulization on the integrity and transduction efficacy of the AAV vector was then examined. Subsequent in vivo studies examined the efficacy of aerosolized rAAV2/6 overexpressing the NF-κB inhibitor IκBα in a rodent endotoxin-induced lung injury model. Initial in vitro investigations indicated that rAAV2/6 was the most effective vector to transduce the lung epithelium, and maintained its integrity and transduction efficacy after repeated nebulization. In our in vivo studies, animals that received aerosolized rAAV2/6-IκBα demonstrated a significant increase in total IκBα levels in lung tissue relative to null vector-treated animals. Aerosolized rAAV2/6-IκBα attenuated endotoxin-induced bronchoalveolar lavage-detected neutrophilia, interleukin-6 and cytokine-induced neutrophil chemoattractant-1 levels, as well as total protein content, and decreased histologic indices of injury. These results demonstrate that aerosolized AAV vectors encoding human IκBα significantly attenuate endotoxin-mediated lung injury and may be a potential therapeutic candidate in the treatment of acute lung injury.

FIG. 1.

(A) In vitro transduction efficiency of rAAV2, rAAV2/5, and rAAV2/6 in PT2SD, A549, and L2 bronchial epithelial cell types, 48 hr postadministration (n=10 per group). (B) Transduction efficiency of rAAV2, rAAV2/5, and rAAV2/6 in whole lung 5 days postadministration (n=5 animals per group). Significance established at ***p<0.0001. All significance was established relative to rAAV2/6. Data are presented as means±standard deviation.

FIG. 2.

(A) Effect of two rounds of nebulization on the titer of rAAV2, rAAV2/5, and rAAV2/6 (n=5 for each group). (B) Effect of nebulization on the transduction efficiency of rAAV2-, rAAV2/5-, and rAAV2/6-β-galactosidase in A549 cells (n=5 per group). Significance established at *p<0.05, **p<0.01, and ***p<0.0001. Significance was established relative to unnebulized sample. Data are presented as means±standard deviation.

FIG. 3.

(A) RT-PCR detection of the exogenous IκBα transgene in the lung 24 hr after LPS or saline sham administration. Each column represents three pools of two animals (n=6 animals) and is the mean of three experimental replicates±standard deviation. (B) Total IκBα expression levels 24 hr after LPS or saline sham administration to the lung and Western blot of IκBα protein expression. Data are presented as the IκBα-to-actin signal ratio normalized to sham control. Significance established at *p<0.05 and **p<0.01. Significance was established relative to the IκBα gene+LPS injury treatment group.

FIG. 4.

FACS analysis of rat pulmonary epithelial cells transduced with AAV expressing IκBα-GFP construct. (A–C) Control; (D–F) IκBα and GFP (1×10¹⁰ drp in 0.3 ml). After 96 hr a PE-labeled epithelial specific antibody (pan-cytokeratin) was used to identify epithelial cells from a single-cell suspension of the lung by FACS. FITC excitation was used to calculate the percentage of cells that were transduced with GFP. Of the epithelial cells stained with the cytokeratin–PE antibody, 21% were positive for GFP (F), whereas only 1.2% were positive in the control (C). Data are presented as percentage of live cells present in the suspension. (G) summarizes the increased FITC excitation and hence IκBα GFP expression in the transduced epithelial lung cells compared with the nontransduced control. Color images available online at www.liebertpub.com/hum

FIG. 5.

(A) IL-6 levels in BAL 24 hr after LPS administration. (B) CINC-1 levels in BAL 24 hr after LPS administration. (C) Total BAL cell counts recorded 24 hr after LPS administration. (D) Absolute neutrophil counts recorded 24 hr after LPS or vehicle administration. Significance established at **p<0.01 and ***p<0.0001. Significance was established relative to the IκBα gene+LPS injury treatment group. Data are presented as minimal value, median, interquartile ranges, and maximal value.

FIG. 6.

(A) Total protein concentration in BAL samples recorded 24 hr after LPS administration. Significance was established relative to the IκBα gene+LPS injury treatment group. Data are presented as minimal value, median, and maximal value. (B) Stereologic assessment of the extent of histologic injury across the treatment groups in LPS and sham injured lung. Significance established at *p<0.05, **p<0.01, and ***p<0.0001. Significance was established relative to the IκBα gene+LPS injury treatment group. Data are presented as means±standard deviation.

FIG. 7.

Photomicrographs (original magnification, ×10) of representative sections of hematoxylin–eosin (H&E)-stained lung tissue from each of the treatment protocols demonstrating the histologic level of ALI. (A) Normal lung (sham). (B) LPS-induced ALI demonstrating inflammation of tissue, and infiltration of macrophages and neutrophils (LPS injury). (C) Vector control treatment (null gene+LPS injury). (D) Attenuation of ALI as indicated by a reduced level of tissue inflammation concomitant with reduced neutrophil and macrophage counts (IκBα gene+LPS injury). Color images available online at www.liebertpub.com/hum