Perfluorochemical (PFC) liquid enhances recombinant adenovirus vector-mediated viral interleukin-10 (AdvIL-10) expression in rodent lung

Abstract

Adenovirus and cationic liposome mediated transfer of Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, has been shown to decrease pro-inflammatory cytokine levels and overall lung inflammation in models of lung transplantation and injury. Limitations to current approaches of IL-10 gene therapy include poor vector delivery methods and pro-inflammatory properties of human IL-10 under certain conditions. We hypothesize that using perfluorochemical (PFC) liquid to deliver the highly homologous viral IL-10 (vIL-10), which is predominantly anti-inflammatory with minimal pro-inflammatory activities, can potentially be a more effective strategy to combat inflammatory lung diseases. In this study, we compare the use of PFC liquid versus aerosolized method to deliver adenovirus encoding the vIL-10 gene (AdvIL-10) in C57Bl6 mice. Detectable vIL-10 levels were measured from bronchoalveolar lavage fluid and lung homogenates at one, four, ten and thirty days after AdvIL-10. Furthermore, we determined if use of PFC liquid could allow for the use of a lower dose of AdvIL-10 by comparing the levels of detectable vIL-10 at different doses of AdvIL-10 delivered +/- PFC liquid. Results showed that PFC liquid enhanced detectable vIL-10 by up to ten fold and that PFC liquid allowed the use of ten-fold less vector. PFC liquid increased detectable vIL-10 in lung homogenates at all time points; however, the increase in detectable vIL-10 in BAL fluid peaked at four days and was no longer evident by thirty days after intratracheal instillation. In summary, this is the first report utilizing PFC liquid to enhance the delivery of a potentially therapeutic molecule, vIL-10. We believe this strategy can be used to perform future studies on the use of the predominantly anti-inflammatory vIL-10 to treat inflammatory lung diseases.

Figure 1

AdvIL-10 infects lung A549 epithelial cells to produce detectable vIL-10 protein in the culture media and addition of protease inhibitor cocktail preserves detectable vIL-10. A549 cells at were infected with 1000:1 MOI of either Adlac-Z or AdvIL-10. Supernatants collected +/- protease inhibitor cocktail and assayed for vIL-10 by ELISA. Values represent average of two experiments.

Figure 2

Use of PFC liquid increases detectable viral IL-10 in BAL fluid (A) and lung homogenate (B). Viral IL-10 levels from BAL fluid or right lower lobe lung homogenate were collected at 1, 4, 10 and 30 days after intratracheal instillation of AdvIL-10 with PFC liquid (■) or AdvIL-10 with air (●). Each time point represents the mean ± SEM (* p-value < 0.05). Representative experiments from repeated experiments (n = 3–5/group).

Figure 3

PFC liquid allows for use of a lower dose of AdvIL-10. BAL fluid viral IL-10 levels fluid from mice treated with lower dose 1 × 10⁸ particles of AdvIL-10 with PFC liquid (≡), lower dose 1 × 10⁸ particles AdvIL-10 with air (□), or higher dose 1 × 10⁹ particles AdvIL-10 with air (■). Control animals did not receive AdvIL-10. Values represent mean ± SEM (* p-value < 0.05 when compared with lower dose 1 × 10⁸ particles AdvIL-10 alone group, n = 4 per group).

Figure 4

Use of PFC liquid does not potentiate transient lung inflammation provoked by adenovirus delivery. (A) Total cell count from BAL fluid collected 1 day after intratracheal instillation of AdvIL-10 with PFC liquid or AdvIL-10 with air. (B) Cell differential (□ %macrophage, ≡ %neutrophil, ■ %lymphocyte) from BAL fluid collected at 1 day after intratracheal delivery of AdvIL-10 with PFC liquid or AdvIL-10 with air. (C) Total cell count from BAL fluid collected 4 days after intratracheal instillation of AdvIL-10 with PFC liquid or AdvIL-10 with air. (D) Cell differential (□ %macrophage, ≡ %neutrophil, ■ %lymphocyte) from BAL fluid collected at 4 days after intratracheal delivery of AdvIL-10 with PFC liquid or AdvIL-10 with air. Control animals did not receive adenovirus instillation. Values represent mean ± SEM.