The intratracheal administration of locked nucleic acid containing antisense oligonucleotides induced gene silencing and an immune-stimulatory effect in the murine lung

Abstract

Locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) have the potential to modulate the disease-related gene expression by the RNaseH-dependent degradation of mRNAs. Pulmonary drug delivery has been widely used for the treatment of lung disease. Thus, the inhalation of LNA-ASOs is expected to be an efficient therapy that can be applied to several types of lung disease. Because the lung has a distinct immune system against pathogens, the immune-stimulatory effect of LNA-ASOs should be considered for the development of novel inhaled LNA-ASOs therapies. However, there have been no reports on the relationship between knock-down (KD) and the immune-stimulatory effects of inhaled LNA-ASOs in the lung. In this report, LNA-ASOs targeting Scarb1 (Scarb1-ASOs) or negative control LNA-ASOs targeting ApoB (ApoB-ASOs) were intratracheally administered to mice to investigate the KD of the gene expression and the immune-stimulatory effects in the lung. We confirmed that the intratracheal administration of Scarb1-ASOs exerted a KD effect in the lung without a drug delivery system. On the other hand, both Scarb1-ASOs and ApoB-ASOs induced neutrophilic infiltration in the alveoli and increased the expression levels of G-CSF and CXCL1 in the lung. The dose required for KD was the same as the dose that induced the neutrophilic immune response. In addition, in our in vitro experiments, Scarb1-ASOs did not increase the G-CSF or CXCL1 expression in primary lung cells, even though Scarb1-ASOs exerted a strong KD effect. Hence, we hypothesize that inhaled LNA-ASOs have the potential to exert a KD effect in the lung, but that they may be associated with a risk of immune stimulation. Further studies about the mechanism underlying the immune-stimulatory effect of LNA-ASOs is necessary for the development of novel inhaled LNA-ASO therapies.

Fig 1. The KD effects of Scarb1-ASOs and ApoB-ASOs on mouse hepatocytes in vitro.

Mouse hepatocytes were treated with the indicated concentrations of Scarb1-ASOs (A) or ApoB-ASOs (B). After the treatment, the expression levels of Scarb1 or ApoB mRNA were measured. The values represent the mean ± SD of triplicate experiments. ***, P < 0.001, **, P < 0.01, *, P < 0.05 versus Medium group (Williams test).

Fig 2. The KD effect of Scarb1-ASOs on the mouse whole lung in vivo.

Scarb1-ASOs (A) or ApoB-ASOs (B) were intratracheally administered to C57BL/6 mice once a day, for 2 days. One day after the final administration, the right lung was collected and the Scarb1 mRNA expression was measured. The dots indicate each measurement in mice (n = 4). Horizontal bars indicate the mean values. **, P < 0.01 versus the Saline group (Williams test).

Fig 3. The effect of Scarb1-ASOs on the total cell count and the number of neutrophils in bronchoalveolar lavage fluid (BALF).

Scarb1-ASOs were intratracheally administered to C57BL/6 mice once a day, for 2 days. One day after the final administration, BALF was harvested and the total cells (A) and neutrophils (B) in the BALF were analyzed. The dots indicate each measurement in mice (n = 4). Horizontal bars indicate the means. *, P < 0.05 (Wilcoxon rank sum test).

Fig 4. The effect of Scarb1-ASOs on the G-CSF and CXCL1 expression in the lung.

Scarb1-ASOs were intratracheally administered to C57BL/6 mice once a day, for 2 days. One day after the final administration, the right lung and BALF were collected and the G-CSF (A) and Cxcl1 mRNA (B) expression in the lung, and the G-CSF protein (C) and CXCL1 protein (D) expression in the BALF were measured. Dots indicate each measurement in mice (n = 4). Horizontal bars indicate the mean value. *, P < 0.05 (Wilcoxon rank sum test).

Fig 5. The expression of cell surface markers in primary lung cell suspension derived from the mouse lung.

The suspensions were harvested and alveolar macrophages and lung epithelial cells were identified using flow cytometry. Alveolar macrophages were identified as CD45+, CD11b-, F4/80+, and CD11c+. The lung epithelial cells were identified as CD45-, CD326+.

Fig 6. The KD effect of Scarb1-ASOs on primary lung cells in vitro.

Primary lung cells were treated with the indicated concentrations of Scarb1-ASOs for 24 h. The Scarb1 mRNA expression was measured after the treatment. The left Y axis shows the values for the Scarb1-ASO or medium-treated cells. The right Y axis shows the value for the R848-treated cells. The values represent the mean ± SD of triplicate experiments. ***, P < 0.001, versus Medium group (Williams test).

Fig 7. The effect of Scarb1-ASOs on the G-CSF and CXCL1 expression of primary lung cells.

Primary lung cells were treated with the indicated concentrations of Scarb1-ASOs or R848 for 24 h. After the treatment, the G-CSF (A) and CXCL1 (B) mRNA expression in primary lung cells, and the G-CSF (C) and CXCL1 (D) protein expression in the supernatant were measured. The left Y axis shows the value for the Scarb1-ASO- or medium-treated cells. The right Y axis shows the value for the R848-treated cells. The values represent the mean ± SD of triplicate experiments. **, P < 0.01, *, P < 0.05 versus Medium group (Aspin-Welch test).