Targeting murine alveolar macrophages by the intratracheal administration of locked nucleic acid containing antisense oligonucleotides

Abstract

The pulmonary delivery of locked nucleic acid containing antisense oligonucleotides (LNA-ASOs) is expected to be a novel therapeutic approach for lung diseases. However, there are two concerns to be considered: immune responses, as the lung has a distinct immune mechanism to protect it from inhaled pathogens; and leakage into blood, since the lung is permeable to small molecules. As phagocytic alveolar macrophages reside in the alveolar space, it is hypothesized that inhaled LNA-ASOs effectively accumulate and exert a knockdown (KD) effect on these cells at low doses. Thus, targeting alveolar macrophages by inhaled LNA-ASOs may reduce these risks. To test this hypothesis, LNA-ASOs targeting Scarb1 or Hprt1 were intratracheally administered to mice. We confirmed the remarkable accumulation of intratracheally administered LNA-ASOs in murine alveolar macrophages and found that they exerted a significant and sequence-dependent KD effect. The dose required for KD in alveolar macrophages was lower than that required to induce KD in the whole lung. Furthermore, when a KD effect was observed in alveolar macrophages, no KD effect was observed in the liver or kidney; however, several inflammatory cytokines were increased in the lung. These results suggest the potential application of LNA-ASOs as an inhaled drug specific to alveolar macrophages. However, further studies on the immuno-stimulatory effects of LNA-ASOs will be necessary for the development of novel inhaled therapeutic agents.

Keywords: Pulmonary delivery; alveolar macrophages; antisense oligonucleotides; locked nucleic acid.

Figure 1.

The uptake and KD effect of LNA-ASOs in murine peritoneal macrophages in vitro. Thioglycollate-elicited murine peritoneal macrophages were harvested and the expression of CD11b and F4/80 was analyzed using flow cytometry (A). Macrophages were co-cultured with 8, 40, or 200 pg/mL of AF647-conjugated Scarb1-ASOs for 2 h. After which, the cells were harvested and analyzed using flow cytometry (B). The filled histogram shows cells cultured with medium. Representative figures are shown from four independent experiments. Thioglycollate-elicited murine peritoneal macrophages were co-cultured with Scarb1-ASOs or Hprt1-ASOs. After one-day culture, the expression of Scarb1 mRNA (C) or Hprt1 mRNA (D) was measured. The values represent the mean ± SD of four independent experiments. ***p<.001 versus the Medium group (Williams’ test).

Figure 2.

The accumulation of intratracheally administered LNA-ASOs in murine alveolar macrophages. AF647-conjugated Scarb1-ASOs were intratracheally administered to C57BL/6 mice. Two hours later, the lung was collected and AF647-positive cells were detected using flow cytometry. Alveolar macrophages were identified as CD45+, F4/80+, and CD11c+.

Figure 3.

The KD effect of Scarb1-ASOs and Hprt1-ASOs in murine alveolar macrophages in vivo. Scarb1-ASOs or Hprt1-ASOs were intratracheally administered to C57BL/6 mice. One day after the administration, bronchoalveolar cells were collected, and the expression of Scarb1 mRNA (A) and Hprt1 mRNA (B) was measured. The dots indicate each measurement in mice (n = 5). Horizontal bars indicate the mean values. *p<.05 vs. the PBS group (Wilcoxon’s rank sum test).

Figure 4.

The KD effect of Scarb1-ASOs and Hprt1-ASOs in the murine whole lung in vivo. Scarb1-ASOs or Hprt1-ASOs were intratracheally administered to C57BL/6 mice. One day after the administration, the lung was collected, and the expression of Scarb1 mRNA (A) and Hprt1 mRNA (B) was measured. The dots indicate each measurement in mice (n = 5). Horizontal bars indicate the mean values.

Figure 5.

The effect of Scarb1-ASOs and Hprt1-ASOs on the inflammatory cytokine mRNA expression in the lungs. Scarb1-ASOs or Hprt1-ASOs were intratracheally administered to C57BL/6 mice. One day after the administration, the lung was collected, and the expression of G-CSF (A), Cxcl1 (B), TNF-α (C), and IL-6 (D) mRNAs was measured. The dots indicate each measurement in mice (n = 5). Horizontal bars indicate the mean values. *p<.05 by Wilcoxon’s rank sum test.

Figure 6.

The KD effect of Scarb1-ASOs and Hprt1-ASOs in murine liver and kidney in vivo. Scarb1-ASOs or Hprt1-ASOs were intratracheally administered to C57BL/6 mice. One day after the administration, the liver (A and B) and kidney (C and D) were collected and the expression of Scarb1 mRNA and Hprt1 mRNA was measured. The dots indicate each measurement in mice (n = 5). Horizontal bars indicate the mean values.