RANKL confers protection against cell death in precision-cut lung slices

Abstract

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death globally and constitutes a major health problem. The disease is characterized by airflow obstructions due to chronic bronchitis and/or emphysema. Emerging evidence suggests that COPD is the result of impaired epithelial repair. Motivated by the need for more effective treatments, we studied whether receptor activator of nuclear factor κ-Β ligand (RANKL) contributed to epithelial repair, as this protein has been implicated in epithelial regeneration of breast and thymus. To do so, we used precision-cut lung slices prepared from mouse tissue-viable explants that can be cultured ex vivo for up to a few days while retaining features of lung tissue. Slices were cultured with 10, 100, or 500 ng/ml of mouse RANKL for 24 h. We first found RANKL activated nuclear factor κ-Β signaling, which is involved in cellular stress responses, without affecting the general viability of slices. Cell proliferation, however, was not altered by RANKL treatment. Interestingly, RANKL did reduce cell death, as revealed by TUNEL stainings and profiling of apoptosis-related proteins, indicating that it contributes to repair by conferring protection against cell death. This study improves our understanding of lung repair and could create new opportunities for developing COPD treatments.

Keywords: OPGL; TRANCE; chronic bronchitis; emphysema; explant culture; tissue regeneration.

FIGURE 1

Slice viability and morphology. Slices were sampled after slicing (0 h) and after 24 h of incubation without or with 10, 100, or 500 ng/ml RANKL (n = 4). The protein, ATP/protein, and RNA/protein content (A) was determined to assess general slice viability, and H&E stainings (B) were carried out to visualize the morphology. Values represent individual experiments performed in triplicate and are accompanied with the arithmetic mean (horizontal line) ± standard error of the mean (error bars). (^# indicates p < 0.05 between untreated slices at 0 and 24 h).

FIGURE 2

NF-κB signaling. Slices were sampled after slicing (0 h) and after 24 h of incubation without or with 10, 100, or 500 ng/ml RANKL (n = 4). Expression of IκBα and Nfkbia (A), expression of Tnfsf11, Tnfrsf11b, and Tnfrsf11a (B), and concentrations of RANKL, OPG, and RANKL-OPG (C) were analyzed to detect activation of NF-κΒ signaling. Values represent individual experiments performed in triplicate and are accompanied with the arithmetic mean (horizontal line) ± standard error of the mean (error bars). (^# indicates p < 0.05 between untreated slices at 0 and 24 h; *, **, and **** indicate p < 0.05, <0.01, and <0.0001, respectively, between untreated slices and slices treated with exogenous RANKL at 24 h).

FIGURE 3

Cell proliferation. Slices were sampled after slicing (0 h) and after 24 h of incubation without or with 10, 100, or 500 ng/ml RANKL (n = 4). Cyclin D1 mRNA and protein expression (A) were analyzed to assess whether cell proliferation was affected by RANKL. To visualize proliferating cells and ATII cells (arrows), we stained sections for KI67 (B) and SFTPC (C), respectively. Values depict individual experiments performed in triplicate and are accompanied with the arithmetic mean (horizontal line) ± standard error of the mean (error bars). (^# indicates p < 0.05 between untreated slices at 0 and 24 h).

FIGURE 4

Cell death and inflammation. Slices were sampled after slicing (0 h) and after 24 h of incubation without or with 10, 100, or 500 ng/ml RANKL (n = 4). TUNEL stainings (A) were carried out to visualize DNA fragmentation (arrows), and mRNA expression of pro-inflammatory cytokines (B) was measured to study inflammation. Values represent individual experiments performed in triplicate and are accompanied with the arithmetic mean (horizontal line) ± standard error of the mean (error bars). (^# indicates p < 0.05 between untreated slices at 0 and 24 h; * indicates p < 0.05 between untreated slices and slices treated with exogenous RANKL at 24 h).

FIGURE 5

PI3K/Akt signaling and apoptosis. Slices were sampled after slicing (0 h) and after 24 h of incubation without or with 10, 100, or 500 ng/ml RANKL (n = 4). The phosphorylation of PI3K and Akt (A) was analyzed to determine whether the tissue adopted a pro-survival phenotype. We profiled apoptosis-related proteins (B) to obtain more information about stressors and effectors (row z-score). Values represent individual experiments performed in triplicate and are accompanied with the arithmetic mean (horizontal line) ± standard error of the mean (error bars). (^# indicates p < 0.05 between untreated slices at 0 and 24 h).