Loss of Nrf2 promotes alveolar type 2 cell loss in irradiated, fibrotic lung

Abstract

The development of radiation-induced pulmonary fibrosis represents a critical clinical issue limiting delivery of therapeutic doses of radiation to non-small cell lung cancer. Identification of the cell types whose injury initiates a fibrotic response and the underlying biological factors that govern that response are needed for developing strategies that prevent or mitigate fibrosis. C57BL/6 mice (wild type, Nrf2 null, Nrf2flox/flox, and Nrf2Δ/Δ; SPC-Cre) were administered a thoracic dose of 12 Gy and allowed to recover for 250 days. Whole slide digital and confocal microscopy imaging of H&E, Masson’s trichrome and immunostaining were used to assess tissue remodeling, collagen deposition and cell renewal/mobilization during the regenerative process. Histological assessment of irradiated, fibrotic wild type lung revealed significant loss of alveolar type 2 cells 250 days after irradiation. Type 2 cell loss and the corresponding development of fibrosis were enhanced in the Nrf2 null mouse. Yet, conditional deletion of Nrf2 in alveolar type 2 cells in irradiated lung did not impair type 2 cell survival nor yield an increased fibrotic phenotype. Instead, radiation-induced ΔNp63 stem/progenitor cell mobilization was inhibited in the Nrf2 null mouse while the propensity for radiation-induced myofibroblasts derived from alveolar type 2 cells was magnified. In summary, these results indicate that Nrf2 is an important regulator of irradiated lung’s capacity to maintain alveolar type 2 cells, whose injury can initiate a fibrotic phenotype. Loss of Nrf2 inhibits ΔNp63 stem/progenitor mobilization, a key event for reconstitution of injured lung, while promoting a myofibroblast phenotype that is central for fibrosis.

The Role of Nrf2 in the Response to Normal Tissue Radiation Injury

“…Loss of Nrf2 increases radiation-mediated pulmonary tissue remodeling and collagen deposition (78). Loss of Nrf2 in irradiated lung impairs ΔNp63 stem/progenitor cell mobilization and proliferation, regeneration of alveolar type 2 cells (78), with an outcome of elevated FSP1 expression (79) and enhanced Masson’s trichrome staining of collagen deposition (78), two markers of fibrosis. In injured lung Δp63+/Krt5+ stem cells can be mobilized to proliferate significantly and differentiate into alveolar type 1 and 2 epithelial cells in vitro and in vivo.…”

“…Diphtheria toxin targeting of these stem cells impairs regeneration of injured lung and pulmonary oxygenation while promoting fibrosis, as measured by FSP-1, Masson’s trichrome, and α-smooth muscle staining (80, 81). Thus, it has been hypothesized that ΔNp63 + stem/progenitor cell mobilization contributes to alveolar type 2 cell regeneration after radiation injury (78). However, it is important to note that if there are hypoxic regions in injured lung then HIFα/ Notch signaling reprograms Δp63+/Krt5 + cells to form basal-like metaplasia (82).…”

“…However, it is important to note that if there are hypoxic regions in injured lung then HIFα/ Notch signaling reprograms Δp63+/Krt5 + cells to form basal-like metaplasia (82). Additionally, an Nrf2 deficiency promotes radiation-induced alveolar type 2 cell epithelial-mesenchymal transitions into myofibroblasts (78). At a molecular level it has been shown that Nrf2 can form a nuclear complex with nuclear pSmad3 at CAGA sites located in the proximal promoters of TGF-β target genes (79), suppressing gene expression (79, 83).…”

“…IsoLG adduction of collagen impairs the ability of MMP1 to degrade it, thus contributing to collagen accumulation. Chronic oxidative stress results in accumulation of IsoLG-adducted protein that can evolve into proteotoxic/apoptotic events (78), i.e., recurrent injury.…”

Downregulation of HMGB1 is required for the protective role of Nrf2 in EMT‐mediated PF

“…We and others have previously shown that Nrf2 protects against lung fibrosis in vivo and in vitro (Traver et al, 2017;Zhou et al, 2016). However, the molecular mechanism of Nrf2 in development of EMTmediated PF remains elusive.…”