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. 2017 Nov:112:578-586.
doi: 10.1016/j.freeradbiomed.2017.08.026. Epub 2017 Sep 1.

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

Geri Traver  1 , Stacey Mont  1 , David Gius  2 , William E Lawson  3 , George X Ding  1 , Konjeti R Sekhar  1 , Michael L Freeman  4
Affiliations

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

Geri Traver et al. Free Radic Biol Med. 2017 Nov.

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 12Gy 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.

Keywords: Alveolar type 2 cell; Nrf2; Pulmonary fibrosis; Radiation; ΔNp63.

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Conflict of interest statement

None of the authors has a conflict of interest.

Figures

Figure 1
Figure 1
Loss of Nrf2 potentiates radiation-induced tissue remodeling and collagen deposition. Male wild type (A, C, E, G) and Nrf2 null (B, D, F, H) mice were administered 0 (A, B, E, F) or 12 Gy (C, D, G, H) to the thorax and allowed to recover for 250 days. The degree of tissue remodeling was quantified from H&E sections (I, N = 112 fields, 10 mice) while collagen deposition was quantified from Masson’s trichrome staining (J, N = 71 fields, 10 mice). Black bar = 100 μm. Red arrow in panel C identifies tissue remodeling.
Figure 2
Figure 2
Loss of Nrf2 potentiates radiation-induced injury to alveolar type 2 cells. The thorax of male wild type and Nrf2 null mice was administered 0 (A, B) or 12 Gy (C, D) and the mice allowed to recover for 250 days. Wide field whole slide scanning microscopy was used to image Pdpn immunofluorescence (green) in order to identify alveolar type 1 cells. Spc immunofluorescence (red) in alveoli was used to identify and quantify alveolar type 2 cells. DAPI staining identifies nuclei. E) Quantification of Spc immunofluorescent cells per field, corrected for DAPI staining. White bar 100 μm.
Figure 3
Figure 3
Tissue remodeling and Spc immunofluorescence in sham (0 Gy) and irradiated (12 Gy) lung of Nrf2flox/flox and Nrf2Δ/Δ; SPC-Cre mice 250 days after irradiation. A) Representative H&E stained sections. B) Quantification of tissue remodeling in irradiated ROSAmT/mG SFTPC-Cre, Nrf2flox/flox and Nrf2Δ/Δ; SPC-Cre mice. C) Representative sections immunostained for Spc (red) and Pdpn (green), and counter stained with DAPI, imaged using wide field whole slide scanner. microscopy. D) Quantification of relative Spc immunofluorescent cells per field corrected for DAPI staining. Black and white bars = 100 μm.
Figure 4
Figure 4
Loss of Nrf2 suppresses ΔNp63 progenitor cell mobilization in irradiated lung. The thorax of male wild type and Nrf2 null mice was administered 0 (A, B) or 12 Gy (C, D) and the mice allowed to recover for 250 days. Wide field whole slide scanning microscopy was used to image ΔNp63 immunofluoresence with DAPI counter staining. E) Quantification of ΔNp63 immunofluorescent cells per field corrected for DAPI staining. White bar = 10 μM 40x.
Figure 5
Figure 5
Loss of Nrf2 increases EMT events in irradiated lung. The thorax of male wild type and Nrf2 null mice was administered 0 or 12 Gy and the mice were allowed to recover for 250 days. Wide field whole slide scanning microscopy was used to image Spc and αSMA immunofluorescence surrounding DAPI stained nuclei. A) A representative section from a sham treated wild type lung. B) A representative section from an irradiated wild type lung illustrating cells with only Spc immunofluorescence (white arrow). C) A representative section from an irradiated wild type lung illustrating cells with only αSMA immunofluorescence (white arrow). D) A representative section from an irradiated wild type lung illustrating a cell with both Spc and αSMA immunofluorescence (white arrow). E) The frequency of EMT events in wild type and Nrf2 null lung sections obtained from 8 mice. EMT events are defined as DAPI stained nuclei surround by both Spc and αSMA immunofluorescence. 40x, White bar = 10 μm.
Figure 6
Figure 6
Alveolar type 2 cell EMT. Thoraxes of Nrf2 null mice were administered 12 Gy and the mice were allowed to recover for 250 days. Confocal microscopy with Z stack imaging was used to capture Spc immunofluorescent (green) and αSMA immunofluorescent (red) cells. 40x with an additional 3x optical zoom. Panel A illustrates 4 of 9 - Z stack slices (0.58 μm each) covering a total of 4.06 μm. Six nuclei (blue, DAPI) are shown. None of the nuclei in Panel A co-express Spc and αSMA. Panel B illustrates two nuclei (blue, DAPI, white arrows) surrounded by both Spc (green) and αSMA (red), optical section Z5357.96 μm, 40x with an additional 3x optical zoom. Panel C illustrates the same two nuclei as Panel B but at optical section Z5359.12 μm, 40x with an additional 3x optical zoom.
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