Rare SOX2+ Airway Progenitor Cells Generate KRT5+ Cells that Repopulate Damaged Alveolar Parenchyma following Influenza Virus Infection

Abstract

Recent studies have implicated keratin 5 (KRT5)⁺ cells in repopulation of damaged lung tissue following severe H1N1 influenza virus infection. However, the origins of the cells repopulating the injured alveolar region remain controversial. We sought to determine the cellular dynamics of lung repair following influenza infection and define whether nascent KRT5⁺ cells repopulating alveolar epithelium were derived from pre-existing alveolar or airway progenitor cells. We found that the wound-healing response begins with proliferation of SOX2⁺ SCGB1A1^- KRT5^- progenitor cells in airways. These cells generate nascent KRT5⁺ cells as an early response to airway injury and yield progeny that colonize damaged alveolar parenchyma. Moreover, we show that local alveolar progenitors do not contribute to nascent KRT5⁺ cells after injury. Repopulation of injured airway and alveolar regions leads to proximalization of distal airways by pseudostratified epithelium and of alveoli by airway-derived epithelial cells that lack the normal characteristics of mature airway or alveolar epithelium.

Keywords: SCGB1A1 lineage; SOX2 lineage; influenza virus; keratin 5; lung injury; lung repair; progenitor cells; wound repair.

Graphical abstract

Figure 1

Nascent KRT5⁺ Cell Appearance Occurs in a Proximal to Distal Pattern following PR8 Influenza Virus Infection in Mice and Results in Remodeling of the Distal Lung Photomicrographs of lung tissue sections from control mice (A–C) and mice recovered for 7 days (D–F), 10 days (G–I), or 14 days (J–L) following infection with PR8 influenza virus. For each time point the distal conducting airways and alveolar parenchyma panels are serial sections. Sections are immunostained for the basal cell marker KRT5 (green) and the pan-epithelial cell marker, E-cadherin (red), or the AT1 cell marker, PDPN (red), and the AT2 cell marker PROSP-C (green). For each panel the zoomed insert shows magnified boxed region without DAPI staining to highlight co-stained regions. At least three animals were analyzed per time point. Scale bar, 100 μm.

Figure 2

Contribution of Pre-existing SFTPC, HOPX, KRT5, and SCGB1A1 Lineage-Labeled Cells to KRT5⁺ Cells after PR8 Infection Sftpc-CreER, Rosa-tdtomato (A), Hopx-CreER, Rosa-mTmG (B), Krt5-CreER, Rosa-mTmG (C), and Scgb1a1-CreER, Rosa-mTmG (D) mice treated with tamoxifen to lineage label AT2, AT1, basal, or club cells, respectively, were infected with PR8 influenza virus and killed at day 14 post infection. Photomicrographs showing representative lung tissue sections immunostained for KRT5 (red), lineage markers (green), and DAPI (blue). In (A) RFP (green) and KRT5 (red) staining was performed on serial sections due to the incompatibility of primary antibodies. For each panel, the boxed region is magnified to highlight representative KRT5⁺ airway cells and alveolar pods. At least three animals were analyzed per time point. Scale bars, 100 μm. See also Figure S1.

Figure 3

Pre-existing SOX2⁺ Cells Are the Major Cellular Source of KRT5⁺ Cells after PR8 Infection Sox2-CreER, Rosa-mTmG mice treated with tamoxifen to lineage label airway epithelial cells were infected with PR8 influenza virus and killed 14 and 21 post infection. Photomicrographs (A–D) showing representative lung tissue sections immunostained for KRT5 (red), lineage markers (green), and DAPI (blue) (A–D). Scale bars, 100 μm. Scatterplots and pie charts show the quantitation and relative contribution, respectively, of HOPX, SFTPC, SCGB1A1, KRT5, and SOX2 cell lineages toward airway (E) and alveolar (F) KRT5⁺ cells after PR8 infection. Individual dots represent cells per animal (E) or cells per pod (F). Data include means ± SEM. Photomicrographs (G and H) show representative lung tissue sections from uninjured animals immunostained for KRT5, SCGB1A1, and FOXJ1 (red), lineage marker (green), and DAPI (blue). Arrows identify SOX2⁺ Lin⁻ cells. At least three animals were analyzed per time point. See also Figures S1–S3 and Table S1.

Figure 4

Activation of Airway-Resident SOX2⁺ Lin⁻ Progenitor Cells in Response to Influenza-Induced Severe Lung Injury The schematic shows that following PR8 influenza virus infection-induced lung injury SOX2⁺ Lin⁻ progenitor cells are the major source of nascent KRT5⁺ cells in the airway and alveolar zones of cell loss. The percent contribution of SOX2⁺ Lin⁺ and SOX2⁺ Lin⁻ progenitor cells has been included.