Airway Epithelial Progenitors Are Region Specific and Show Differential Responses to Bleomycin-Induced Lung Injury

Mechanisms that regulate regional epithelial cell diversity and pathologic remodeling in airways are poorly understood. We hypothesized that regional differences in cell composition and injury-related tissue remodeling result from the type and composition of local progenitors. We used surface markers and the spatial expression pattern of an SFTPC-GFP transgene to subset epithelial progenitors by airway region. Green fluorescent protein (GFP) expression ranged from undetectable to high in a proximal-to-distal gradient. GFPhi cells were subdivided by CD24 staining into alveolar (CD24neg) and conducting airway (CD24low) populations. This allowed for the segregation of three types of progenitors displaying distinct clonal behavior in vitro. GFPneg and GFPlow progenitors both yielded lumen containing colonies but displayed transcriptomes reflective of pseudostratified and distal conducting airways, respectively. CD24lowGFPhi progenitors were present in an overlapping distribution with GFPlow progenitors in distal airways, yet expressed lower levels of Sox2 and expanded in culture to yield undifferentiated self-renewing progeny. Colony-forming ability was reduced for each progenitor cell type after in vivo bleomycin exposure, but only CD24lowGFPhi progenitors showed robust expansion during tissue remodeling. These data reveal intrinsic differences in the properties of regional progenitors and suggest that their unique responses to tissue damage drive local tissue remodeling. Disclosure of potential conflicts of interest is found at the end of this article

β-Catenin Is Not Necessary for Maintenance or Repair of the Bronchiolar Epithelium

Signaling by Wnt/β-catenin regulates self-renewal of tissue stem cells in the gut and, when activated in the embryonic bronchiolar epithelium, leads to stem cell expansion. We have used transgenic and cell type–specific knockout strategies to determine roles for β-catenin–regulated gene expression in normal maintenance and repair of the bronchiolar epithelium. Analysis of TOPGal transgene activity detected β-catenin signaling in the steady-state and repairing bronchiolar epithelium. However, the broad distribution and phenotype of signaling cells precluded establishment of a clear role for β-catenin in the normal or repairing state. Necessity of β-catenin signaling was tested through Cre-mediated deletion of Catnb exons 2–6 in airway epithelial cells. Functional knockout of β-catenin had no impact on expression of Clara cell differentiation markers, mitotic index, or sensitivity of these cells to the Clara cell–specific toxicant, naphthalene. Repair of the naphthalene-injured airway proceeded with establishment of focal regions of β-catenin–null epithelium. The size of regenerative epithelial units, mitotic index, and restoration of the ciliated cell population did not vary between wild-type and genetically modified mice. Thus, β-catenin was not necessary for maintenance or efficient repair of the bronchiolar epithelium

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

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

An Optimal cis-Replication Stem-Loop IV in the 5′ Untranslated Region of the Mouse Coronavirus Genome Extends 16 Nucleotides into Open Reading Frame 1▿

The 288-nucleotide (nt) 3′ untranslated region (UTR) in the genome of the bovine coronavirus (BCoV) and 339-nt 3′ UTR in the severe acute respiratory syndrome (SARS) coronavirus (SCoV) can each replace the 301-nt 3′ UTR in the mouse hepatitis coronavirus (MHV) for virus replication, thus demonstrating common 3′ cis-replication signals. Here, we show that replacing the 209-nt MHV 5′ UTR with the ∼63%-sequence-identical 210-nt BCoV 5′ UTR by reverse genetics does not yield viable virus, suggesting 5′ end signals are more stringent or possibly are not strictly 5′ UTR confined. To identify potential smaller, 5′-common signals, each of three stem-loop (SL) signaling domains and one inter-stem-loop domain from the BCoV 5′ UTR was tested by replacing its counterpart in the MHV genome. The SLI/II domain (nucleotides 1 to 84) and SLIII domain (nucleotides 85 to 141) each immediately enabled near-wild-type (wt) MHV-like progeny, thus behaving similarly to comparable 5′-proximal regions of the SCoV 5′ UTR as shown by others. The inter-stem-loop domain (nt 142 to 173 between SLs III and IV) enabled small plaques only after genetic adaptation. The SLIV domain (nt 174 to 210) required a 16-nt extension into BCoV open reading frame 1 (ORF1) for apparent stabilization of a longer BCoV SLIV (nt 174 to 226) and optimal virus replication. Surprisingly, pleiomorphic SLIV structures, including a terminal loop deletion, were found among debilitated progeny from intra-SLIV chimeras. The results show the inter-stem-loop domain to be a potential novel species-specific cis-replication element and that cis-acting SLIV in the viral genome extends into ORF1 in a manner that stabilizes its lower stem and is thus not 5′ UTR confined