The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6th generation of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq).

Rationale: Remodeling and loss of distal conducting airways, including preterminal and terminal bronchioles (pre-TBs/TBs), underlie progressive airflow limitation in chronic obstructive pulmonary disease (COPD). The cellular basis of these structural changes remains unknown. Objectives: To identify biological changes in pre-TBs/TBs in COPD at single-cell resolution and determine their cellular origin. Methods: We established a novel method of distal airway dissection and performed single-cell transcriptomic profiling of 111,412 cells isolated from different airway regions of 12 healthy lung donors and pre-TBs of 5 patients with COPD. Imaging CyTOF and immunofluorescence analysis of pre-TBs/TBs from 24 healthy lung donors and 11 subjects with COPD were performed to characterize cellular phenotypes at a tissue level. Region-specific differentiation of basal cells isolated from proximal and distal airways was studied using an air–liquid interface model. Measurements and Main Results: The atlas of cellular heterogeneity along the proximal–distal axis of the human lung was assembled and identified region-specific cellular states, including SCGB3A2+ SFTPB+ terminal airway-enriched secretory cells (TASCs) unique to distal airways. TASCs were lost in COPD pre-TBs/TBs, paralleled by loss of region-specific endothelial capillary cells, increased frequency of CD8+ T cells normally enriched in proximal airways, and augmented IFN-γ signaling. Basal cells residing in pre-TBs/TBs were identified as a cellular origin of TASCs. Regeneration of TASCs by these progenitors was suppressed by IFN-γ. Conclusions: Altered maintenance of the unique cellular organization of pre-TBs/TBs, including loss of the region-specific epithelial differentiation in these bronchioles, represents the cellular manifestation and likely the cellular basis of distal airway remodeling in COPD.

Rationale: Small airways are the primary site of pathologic changes in chronic obstructive pulmonary disease (COPD), the major smoking-induced lung disorder. Objectives: On the basis of the concept of proximal–distal patterning that determines regional specialization of the airway epithelium during lung development, we hypothesized that a similar program operates in the adult human lung being altered by smoking, leading to decreased regional identity of the small airway epithelium (SAE). Methods: The proximal and distal airway signatures were identified by comparing the transcriptomes of large and small airway epithelium samples obtained by bronchoscopy from healthy nonsmokers. The expression of these signatures was evaluated in the SAE of healthy smokers and smokers with COPD compared with that of healthy nonsmokers. The capacity of airway basal stem cells (BCs) to maintain region-associated phenotypes was evaluated using the air–liquid interface model. Measurements and Main Results: The distal and proximal airway signatures, containing 134 and 233 genes, respectively, were identified. These signatures included known developmental regulators of airway patterning, as well as novel regulators such as epidermal growth factor receptor, which was associated with the proximal airway phenotype. In the SAE of smokers with COPD, there was a dramatic smoking-dependent loss of the regional transcriptome identity with concomitant proximalization. This repatterning phenotype was reproduced by stimulating SAE BCs with epidermal growth factor, which was up-regulated in the SAE of smokers, during differentiation of SAE BCs in vitro. Conclusions: Smoking-induced global distal-to-proximal reprogramming of the SAE represents a novel pathologic feature of COPD and is mediated by exaggerated epidermal growth factor/epidermal growth factor receptor signaling in SAE BCs.

The airway epithelium of cigarette smokers undergoes dramatic remodeling with hyperplasia of basal cells (BC) and mucus-producing cells, squamous metaplasia, altered ciliated cell differentiation and decreased junctional barrier integrity, relevant to chronic obstructive pulmonary disease and lung cancer. In this study, we show that epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) is induced by smoking in human airway epithelium as a result of epidermal growth factor (EGF)-driven squamous differentiation of airway BC stem/progenitor cells. In turn, AREG induced a unique EGFR activation pattern in human airway BC, distinct from that evoked by EGF, leading to BC- and mucous hyperplasia, altered ciliated cell differentiation and impaired barrier integrity. Further, AREG promoted its own expression and suppressed expression of EGF, establishing an autonomous self-amplifying signaling loop in airway BC relevant for promotion of EGF-independent hyperplastic phenotypes. Thus, EGF-AREG interplay in airway BC stem/progenitor cells is one of the mechanisms that mediates the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium.

Epithelial cells (ECs) are usually hypore‐sponsive to various microbial products. Detection of lipopolysaccharide (LPS), the major component of gram‐negative bacteria, is impeded, at least in part, by intracellular sequestration of its receptor, Toll‐like re‐ceptor‐4 (TLR4). In this study, using human bronchial ECs (hBECs) as a model of mucosal epithelium, we tested the hypothesis that the human LPS‐binding, membrane‐active cationic host defense peptide catheli‐cidin LL‐37 augments epithelial response to LPS by facilitating its delivery to TLR4‐containing intracellular compartments. We found that LL‐37 significantly increases uptake of LPS by ECs with subsequent targeting to cholera toxin subunit B‐labeled structures and lyso‐somes. This uptake is peptide specific, dose and time dependent, and involves the endocytotic machinery, functional lipid rafts, and epidermal growth factor receptor signaling. Cathelicidin‐dependent LPS inter‐nalization resulted in significant increased release of the inflammatory cytokines IL‐6 and IL‐8. This indicates that, in ECs, this peptide may replace LPS‐binding protein functions. In polarized ECs, the effect of LL‐37 was restricted to the basolateral compartment of the epithelial membrane, suggesting that LL‐37‐mediated activation of ECs by LPS may be relevant to disease conditions associated with damage to the epithelial barrier. In summary, our study identified a novel role of LL‐37 in host‐microbe interactions as a host factor that licenses mucosal ECs to respond to LPS.— Shaykhiev, R., Sierigk, J., Herr, C., Krasteva, G., Kummer, W., Bals, R. The antimicrobial peptide cathelicidin enhances activation of lung epithelial cells by LPS. FASEB J. 24, 4756–4766 (2010). www.fasebj.org