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While airway epithelial biorepositories have established roles in the study of bronchial progenitor stem (basal) cells, the utility of a bank of tracheal basal cells from pediatric patients, who have or are suspected of having an airway disease, has not been established.Pediatric patients undergoing bronchoscopy were identified and endotracheal brush (No adverse events were associated with biopsy collection. Of 29 brush biopsies, 16 (55%) were successfully cultured to passage 1/cryopreserved. Samples with higher initial cell yields were more likely to achieve this benchmark. Ten unique donors were then thawed for analysis of differentiation. The average age was 2.2 ± 2.2 years with five donors (50%) having laryngotracheal pathology. Nine donors (90%) demonstrated differentiation capacity at 21 days of culture, as indicated by detection of ciliated cells (ACT+) and mucous cells (MUC5B+).Pediatric tracheal basal cells can be successfully collected and cryopreserved. Recovered cells retain the ability to differentiate into epithelial cell typesLevel 3.

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. We investigated these mechanisms using mouse and human tracheobronchial tissue‐specific stem cells (TSC). In mouse, chromatin labeling and flow cytometry demonstrated that naphthalene (NA) injury activated a subset of TSC. These activated TSC continued to proliferate after the epithelium was repaired and a clone study demonstrated that ~96% of activated TSC underwent terminal differentiation. Despite TSC attrition, epithelial repair after a second NA injury was normal. The second injury accelerated proliferation of previously activated TSC and a nucleotide‐label retention study indicated that the second injury recruited TSC that were quiescent during the first injury. These mouse studies indicate that (a) injury causes selective activation of the TSC pool; (b) activated TSC are predisposed to further proliferation; and (c) the activated state leads to terminal differentiation. In human TSC, repeated proliferation also led to terminal differentiation and depleted the TSC pool. A clone study identified long‐ and short‐lived TSC and showed that short‐lived TSC clones had significantly shorter telomeres than their long‐lived counterparts. The TSC pool was significantly depleted in dyskeratosis congenita donors, who harbor mutations in telomere biology genes. The remaining TSC had short telomeres and short lifespans. Collectively, the mouse and human studies support a model in which epithelial injury increases the biological age of the responding TSC. When applied to chronic lung disease, this model suggests that repeated injury accelerates the biological aging process resulting in abnormal repair and disease initiation., Biological aging of tracheobronchial tissue specific stem cells (TSC) and their trophic unit, the pseudostratified conducting airway epithelium. Each injury activates a subset of TSC which proliferate and undergo terminal differentiation. Thus, each injury cycle depletes the TSC pool and many injuries compromise epithelial regeneration. Over time, biological age exceeds chronological age and increases the risk of chronic lung disease.

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. Goblet and ciliated cell types are derived from tracheobronchial stem/progenitor cells via a Notch-dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and JAG2 is unclear. This study examined JAG1 and JAG2 function using human air-liquid-interface cultures that were treated with γ-secretase complex (GSC) inhibitors, neutralizing peptides/antibodies, or WNT/β-catenin pathway antagonists/agonists. These experiments revealed that JAG1 and JAG2 regulated cell fate determination in the tracheobronchial epithelium; however, their roles did not adhere to simple necessity and sufficiency rules. Biochemical studies indicated that JAG1 and JAG2 underwent posttranslational modifications that resulted in generation of a JAG1 C-terminal peptide and regulated the abundance of full-length JAG2 on the cell surface. GSC and glycogen synthase kinase 3 were implicated in these posttranslational events, but WNT agonist/antagonist studies and RNA-Seq indicated a WNT-independent mechanism. Collectively, these data suggest that posttranslational modifications create distinct assemblies of JAG1 and JAG2, which regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient’s immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

Synthetic scaffolds for the repair of long-segment tracheal defects are hindered by insufficient biocompatibility and poor graft epithelialization. In this study, we determined if extracellular matrix (ECM) coatings improved the biocompatibility and epithelialization of synthetic tracheal grafts (syn-TG). Porcine and human ECM substrates (pECM and hECM) were created through the decellularization and lyophilization of lung tissue. Four concentrations of pECM and hECM coatings on syn-TG were characterized for their effects on scaffold morphologies and on in vitro cell viability and growth. Uncoated and ECM-coated syn-TG were subsequently evaluated in vivo through the orthotopic implantation of segmental grafts or patches. These studies demonstrated that ECM coatings were not cytotoxic and, enhanced the in vitro cell viability and growth on syn-TG in a dose-dependent manner. Mass spectrometry demonstrated that fibrillin, collagen, laminin, and nephronectin were the predominant ECM components transferred onto scaffolds. The in vivo results exhibited similar robust epithelialization of uncoated and coated syn-TG patches, however, the epithelialization remained poor with either uncoated or coated scaffolds in the segmental replacement models. Overall, these findings demonstrated that ECM coatings improve the seeded cell biocompatibility of synthetic scaffolds in vitro, however, they do not improve graft epithelialization in vivo.

Objectives/hypothesis Tissue-engineered tracheal grafts (TETGs) offer a potential solution for repair of long-segment airway defects. However, preclinical and clinical TETGs have been associated with chronic inflammation and macrophage infiltration. Macrophages express great phenotypic heterogeneity (generally characterized as classically activated [M1] vs. alternatively activated [M2]) and can influence tracheal repair and regeneration. We quantified and characterized infiltrating host macrophages using mouse microsurgical tracheal replacement models. Study design Translational research, animal model. Methods We assessed macrophage infiltration and phenotype in animals implanted with syngeneic tracheal grafts, synthetic TETGs, or partially decellularized tracheal scaffolds (DTSs). Results Macrophage infiltration was observed following tracheal replacement with syngeneic trachea. Both M1 and M2 macrophages were present in native trachea and increased during early tracheal repair (P = .014), with an M1/M2 ratio of 0.48 ± 0.15. In contrast, orthotopic implantation of synthetic TETGs resulted in a shift to M1 predominant macrophage phenotype with an increased M1/M2 ratio of 1.35 ± 0.41 by 6 weeks following implant (P = .035). Modulation of the synthetic scaffold with the addition of polyglycolic acid (PGA) resulted in a reduction of M1/M2 ratio due to an increase in M2 macrophages (P = .006). Using systemic macrophage depletion, the M1/M2 ratio reverted to native values in synthetic TETG recipients and was associated with an increase in graft epithelialization. Macrophage ratios seen in DTSs were similar to native values. Conclusions M1 and M2 macrophages are present during tracheal repair. Poor epithelialization with synthetic TETG is associated with an elevation of the M1/M2 ratio. Macrophage phenotype can be altered with scaffold composition and host-directed systemic therapies. DTSs exhibit M1/M2 ratios similar to those seen in native trachea and syngeneic tracheal replacement. Level of evidence NA Laryngoscope, 2021.

Background There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes. Methods Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds. Results AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells. Conclusions The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.

Objective Significant morbidity and mortality are associated with clinical use of synthetic tissue-engineered tracheal grafts (TETG). Our previous work focused on an electrospun polyethylene terephthalate and polyurethane (PET/PU) TETG that was tested in sheep using a long-segment tracheal defect model. We reported that graft stenosis and limited epithelialization contributed to graft failure. The present study determined if the epithelialization defect could be attributed to: 1) postsurgical depletion of native airway basal stem/progenitor cells; 2) an inability of the PET/PU-TETG to support epithelial migration; or 3) compromised basal stem/progenitor cell proliferation within the PET/PU environment. Study design Experimental. Methods Basal stem/progenitor cell frequency in sheep that underwent TETG implantation was determined using the clone-forming cell frequency (CFCF) method. A novel migration model that mimics epithelial migration toward an acellular scaffold was developed and used to compare epithelial migration toward a control polyester scaffold and the PET/PU scaffold. Basal stem/progenitor cell proliferation within the PET/PU scaffold was evaluated using the CFCF assay, doubling-time analysis, and mitotic cell quantification. Results We report that TETG implantation did not decrease basal stem/progenitor cell frequency. In contrast, we find that epithelial migration toward the PET/PU scaffold was significantly less extensive than migration toward a polyester scaffold and that the PET/PU scaffold did not support basal stem/progenitor cell proliferation. Conclusions We conclude that epithelialization of a PET/PU scaffold is compromised by poor migration of native tissue-derived epithelial cells and by a lack of basal stem/progenitor cell proliferation within the scaffold. Level of evidence NA.

The wingless/integrase-1 (WNT)/β-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/β-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of β-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of β-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air–liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the β-catenin agonist CHIR99021 (CHIR) and antagonists to β-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that β-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a β-catenin/P300 complex promotes mucus cell specification and that β-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of β-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the β-catenin signal to favor mucus cell differentiation.

Decellularized tracheal scaffolds offer a potential solution for the repair of long-segment tracheal defects. However, complete decellularization of trachea is complicated by tracheal collapse. We created a partially decellularized tracheal scaffold (DTS) and characterized regeneration in a mouse model of tracheal transplantation. All cell populations except chondrocytes were eliminated from DTS. DTS maintained graft integrity as well as its predominant extracellular matrix (ECM) proteins. We then assessed the performance of DTS in vivo. Grafts formed a functional epithelium by study endpoint (28 days). While initial chondrocyte viability was low, this was found to improve in vivo. We then used atomic force microscopy to quantify micromechanical properties of DTS, demonstrating that orthotopic implantation and graft regeneration lead to the restoration of native tracheal rigidity. We conclude that DTS preserves the cartilage ECM, supports neo-epithelialization, endothelialization and chondrocyte viability, and can serve as a potential solution for long-segment tracheal defects.

SUMMARYChronic lung disease has been attributed to stem cell aging and/or exhaustion. To address this issue, we investigated the lifespan of tracheobronchial tissue stem cells (TSC) over time and in response to repeated injury. Chromatin and nucleotide labeling studies in mice indicated that: 1) injury activated a subset of the TSC pool and that this process conserved TSC over time; and 2) activated TSC were predisposed to further proliferation and this activated state lead to terminal differentiation. Analysis of human TSC and clonal isolates indicated that repeated TSC proliferation led to telomere shortening and analysis of TSC from Dyskeratosis Congenita donors indicated that mutations in telomere biology genes accelerated TSC depletion. RNAseq and functional studies indicated that human TSC terminated as a secretory committed cell. These data support a model in which a repeated epithelial injury depletes the TSC pool and initiates the abnormal repair associated with chronic lung disease.

Cystic fibrosis (CF) is an autosomal recessive disease caused by variations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although CF affects multiple organs, the primary cause of mortality is respiratory failure resulting from poor clearance of hyperviscous secretions and subsequent airway infection. Recently developed CFTR modulators provide significant therapeutic benefit to the majority of CF individuals. However, treatments directed at the underlying cause are needed for the ∼7% of CF patients who are not expected to be responsive to these modulators. Genome editing can restore the native CFTR genetic sequence and function to mutant cells, representing an approach to establish durable physiologic CFTR correction. Although editing the CFTR gene in various airway cell types may transiently restore CFTR activity, effort is focused on editing airway basal stem/progenitor cells, since their correction would allow appropriate and durable expression of CFTR in stem cell-derived epithelial cell types. Substantial progress has been made to directly correct airway basal cells in vitro, theoretically enabling transplantation of autologous corrected cells to regenerate an airway with CFTR functional cells. Another approach to create autologous, gene-edited airway basal cells is derivation of CF donor-specific induced pluripotent stem cells, correction of the CFTR gene, and subsequent directed differentiation to airway basal cells. Further work is needed to translate these advances by developing effective transplantation methods. Alternatively, gene editing in vivo may enable CFTR correction. However, this approach will require robust delivery methods ensuring that basal cells are efficiently targeted and corrected. Recent advances in gene editing-based therapies provide hope that the genetic underpinning of CF can be durably corrected in airway epithelial stem cells, thereby preventing or treating lung disease in all people with CF.

OBJECTIVES/HYPOTHESIS The ideal trachea replacement would be a living graft that is genetically identical to the host, avoiding the need for immunosuppression. We have developed a mouse model of syngeneic tracheal transplant that results in long-term survival without graft stenosis or delayed healing. To understand how host cells contribute to tracheal transplant integration, we quantified the populations of host cells in the graft and native trachea following implant. STUDY DESIGN Tracheal transplant, tracheal replacement, regenerative medicine, animal model. METHODS Tracheal grafts were obtained from female C57BL/6 mice and orthotopically transplanted into syngeneic male recipients. Cohorts were euthanized on day 14, day 45, and day 90 post-transplantation. Host and graft tracheas were explanted and analyzed by histology. Male host cells were quantified using fluorescence in situ hybridization, and macrophages were quantified with immunofluorescence. RESULTS Evidence of host-derived cells was found in the midgraft at the earliest time point (14 days). Host-derived cells transiently increased in the graft on day 45 and were predominantly found in the submucosa. By day 90, the population of host-derived cells population declined to a similar level on day 14. Macrophage infiltration of host and graft tissue was observed at all time points and was greatest on day 90. CONCLUSIONS Tracheal graft integration occurs by way of subacute transient host-cell infiltration and is primarily inflammatory in nature. Host-cell contribution to the graft epithelium is limited. These data indicate that creation of living, nonimmunogenic tracheal graft could serve as a viable solution for long-segment tracheal defects. LEVEL OF EVIDENCE 3 Laryngoscope, 131:E340-E345, 2021.

Background The conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury, each TSC repairs a discrete area of the epithelium. In contrast, severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis, LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients. Methods Airway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity. Results LTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91% of anastomotic sites and 93% of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33% of allograft regions were at risk for abnormal CFTR activity. Conclusions LTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium.

Introduction Congenital or acquired tracheal lesions alter airway epithelial structure and can lead to long-segment tracheal defects. Tissue engineered tracheal grafts (TETG) have the potential to cure such defects; however, clinical applications have been plagued with numerous complications including delayed graft epithelialization. The knowledge that epithelial cells migrate from native tissue to the TETG raises the possibility that TETG performance can be improved by increasing the rate of epithelialization. Objectives We developed a model that can be used quantify epithelial migration in clinically-relevant conditions. Methods Existing histological analyses determined the differentiation status of the normal and injured human tracheal epithelium and were used to identify in vitro culture conditions that mimic these parameters. The classical scratch assay was adapted to permit analysis of migratory velocity as a function of differentiation status. Migration of undifferentiated (UD), partially-differentiated (PD), and well-differentiated (WD) epithelia was quantified. Results The normal and injured epithelium can be modeled using human cells that are cultured using a modified air-liquid-interface culture system. PD cell cultures are similar to the remodeled epithelium; whereas; WD cultures are similar to the normal epithelium. Preliminary results indicate that PD cells migrate more rapidly than WD cells and that PD and WD cells migrate more rapidly than UD cells. Conclusion Pending verification of these results, we suggest that epithelial migration is significantly altered by differentiation status. Thus, efforts to improve TETG epithelialization should use model systems that faithfully-represent the differentiation state of the native tissue.

We report that the quantitative western blot (qWB) analysis requires a target protein-specific approach, and we provide a workflow that streamlines development of this process. First, the optimal primary antibody dilution is determined. Blots containing 15 μg total protein per lane are probed with the primary antibody at three concentrations and a secondary antibody concentration that is defined by the manufacturer. The lowest primary antibody concentration that detects a discrete band at the correct molecular weight is used in the remaining two steps. Secondly, the optimal protein load is determined. Blots containing 3.75 to 60 μg protein per lane are probed using the antibody concentrations defined in step 1. A target protein band intensity vs. protein load plot is used to determine the linear dynamic range (LDR) for the target protein. The midpoint of the LDR is defined as the optimal protein load. Finally, an appropriate loading control (LC) is identified. We found that the LDR for β-actin, a commonly used LC, exhibited a narrow range, 3.75 to 15 μg. In contrast, the total protein assessed by a Ponceau staining method exhibited a broader LDR, 3.75 to 60 μg. Thus, the total protein is used as a LC. We conclude that the sensitivity and accuracy of the qWB method is dependent on the use of an optimal: 1) primary antibody dilution; 2) total protein load; 3) and LC. Our workflow simplifies the identification of these values.

OBJECTIVES. Humans receiving tissue-engineered tracheal grafts have experienced poor outcomes ultimately resulting in death or the need for graft explantation. We assessed the performance of the synthetic scaffolds used in humans with an ovine model of orthotopic tracheal replacement, applying standard postsurgical surveillance and interventions to define the factors that contributed to the complications seen at the bedside. STUDY DESIGN. Large animal model. SETTING. Pediatric academic research institute. SUBJECTS AND METHODS. Human scaffolds were manufactured with an electrospun blend of polyethylene terephthalate and polyurethane reinforced with polycarbonate rings. They were seeded with autologous bone marrow–derived mononuclear cells and implanted in sheep. Animals were evaluated with routine bronchoscopy and fluoroscopy. Endoscopic dilation and stenting were performed to manage graft stenosis for up to a 4-month time point. Grafts and adjacent native airway were sectioned and evaluated with histology and immunohistochemistry. RESULTS. All animals had signs of graft stenosis. Three of 5 animals (60%) designated for long-term surveillance survived until the 4-month time point. Graft dilation and stent placement resolved respiratory symptoms and prolonged survival. Necropsy demonstrated evidence of infection and graft encapsulation. Granulation tissue with signs of neovascularization was seen at the anastomoses, but epithelialization was never observed. Acute and chronic inflammation of the native airway epithelium was observed at all time points. Architectural changes of the scaffold included posterior wall infolding and scaffold delamination. CONCLUSIONS. In our ovine model, clinically applied synthetic tissue-engineered tracheas demonstrated infectious, inflammatory, and mechanical failures with a lack of epithelialization and neovascularization.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

On behalf of the Clinical Care Consortium for Telomere-associated Ailments (CCCTAA)., Vascular complications such as bleeding due to gastrointestinal telangiectatic anomalies, pulmonary arteriovenous malformations, hepatopulmonary syndrome, and retinal vessel abnormalities are being reported in patients with telomere biology disorders (TBDs) more frequently than previously described. The international clinical care consortium of telomere-associated ailments and family support group Dyskeratosis Congenita Outreach, Inc. held a workshop on vascular abnormalities in the TBDs at the National Cancer Institute in October 2017. Clinicians and basic scientists reviewed current data on vascular complications, hypotheses for the underlying biology and developed new collaborations to address the etiology and clinical management of vascular complications in TBDs.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 10(10) cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632-treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell-cell junctions, and cell-extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell-cell and cell-ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.

Respiratory syncytial virus (RSV) is a common virus that can have varying effects ranging from mild cold-like symptoms to mortality depending on the age and immune status of the individual. We combined mathematical modelling using ordinary differential equations (ODEs) with measurement of RSV infection kinetics in primary well-differentiated human bronchial epithelial cultures in vitro and in immunocompetent and immunosuppressed cotton rats to glean mechanistic details that underlie RSV infection kinetics in the lung. Quantitative analysis of viral titre kinetics in our mathematical model showed that the elimination of infected cells by the adaptive immune response generates unique RSV titre kinetic features including a faster timescale of viral titre clearance than viral production, and a monotonic decrease in the peak RSV titre with decreasing inoculum dose. Parameter estimation in the ODE model using a nonlinear mixed effects approach revealed a very low rate (average single-cell lifetime > 10 days) of cell lysis by RSV before the adaptive immune response is initiated. Our model predicted negligible changes in the RSV titre kinetics at early times post-infection (less than 5 dpi) but a slower decay in RSV titre in immunosuppressed cotton rats compared to that in non-suppressed cotton rats at later times (greater than 5 dpi) in silico. These predictions were in excellent agreement with the experimental results. Our combined approach quantified the importance of the adaptive immune response in suppressing RSV infection in cotton rats, which could be useful in testing RSV vaccine candidates.

The human airway epithelium is regenerated by basal cells. Thus, basal cell therapy has the potential to cure cystic fibrosis (CF) lung disease. We previously reported that the human basal cells repopulated the mouse airway epithelium after transplantation, and we estimated that 60 million cells would be needed to treat a human patient. To further develop cell therapy, we compared the proliferation potential of non-CF and CF tissue-derived bronchial basal cells. Three methods were used: regenerative cell frequency, burst size, and cell division frequency. Second, we used a serial passage strategy to determine if CF basal cells could be amplified to the estimated therapeutic dose. These studies evaluated that tissue-derived bronchial basal cells and the basal cells that were recovered by brushing bronchial airways or the nasal respiratory epithelium. Finally, we used the limiting dilution method to isolate non-CF and CF basal cell clones. The proliferation assays and the air-liquid-interface differentiation method were used to determine if cell amplification altered the proliferation and/or differentiation potential of clonal isolates. We demonstrate that: (a) non-CF and CF basal cell proliferation is similar, (b) CF basal cells can be amplified to a therapeutic cell dose, and (c) amplified non-CF and CF basal cell clones differentiate normally. Despite these encouraging findings, we also find that the cell amplification process depletes the regenerative basal cell pool. Analysis of basal cell clones indicates that serial passage selects for long-lived basal cells and raise the possibility that prospective isolation of these stem-like cells will improve the efficacy of cell replacement therapy. Stem Cells Translational Medicine 2019;8:225&235

ERB family receptors (EGFR, ERB-B2, ERB-B3, and ERB-B4) regulate epithelial cell function in many tissue types. In the human airway epithelium, changes in ERB receptor expression are associated with epithelial repair defects. However, the specific role(s) played by ERB receptors in repair have not been determined. We aimed to determine whether ERB receptors regulate proliferation of the tracheobronchial progenitor, the basal cell. Receptor tyrosine kinase arrays were used to evaluate ERB activity in normal and naphthalene (NA)-injured mouse trachea and in air-liquid interface cultures. Roles for epidermal growth factor (EGF), EGFR, and ERB-B2 in basal cell proliferation were evaluated in vitro. NA injury and transgenic expression of an EGFR-dominant negative (DN) receptor were used to evaluate roles for EGFR signaling in vivo. EGFR and ERB-B2 were active in normal and NA-injured trachea and were the only active ERB receptors detected in proliferating basal cells in vitro. EGF was necessary for basal cell proliferation in vitro. The EGFR inhibitor, AG1478, decreased proliferation by 99, and the Erb-B2 inhibitor, AG825, decreased proliferation by ∼66%. In vivo, EGFR-DN expression in basal cells significantly decreased basal cell proliferation after NA injury. EGF and EGFR are necessary for basal cell proliferation. The EGFR/EGFR homo- and the EGFR/ERB-B2 heterodimer account for ∼34 and 66%, respectively, of basal cell proliferation in vitro. Active EGFR is necessary for basal cell proliferation after NA injury. We conclude that EGFR activation is necessary for mouse basal cell proliferation and normal epithelial repair.

In the early stages of cilia formation in the mouse airway epithelium, Chibby is recruited to the distal appendages of centrioles and is necessary for efficient ciliary vesicle formation and basal body docking at the apical cell membrane., Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.

Tissue-specific stem cell (TSC) number is tightly regulated in normal individuals but can change following severe injury. We previously showed that tracheobronchial epithelial TSC number increased after severe naphthalene (NA) injury and then returned to normal. This study focused on the fate of the supernumerary TSC and the signals that regulate TSC pool size. We used the Keratin 5-rTA/Histone 2B:green fluorescent protein (GFP) model to purify basal cells that proliferated infrequently (GFPbright) or frequently (GFPdim) after NA injury. Both populations contained TSC but TSCs were 8.5-fold more abundant in the GFPbright population. Interestingly, both populations also contained a unipotential basal progenitor (UPB), a mitotic basal cell subtype whose daughters were terminally differentiated basal cells. The ratio of TSC to UPB was 5:1 in the GFPbright population and 1:5 in the GFPdim population. These data suggested that TSC proliferation in vivo promoted TSC-to-UPB differentiation. To evaluate this question, we cloned TSC from the GFPbright and GFPdim populations and passaged the clones seven times. We found that TSC number decreased and UPB number increased at each passage. Reciprocal changes in TSC and UPB frequency were more dramatic in the GFPdim lineage. Gene expression analysis showed that β-catenin and Notch pathway genes were differentially expressed in freshly isolated TSC derived from GFPbright and GFPdim populations. We conclude that (a) TSC and UPB are members of a single lineage; (b) TSC proliferation in vivo or in vitro promotes TSC-to-UPB differentiation; and (c) an interaction between the β-catenin and Notch pathways regulates the TSC-to-UPB differentiation process. Stem Cells 2013;31:2767–2778

Tracheal brush cells (BCs) are specialized epithelial chemosensors that use the canonical taste transduction cascade to detect irritants. To test whether BCs are replaced at the same rate as other cells in the surrounding epithelium of adult mice, we used 5-bromo-2′-deoxyuridine (BrdU) to label dividing cells. Although scattered BrdU-labeled epithelial cells are present 5–20 days after BrdU, no BCs are labeled. These data indicate that BCs comprise a relatively static population. To determine how BCs are generated during development, we injected 5-day-old mice with BrdU and found labeled BCs and non-BC epithelial cells 5 days after BrdU. During the next 60 days, the percentage of labeled BCs increased, whereas the percentage of other labeled cell types decreased. These data suggest that BCs are generated from non-BC progenitor cells during postnatal tracheal growth. To test whether the adult epithelium retains the capacity to generate BCs, tracheal epithelial cells were recovered from adult mice and grown in an air–liquid interface (ALI) culture. After transition to differentiation conditions, BCs are detected, and comprise 1% of the total cell population by Day 14. BrdU added to cultures before the differentiation of BCs was chased into BCs, indicating that the increase in BC density is attributable to the proliferation of a non-BC progenitor. We conclude that: (1) BCs are normally a static population in adult mice; (2) BC progenitors proliferate and differentiate during neonatal development; and (3) BCs can be regenerated from a proliferative population resident in adult epithelium.

Cell therapy has the potential to cure disease through replacement of malfunctioning cells. Although the tissue stem cell (TSC) is thought to be the optimal therapeutic cell, transplantation of TSC/progenitor cell mixtures has saved lives. We previously purified the mouse tracheobronchial epithelial TSCs and reported that in vitro amplification generated numerous TSCs. However, these cultures also contained TSC-derived progenitor cells and TSC repurification by flow cytometry compromised TSC self-renewal. These limitations prompted us to determine if a TSC/progenitor cell mixture would repopulate the injured airway epithelium. We developed a cell transplantation protocol and demonstrate that transplanted mouse and human tracheobronchial epithelial TSC/progenitor cell mixtures are 20-25% of airway epithelial cells, actively contribute to epithelial repair, and persist for at least 43 days. At 2 weeks after transplantation, TSCs/progenitor cells differentiated into the three major epithelial cell types: basal, secretory, and ciliated. We conclude that cell therapy that uses adult tracheobronchial TSCs/progenitor cells is an effective therapeutic option.

Airway epithelial healing is defined as restoration of health or soundness; to cure. Our research indicates that two types of progenitor cells participate in this process: the tissue-specific stem cell (TSC) and the facultative basal progenitor (FBP). The TSC restores the epithelium to its normal structure and function. Thus, the TSC regenerates the epithelium. In contrast, the FBP-derived epithelium is characterized by regions of cellular hyperplasia and hypoplasia. Since the FBP-derived epithelium deviates from normal, we term the FBP-mediated process repair. Our work indicates that the TSC responds to signals from other epithelial cells, including the FBP. These signals instruct the TSC to proliferate or to select one of several differentiation pathways. We interpret these data in the context of Stephen Padget’s “seed and soil” paradigm. Therein, Padget explained that metastasis of a tumor, the seed, to a specific site, the soil, was determined by the growth and differentiation requirements of the tumor cell. By extending the seed and soil paradigm to airway epithelial healing, we suggest that proliferation and differentiation of the TSC, the seed, is determined by its interactions with other cell types, the soil. Based on this concept, we provide a set of suggestions for development of cell-based therapies that are directed toward chronic airways disease.

Injury to the epithelium is integral to the pathogenesis of many inflammatory lung diseases, and epithelial repair is a critical determinant of clinical outcome. However, the signaling pathways regulating such repair are incompletely understood. We used in vitro and in vivo models to define these pathways. Human neutrophils were induced to transmigrate across monolayers of human lung epithelial cells in the physiological basolateral-to-apical direction. This allowed study of the neutrophil contribution not only to the initial epithelial injury, but also to its repair, as manifested by restoration of transepithelial resistance and reepithelialization of the denuded epithelium. Microarray analysis of epithelial gene expression revealed that neutrophil transmigration activated β-catenin signaling, and this was verified by real-time PCR, nuclear translocation of β-catenin, and TOPFlash reporter activity. Leukocyte elastase, likely via cleavage of E-cadherin, was required for activation of β-catenin signaling in response to neutrophil transmigration. Knockdown of β-catenin using shRNA delayed epithelial repair. In mice treated with intratracheal LPS or keratinocyte chemokine, neutrophil emigration resulted in activation of β-catenin signaling in alveolar type II epithelial cells, as demonstrated by cyclin D1 expression and/or reporter activity in TOPGAL mice. Attenuation of β-catenin signaling by IQ-1 inhibited alveolar type II epithelial cell proliferation in response to neutrophil migration induced by intratracheal keratinocyte chemokine. We conclude that β-catenin signaling is activated in lung epithelial cells during neutrophil transmigration, likely via elastase-mediated cleavage of E-cadherin, and regulates epithelial repair. This pathway represents a potential therapeutic target to accelerate physiological recovery in inflammatory lung diseases.

Lipid mediators are important in lung biochemistry and are derived from the enzymatic oxidation of arachidonic and docosahexaenoic acids, which are PUFAs that are present in phospholipids in cell membranes. In this study, MALDI imaging MS was used to determine the localization of arachidonate- and docosahexaenoate-containing phospholipids in mouse lung. These PUFA-containing phospholipids were determined to be uniquely abundant at the lining of small and large airways, which were unequivocally identified by immunohistochemistry. In addition, it was found that the blood vessels present in the lung were characterized by sphingomyelin molecular species, and lung surfactant phospholipids appeared evenly distributed throughout the lung parenchyma, indicating alveolar localization. This technique revealed unexpected high concentrations of arachidonate- and docosahexaenoate-containing phospholipids lining the airways in pulmonary tissue, which could serve as precursors of lipid mediators affecting airways biology.

Analysis of lineage relationships in the naphthalene-injured tracheal epithelium demonstrated that two multipotential keratin 14–expressing cells (K14ECs) function as progenitors for Clara and ciliated cells. These K14EC were distinguished by their self-renewal capacity and were hypothesized to reside at the stem and transit amplifying tiers of a tissue-specific stem cell hierarchy. In this study, we used gene expression and histomorphometric analysis of the steady-state and naphthalene-injured trachea to evaluate the predictions of this model. We found that the steady-state tracheal epithelium is maintained by two progenitor cell pools, secretory and basal cells, and the latter progenitor pool is further divided into two subsets, keratin 14–negative and –positive. After naphthalene-mediated depletion of the secretory and ciliated cell types, the two basal cell pools coordinate to restore the epithelium. Both basal cell types up-regulate keratin 14 and generate a broadly distributed, abundant, and highly mitotic cell pool. Furthermore, basal cell proliferation is associated with generation of differentiated Clara and ciliated cells. The uniform distribution of basal cell progenitors and of their differentiated progeny leads us to propose that the hierarchical organization of tracheal reparative cells be revised to include a facultative basal cell progenitor pool.

Clara cells were first described as a morphologically distinct cell type by Kolliker in 1881, but they take their name from the seminal study of human and rabbit bronchioles by Max Clara in 1937. Since their discovery, Clara cells have been identified as central players in protecting the airway from environmental exposures. The diverse functions of Clara cells in lung homeostasis include roles in xenobiotic metabolism, immune system regulation, and progenitor cell activity. Recent identification of a sub-population of Clara cells as a bronchiolar tissue-specific stem cell and a potential tumor initiating cell has focused the attention of cell and molecular biologists on the Clara cell and its behavior under normal and disease conditions.

Signaling by Wnt/beta-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 beta-catenin-regulated gene expression in normal maintenance and repair of the bronchiolar epithelium. Analysis of TOPGal transgene activity detected beta-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 beta-catenin in the normal or repairing state. Necessity of beta-catenin signaling was tested through Cre-mediated deletion of Catnb exons 2-6 in airway epithelial cells. Functional knockout of beta-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 beta-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, beta-catenin was not necessary for maintenance or efficient repair of the bronchiolar epithelium.

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.

Bronchiolar Clara cells undergo phenotypic changes during development and in disease. These changes are poorly described due to a paucity of molecular markers. We used chemical and transgenic approaches to ablate Clara cells, allowing identification of their unique gene expression profile. Flavin monooxygenase 3 (Fmo3), paraoxonase 1 (Pon1), aldehyde oxidase 3 (Aox3), and claudin 10 (Cldn10) were identified as novel Clara cell markers. New and existing Clara cell marker genes were categorized into three classes based on their unique developmental expression pattern. Cldn10 was uniformly expressed in the epithelium at Embryonic Day (E)14.5 and became restricted to secretory cells at E18.5. This transition was defined by induction of CCSP. Maturation of secretory cells was associated with progressive increases in the expression of Fmo3, Pon1, Aox3, and Cyp2f2 between late embryonic and postnatal periods. Messenger RNA abundance of all categories of genes was dramatically decreased after naphthalene-induced airway injury, and displayed a sequence of temporal induction during repair that suggested sequential secretory cell maturation. We have defined a broader repertoire of Clara cell–specific genes that allows staging of epithelial maturation during development and repair.

Reporter transgene, knockout, and misexpression studies support the notion that Wnt/beta-catenin signaling regulates aspects of branching morphogenesis, regional specialization of the epithelium and mesenchyme, and establishment of progenitor cell pools. As demonstrated for other foregut endoderm-derived organs, beta-catenin and the Wnt/beta-catenin signaling pathway contribute to control of cellular proliferation, differentiation and migration. However, the contribution of Wnt/beta-catenin signaling to these processes is shaped by other signals impinging on target tissues. In this review, we will concentrate on roles for Wnt/beta-catenin in respiratory system development, including segregation of the conducting airway and alveolar compartments, specialization of the mesenchyme, and establishment of tracheal asymmetries and tracheal glands.

Previous analysis of lung injury and repair has provided evidence for region-specific stem cells that maintain proximal and distal epithelial compartments. However, redundant expression of lineage markers by cells at several levels of the stem cell hierarchy has complicated phenotypic and functional characterization of clonogenic airway cells. Based on the demonstration that rapid efflux of the DNA dye Hoechst 33342 can be used to prospectively purify long-term repopulating hematopoietic stem cells, we hypothesized that lung cells with similar biochemical properties would be enriched for clonogenic progenitors. We demonstrate that Hoechst-dim side population (SP) cells isolated from proximal and distal compartments of the mouse lung were relatively small and agranular, exhibited low red and green autofluorescence, and that the SP fraction was highly enriched in clonogenic cells. Quantitative RT-PCR indicated that vimentin mRNA was enriched and that epithelial markers were depleted in these preparations of SP cells. Bleomycin exposure was associated with decreased clonogenicity among alveolar SP and suggested that SP cell function was compromised under profibrotic conditions. We conclude that the SP phenotype is common to clonogenic cells at multiple airway locations and suggest that Hoechst efflux is a property of cells expressing a wound-repair phenotype.

The trachea, bronchi, and bronchioles constitute the branched conduit for transport of gases to and from the lung parenchyma. The airways regulate the flow and condition the inhaled air. To accomplish these tasks, mammalian airways have acquired a number of adaptive mechanisms and functions, which are reflected in their structure.

Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O{sub 3}). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations.more » Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.« less