Bronchiolar Club Cells Research Articles

Abstract 4299: A lineage-specific role of oncogenic protein kinase Cι in lung adenocarcinoma

Abstract Lung adenocarcinoma (LUAD), the most prevalent subtype of lung cancer, can arise from two different cell populations: alveolar type 2 (AT2) cells and bronchiolar club cells. By utilizing genomic data from the cancer genome atlas program, human LUAD cell lines, and the well-characterized LSL-KrasG12D/Trp53-/- (KP) mouse model, we demonstrated that tumors arising from club cells are much more aggressive and follow a different trajectory of tumorigenesis when compared to those arising from AT2 cells. Intriguingly, our data strongly suggest that expression and signaling activity of protein kinase C iota (PKCι) can profoundly affect the cell of origin and oncogenic signaling pathways employed by resultant tumors. Specifically, KP-mediated transformation of club cells appears to be dependent on PKCι-ELF3-NOTCH3 and PKCι-YAP1 signaling axes, while AT2 cell malignancy is dependent on the PKCι-independent WNT signaling. In addition, club-cell-origin tumors exhibit an enrichment of cancer stem cells and increased infiltration of immunosuppressive myeloid cells in comparison to AT2-cell-origin tumors. This results in accelerated progression and increased likelihood of metastasis by LUAD tumors that originate from club cells. Taken together, our results indicate that PKCι expression and signaling activity can profoundly influence the cells of origin, tumor trajectories, and therapeutic vulnerabilities of LUAD tumors. Citation Format: Duy T. Nguyen, Ning Yin, Capella R. Weems, Lee Jamieson, Kayleah Meneses, Yi Liu, Nicole R. Murray, Alan P. Fields. A lineage-specific role of oncogenic protein kinase Cι in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4299.

Abstract 4294: The emerging role of deubiquitinase enzymes in lung cancer plasticity

Abstract Lung cancer is characterized by a high degree of genetic and molecular heterogeneity. Lineage plasticity has emerged as a source of intratumoral heterogeneity and drug resistance, resulting in poor prognosis and treatment failure. The molecular mechanisms driving lung cancer plasticity remain unclear. Ubiquitin-Specific Peptidase 13 (USP13), a deubiquitinating enzyme, is one of the most amplified genes in lung squamous cell carcinoma (LUSC). We developed a USP13 knock-in overexpressing mouse model in KrasLSL-G12D/+; Trp53fl/fl (KP) background (KPU mice). USP13 overexpression resulted in aggressive tumorigenesis and a shortened survival in Kras/Trp53-driven lung cancer. Notably, while KP mice developed lung adenocarcinoma (LUAD), KPU mice developed LUSC and LUAD. LUSC in KPU mice faithfully recapitulated the key pathohistological, molecular features, and cellular pathways of human LUSC. Bulk RNA-sequencing analysis showed that KPU tumors were heterogeneous and enriched in lineage reprogramming pathways including basal cell signaling, stem cell pluripotency, and epithelial-mesenchymal transition (EMT). Using cell-type-restricted adenoviral Cre to target cells expressing surfactant protein C (SPC) or club cell antigen 10 (CC10), we identified bronchiolar secretory club cells as the predominant cell origin of LUSC. USP13 altered the levels of lineage transcription factors such as TTF-1 and SOX2 in club cells during early tumorigenesis. Altered expression of these factors reinforced the fate of CC10+ club cells to squamous carcinoma development rather than adenocarcinoma. In addition, USP13 directly acted on the K48-linked ubiquitination of c-Myc and increased its protein stability, contributing to the elevation of squamous gene expression (SOX2, CK5, P40) in the primary mouse and advanced human lung cancer cells. These results suggest that USP13 promotes lineage plasticity in club cells during the early stage of cancer development and drives reprogramming into LUSC. We also found a potential functional association between USP13 and EMT pathway. Notably, irrespective of cell origin, USP13 overexpression enhanced EMT marker expression including N-cadherin, SNAI1, and ZEB1 in lung cancer cells, promoting cell migration and invasion. USP13 increased tumorigenic and metastatic abilities of lung cancer cells in 3D organoid culture and syngeneic mouse models. Collectively, our research highlights the pivotal significance of USP13 in unleashing lineage plasticity during lung cancer progression, suggesting the potential role of deubiquitinase enzymes in regulating lineage plasticity, which may lead to novel therapeutics for treating lung cancer. Citation Format: Juntae Kwon, Jinmin Zhang, Boram Mok, Samuel Allsup, Cecil Han. The emerging role of deubiquitinase enzymes in lung cancer plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4294.

Age-associated H3K9me2 loss alters the regenerative equilibrium between murine lung alveolar and bronchiolar progenitors

The lung contains multiple progenitor cell types, but how their responses are choreographed during injury repair and whether this changes with age is poorly understood. We report that histone H3 lysine 9 di-methylation (H3K9me2), mediated by the methyltransferase G9a, regulates the dynamics of distal lung epithelial progenitor cells and that this regulation deteriorates with age. In aged mouse lungs, H3K9me2 loss coincided with fewer alveolar type 2 (AT2) cell progenitors and reduced alveolar regeneration but increased the frequency and activity of multipotent bronchioalveolar stem cells (BASCs) and bronchiolar progenitor club cells. H3K9me2 depletion in young mice decreased AT2 progenitor activity and impaired alveolar injury repair. Conversely, H3K9me2 depletion increased chromatin accessibility of bronchiolar cell genes, increased BASC frequency, and accelerated bronchiolar cell injury repair. These findings indicate that during aging, the epigenetic regulation that coordinates lung progenitor cells' regenerative responses becomes dysregulated, aiding our understanding of age-related susceptibility to lung disease.

Abstract SY33-02: Novel molecular mechanisms of lung cancer plasticity and targeting lung squamous cell carcinoma

Abstract Lung cancer is marked by a high degree of genetic and histopathological heterogeneity. Lineage plasticity, the ability of cells to transform from one cell type to another, has been associated with intratumoral heterogeneity and therapeutic resistance. The molecular drivers and mechanisms of cancer cell plasticity and histological transdifferentiation of lung cancer are largely unknown. A deubiquitinating enzyme, Ubiquitin-Specific Peptidase 13 (USP13), is frequently amplified at chromosome 3q.26 amplicon in lung squamous cell carcinoma. USP13 knock-in overexpressing mouse model was crossed with KrasLSL-G12D; Trp53fl/fl (KP) mouse to generate KrasLSL-G12D; Trp53fl/fl; Usp13LSL (KPU) mouse model. Intratracheal administration of adenovirus expressing Cre recombinase to the lung of the KPU mouse developed aggressive lung tumors and a shortened survival compared to the KP mouse model. Importantly, while the KP mice developed lung adenocarcinoma, 100% of KPU mice developed lung squamous cell carcinoma. The squamous cell carcinoma of the KPU mice faithfully mimicked the molecular signatures, cellular pathways, and tumor microenvironments of human lung squamous cell carcinoma. Delivery of cell type-restricted viruses to the lungs further identified bronchiolar secretory club cells as the predominant cell-of-origin of KPU-induced lung squamous cell carcinoma. USP13 altered the levels of TTF-1 and c-Myc in club cells of the airway and reinforced the fate of CC10+ club cells to squamous carcinoma development instead of adenocarcinoma. USP13 directly acted on the K48-linked ubiquitination of c-Myc and increased the protein stability of c-Myc in human lung cancer cells. Overexpression of USP13 increased the half-life of c-Myc while inhibiting USP13 deubiquitinase activity decreased the c-Myc protein level. USP13 upregulated the expression of squamous lineage markers (Sox2, CK5, P40) in the primary mouse and advanced human lung cancer cells. Our findings revealed that USP13 promotes the lineage plasticity of origin-of-cells of the lung and drives transdifferentiation to squamous cell carcinoma. Finally, our study suggests the critical role of USP13 in unlocking the phenotypic plasticity of lung cancer, which may lead to novel therapeutics targeting lung cancer. Citation Format: Cecil Han. Novel molecular mechanisms of lung cancer plasticity and targeting lung squamous cell carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr SY33-02.

Adipose triglyceride lipase-mediated lipid catabolism is essential for bronchiolar regeneration.

The lung airways are constantly exposed to inhaled toxic substances, resulting in cellular damage that is repaired by local expansion of resident bronchiolar epithelial club cells. Disturbed bronchiolar epithelial damage repair lies at the core of many prevalent lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and lung cancer. However, it is still not known how bronchiolar club cell energy metabolism contributes to this process. Here, we show that adipose triglyceride lipase (ATGL), the rate-limiting enzyme for intracellular lipolysis, is critical for normal club cell function in mice. Deletion of the gene encoding ATGL, Pnpla2 (also known as Atgl), induced substantial triglyceride accumulation, decreased mitochondrial numbers, and decreased mitochondrial respiration in club cells. This defect manifested as bronchiolar epithelial thickening and increased airway resistance under baseline conditions. After naphthalene‑induced epithelial denudation, a regenerative defect was apparent. Mechanistically, dysfunctional PPARα lipid-signaling underlies this phenotype because (a) ATGL was needed for PPARα lipid-signaling in regenerating bronchioles and (b) administration of the specific PPARα agonist WY14643 restored normal bronchiolar club cell ultrastructure and regenerative potential. Our data emphasize the importance of the cellular energy metabolism for lung epithelial regeneration and highlight the significance of ATGL-mediated lipid catabolism for lung health.

Angiotensin-converting enzyme 2 in peripheral lung club cells modulates the susceptibility to SARS-CoV-2 in chronic obstructive pulmonary disease.

Accumulating evidence has confirmed that chronic obstructive pulmonary disease (COPD) is a risk factor for development of severe pathological changes in the peripheral lungs of patients with COVID-19. However, the underlying molecular mechanisms remain unclear. Because bronchiolar club cells are crucial for maintaining small airway homeostasis, we sought to explore whether the altered susceptibility to SARS-CoV-2 infection of the club cells might have contributed to the severe COVID-19 pneumonia in COPD patients. Our investigation on the quantity and distribution patterns of angiotensin-converting enzyme 2 (ACE2) in airway epithelium via immunofluorescence staining revealed that the mean fluorescence intensity of the ACE2-positive epithelial cells was significantly higher in club cells than those in other epithelial cells (including ciliated cells, basal cells, goblet cells, neuroendocrine cells, and alveolar type 2 cells). Compared with nonsmokers, the median percentage of club cells in bronchiolar epithelium and ACE2-positive club cells was significantly higher in COPD patients. In vitro, SARS-CoV-2 infection (at a multiplicity of infection of 1.0) of primary small airway epithelial cells, cultured on air-liquid interface, confirmed a higher percentage of infected ACE2-positive club cells in COPD patients than in nonsmokers. Our findings have indicated the role of club cells in modulating the pathogenesis of SARS-CoV-2-related severe pneumonia and the poor clinical outcomes, which may help physicians to formulate a novel therapeutic strategy for COVID-19 patients with coexisting COPD.

Bronchioalveolar stem cells in lung repair, regeneration and disease.

Bronchioalveolar stem cells (BASCs) are a lung resident stem cell population located at bronchioalveolar duct junctions that contribute to the maintenance of bronchiolar club cells and alveolar epithelial cells of the distal lung. Their transformed counterparts are considered to be likely progenitors of lung adenocarcinomas, which has been a major area of research in relation to BASCs. A critical limitation in addressing the function of BASCs in vivo has been the lack of a unique BASC marker, which has prevented specific targeting of BASCs in animal models of respiratory conditions. Recently, there have been several studies describing genetically modified mice that allow in vivo quantification, tracing, and functional analysis of BASCs to address this long-standing issue. These cutting-edge experimental tools will likely have significant implications for future experimental studies involving BASCs and the elucidation of their role in various lung diseases. To date, this has been largely explored in models of lung injury including naphthalene-induced airway injury, bleomycin-induced alveolar injury, hyperoxia-induced models of bronchopulmonary dysplasia, and influenza virus infection. These novel experimental mouse tools will facilitate the assessment of the impact of BASC loss on additional respiratory conditions including infection-induced severe asthma and chronic obstructive pulmonary disease, as well as respiratory bacterial infections, both in early life and adulthood. These future studies may shed light on the potential broad applicability of targeting BASCs for a diverse range of respiratory conditions during lung development and in promoting effective regeneration and repair of the lung in respiratory diseases. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neonatal endotoxin stimulation is associated with a long-term bronchiolar epithelialexpression of innate immune and anti-allergic markers that attenuates the allergic response.

Allergic asthma is the most common phenotype of the pathology, having an early-onset in childhood and producing a Th2-driven airways remodeling process that leads to symptoms and pathophysiological changes. The avoidance of aeroallergen exposure in early life has been shown to prevent asthma, but without repeated success and with the underlying preventive mechanisms at the beginning of asthma far to be fully recognized. In the present study, we aimed to evaluate if neonatal LPS-induced boost in epithelial host defenses contribute to prevent OVA-induced asthma in adult mice. To this, we focused on the response of bronchiolar club cells (CC), which are highly specialized in maintaining the epithelial homeostasis in the lung. In these cells, neonatal LPS administration increased the expression of TLR4 and TNFα, as well as the immunodulatory/antiallergic proteins: club cell secretory protein (CCSP) and surfactant protein D (SP-D). LPS also prevented mucous metaplasia of club cells and reduced the epidermal growth factor receptor (EGFR)-dependent mucin overproduction, with mice displaying normal breathing patterns after OVA challenge. Furthermore, the overexpression of the epithelial Th2-related molecule TSLP was blunted, and normal TSLP and IL-4 levels were found in the bronchoalveolar lavage. A lower eosinophilia was detected in LPS-pretreated mice, along with an increase in phagocytes and regulatory cells (CD4+CD25+FOXP3+ and CD4+IL-10+), together with higher levels of IL-12 and TNFα. In conclusion, our study demonstrates stable asthma-preventive epithelial effects promoted by neonatal LPS stimulation, leading to the presence of regulatory cells in the lung. These anti-allergic dynamic mechanisms would be overlaid in the epithelium, favored by an adequate epidemiological environment, during the development of asthma.

Club Cell TRPV4 Serves as a Damage Sensor Driving Lung Allergic Inflammation

Airway epithelium is the first body surface to contact inhaled irritants and report danger. Here, we report how epithelial cells recognize and respond to aeroallergen alkaline protease 1 (Alp1) of Aspergillus sp., because proteases are critical components of many allergens that provoke asthma. In a murine model, Alp1 elicits helper T (Th) cell-dependent lung eosinophilia that is initiated by the rapid response of bronchiolar club cells to Alp1. Alp1 damages bronchiolar cell junctions, which triggers a calcium flux signaled through calcineurin within club cells of the bronchioles, inciting inflammation. In two human cohorts, we link fungal sensitization and/or asthma with SNP/protein expression of the mechanosensitive calcium channel, TRPV4. TRPV4 is also necessary and sufficient for club cells to sensitize mice to Alp1. Thus, club cells detect junction damage asmechanical stress, which signals danger via TRPV4, calcium, and calcineurin to initiate allergic sensitization.

Targeted Ablation of AIMP1 in Lipopolysaccharide-induced Acute Lung Injury

Background and Hypothesis: Infants with Bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants, are more susceptible to acute lung injury (ALI). Endothelial-Monocyte Activating Polypeptide II (EMAP II, encoded by Aimp1) is a proinflammatory cytokine that originates from bronchiolar club cells and plays a role in the inflammatory response during BPD development. Prolonged EMAP II exposure has been shown to worsen BPD pathogenesis by activating alveolar macrophages. The targeted ablation of EMAP II in the bronchial club cells of a mouse model (Scgb1a1-ERTCre;Aimp1/fl/fl) is hypothesized to decrease the inflammatory effects of ALI. 
 Experimental Design: Aged-matched littermate mice (ages ranging from 20-25 weeks) with Tamoxifen inducible, Cremediated, bronchial club cell specific ablation of Aimp1 (Scgb1a1-ERTCre;Aimp1/fl/fl, denoted cKO) or with only partial ablation (Scgb1a1-ERT2Cre;Aimp1/fl/wt denoted Ctrl) were given three doses of 120 micro-liters of 20mg/ml tamoxifen over a seven day period. 24 hours after the final dose they were administered a single intratracheal delivery of lipopolysaccharide (LPS) (5mg/kg) 24 hours later immunohistochemistry (IHC) for EMAP II and inflammation was assessed by immunoblotting (IB) for IL-6 in bronchoalveolar lavage (BAL) and cytospin of BAL.
 Results: IHC showed a decrease of EMAP II expression in the bronchioles of the cKO as compared to ctrl. IL-6 was increased 1.95 fold in the BAL fluid of cKO by IB. Cytospin analysis showed: (Cell Type: Ctrl%, cKo%), Macrophages: 4.66%, 4.70%, Mature neutrophils: 44.44%, 60.25%, Banded Neutrophils: 41.93%, 26.20%, Lymphocytes: 4.30%, 5.10%, Eosinophils: 2.51%, 2.10%, Monocytes: 2.15%, 1.64%.
 Conclusions: Ablation of bronchial club cell EMAP II, in LPS-induced ALI increased the amount of IL-6 and percentage of mature neutrophils in bronchoalveolar lavage fluid. No significant difference in macrophage were noted in either group. These findings suggest that EMAP II influences immune response time; however, more experiments would be required to establish a link.

Loss of Polycomb Repressive Complex 2 impairs lung bronchiolar cell growth and underlies chronic obstructive pulmonary disease

EZH2, the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2), is a histone methyl transferase that tri-methylates histone H3 at lysine 27 (H3K27me3). This protein plays a significant role in maintaining tissue specificity by silencing gene programs that control lineage fate. We hypothesized that PRC2 controls cell fate of the lung stem cells. In order to test this hypothesis, we generated mice in which we can conditionally delete Ezh2 upon ingestion of doxycycline. To examine growth, differentiation and self-renewal potentials of different lung epithelial cells, we FACS-isolated distal lung bronchioalveolar stem cells (BASCs) and cultured them in air-liquid-interface Matrigel cultures. We observed a 2fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells and a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. To test in vivo potential of EZH2 depleted epithelium to repair, we performed naphthalene injury, which specifically injures the bronchiolar club cells. We observed that 7 days post naphthalene injury, Ezh2 knock-out lungs had significantly less repair, as measured by repopulation of bronchiolar lung cells. Given that we observed that lung stem cells were impaired when PRC2-mediated gene repression was perturbed, we sought to learn if PRC2 is also dysregulated in Chronic Obstructive Pulmonary Disease (COPD). We first performed Gene Set Enrichment Analysis and found that genes up-regulated in COPD patients relative to healthy smokers were also up-regulated in Ezh2 knock-out adenocarcinoma cells. We next obtained lung tissue from healthy patients and patients with COPD, and performed immunohistochemistry for the PRC2 mark H3K27me3 and immunofluorescence for basal and club cells. There was a significant decrease in H3K27me3, coupled with an increase in basal cells and goblet cells that contained high expressions of club cell markers in COPD lung compared to healthy controls. Finally, to determine if loss of PRC2 function drives basal cell fate and goblet cell differentiation, EZH2 transcripts were knocked down with short hairpins in human bronchiolar epithelial cells, and basal cell differentiation capacity was analyzed. We observed an increased expression of a goblet cell marker and decreased expression of a club cell marker in EZH2 knockdown cultures. Together our results indicate that PRC2 plays an important role in lung stem cell function as well as club cell differentiation fate. These mechanisms may also be perturbed in COPD patients. ChIP-seq and RNA-seq studies to understand the transcriptional and epigenetic changes leading to these phenotypes are underway Support or Funding Information NIH grant K22 CA 20103, Molecular Mechanisms of Toxicity Training Grant T32ES07266 There was a 2-fold decrease in organoid number of EZH2 null vs wild type bronchiolar cells. There was a significant difference in organoid diameter between Ezh2 wild type, heterozygous and null bronchiolar cells. Immunohistochemistry staining of EZH2 shows distinct differences in EZH2 expression between wild type, heterozygous and null organoids. Naphthalene exposure to mouse lung has been shown to kill club cells in the trachea and bronchioles. This injury typically results in club cell regeneration and subsequent lung cell repopulation. Club cells without EZH2 were unable to regenerate and repopulate the airway after naphthalene injury. Club cells that did regenerate in the Ezh2-null lungs lacked the GFP tag, indicating that these cells were likely not responding to doxycycline to delete Ezh2. Gene Set Enrichment Analysis was used to compare gene expression between murine Ezh2-deleted adenocarcinoma cells and COPD patients. It revealed that a significant number of the same genes are highly expressed in both states when compared to healthy lungs or the Ezh2 WT setting. Immunohistochemistry shows significantly lower abundance of H3K27me3 in the nuclei of COPD bronchiolar cells compared to normal. Immunofluorescence staining of club (CCSP and SOX2) and basal cells (KRT5 and SOX2) in normal vs COPD lung airway shows a mixture of metaplasia and hyperplasia along with collapsed airways in COPD airway compared to normal. There is an increase in basal cells and goblet cells that also contain high expressions of CCSP (a club cell marker). EZH2 transcripts were knocked down in immortalized, Human Bronchiolar Epithelial Cells (HBEC) which were, then, prompted to differentiate in air-liquid-interface cultures. Acetylated-Mucin5 was highly expressed while club cell secretory protein (CCSP) was lowly expressed in EZH2 knockdown cells compared to wild type. This suggest that EZH2 plays a role in club and goblet cell differentiation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Physiological Role for a Moonlighting Protein in Lipopolysaccharide-induced Endotoxemia

Background and Hypothesis: The pathogenesis of Bronchopulmonary Dysplasia (BPD) is multifactorial leading to inflammation. In BPD, Endothelial-Monocyte Activating Polypeptide II (EMAP II, encoded by Aimp1), a moonlighting pro-inflammatory cytokine, is initially found in bronchiolar club cells followed by intra-alveolar GAL-3+ macrophages. Sustained EMAP II mimics BPD, invoking inflammation, alveolar simplification, and macrophage recruitment. Targeted ablation of EMAP II in the recruited macrophages may dampen innate immune response. 
 Experimental Design: Gender-matched, aged-matched littermate mice with myeloid-cell specific ablation of Aimp1 (Lyz2-Cre;Aimp1flox/flox, denoted as Aimp1Δ/Δ) or without (control) were subjected to lipopolysaccharide (LPS)-endotoxemia. Survival rates of co-housed or singly housed mice were measured over 72 hours following a lethal dose (15 mg/kg). Clinical scores (0-6) based on the integrity of their locomotion, fur, and eyes were assigned every 2 hours. Blood and bone marrow smears, average bodyweights, spleen-weights to bodyweights and liver-weights to bodyweights were analyzed. 
 Results: There were no baseline differences in bodyweight, spleen weight:bodyweight, liver weight:bodyweight (p-val = 0.42, 0.46, 0.64). Representative bone marrow and blood smears showed no notable difference. Aimp1[Symbol]/[Symbol] male mice co-housed (dose 15 mg/kg) but not singly housed survived longer than their littermates (median survival: hours); Aimp1[Symbol]/[Symbol] female mice showed a survival advantage (median survival: hours) with lower clinical scores than their littermates. The kinetics of NFKBIA/I[Symbol]B degradation was similar between Aimp1[Symbol]/[Symbol] and control peritoneal macrophages in response to LPS, although there was a higher basal amount in Aimp1[Symbol]/[Symbol]. 
 Conclusion and Potential Impact: Aimp1/EMAP II does play a positive feedback role in innate immunity, potentially in a metabolically and gender-specific role of Aimp1 which remain to be explored.

PPARα-mediated peroxisome induction compensates PPARγ-deficiency in bronchiolar club cells.

Despite the important functions of PPARγ in various cell types of the lung, PPARγ-deficiency in club cells induces only mild emphysema. Peroxisomes are distributed in a similar way as PPARγ in the lung and are mainly enriched in club and AECII cells. To date, the effects of PPARγ-deficiency on the overall peroxisomal compartment and its metabolic alterations in pulmonary club cells are unknown. Therefore, we characterized wild-type and club cell-specific PPARγ knockout-mice lungs and used C22 cells to investigate the peroxisomal compartment and its metabolic roles in the distal airway epithelium by means of 1) double-immunofluorescence labelling for peroxisomal proteins, 2) laser-assisted microdissection of the bronchiolar epithelium and subsequent qRT-PCR, 3) siRNA-transfection of PPARγand PPRE dual-luciferase reporter activity in C22 cells, 4) PPARg inhibition by GW9662, 5) GC-MS based lipid analysis. Our results reveal elevated levels of fatty acids, increased expression of PPARα and PPRE activity, a strong overall upregulation of the peroxisomal compartment and its associated gene expression (biogenesis, α-oxidation, β-oxidation, and plasmalogens) in PPARγ-deficient club cells. Interestingly, catalase was significantly increased and mistargeted into the cytoplasm, suggestive for oxidative stress by the PPARγ-deficiency in club cells. Taken together, PPARα-mediated metabolic induction and proliferation of peroxisomes via a PPRE-dependent mechanism could compensate PPARγ-deficiency in club cells.

Postnatal development of the bronchiolar club cells of distal airways in the mouse lung: stereological and molecular biological studies.

Club (Clara) cells are nonciliated secretory epithelial cells present in bronchioles of distal pulmonary airways. So far, no information is available on the postnatal differentiation of club cells by a combination of molecular biological, biochemical, and stereological approaches in the murine lung. Therefore, the present study was designed to investigate the changes in the club cell secretory proteins (CC10, surfactant proteins A, B and D) and club cell abundance within the epithelium of bronchioles of distal airways during the postnatal development of the mouse lung. Perfusion-fixed murine lungs of three developmental stages (newborn, 15-day-old and adult) were used. Frozen, unfixed lungs were used for cryosectioning and subsequent laser-assisted microdissection of bronchiolar epithelial cells and RT-PCR analyses. High resolution analyses of the three-dimensional structures and composition of lung airways were obtained by scanning electron microscopy. Finally, using design-based stereology, the total and average club cell volume and the volume of secretory granules were quantified by light and transmission electron microscopy. Our results reveal that murine club cells are immature at birth and differentiate postnatally. Further, increase of the club cell volume and number of intracellular granules are closely correlated to the total lung volume enlargement. However, secretory granule density was only increased within the first 15days of postnatal development. The differentiation is accompanied by a decrease in glycogen content, and a close positive relationship between CC10 expression and secretory granule abundance. Taken together, our data are consistent with the concept that the morphological and functional differentiation of club cells is a postnatal phenomenon.

Syndecan 4 Mediates Nrf2-dependent Expansion of Bronchiolar Progenitors That Protect Against Lung Inflammation.

The use of lung progenitors for regenerative medicine appears promising, but their biology is not fully understood. Here, we found anti-inflammatory attributes in bronchiolar progenitors that were sorted as a multipotent subset of mouse club cells and found to express secretory leukocyte protease inhibitor (SLPI). Notably, the impaired expression of SLPI in mice increased the number of bronchiolar progenitors and decreased the lung inflammation. We determined a transcriptional profile for the bronchiolar progenitors of Slpi-deficient mice and identified syndecan 4, whose expression was markedly elevated as compared to that of wild-type mice. Systemic administration of recombinant syndecan 4 protein caused a substantial increase in the number of bronchiolar progenitors with concomitant attenuation of both airway and alveolar inflammation. The syndecan 4 administration also resulted in activation of the Keap1-Nrf2 antioxidant pathway in lung cells, which is critically involved in the therapeutic responses to the syndecan 4 treatment. Moreover, in 3D culture, the presence of syndecan 4 induced differentiated club cells to undergo Nrf2-dependent transition into bronchiolar progenitors. Our observations reveal that differentiative switches between bronchiolar progenitors and club cells are under the Nrf2-mediated control of SLPI and syndecan 4, suggesting the possibility of new therapeutic approaches in inflammatory lung diseases.

Repression of CC16 by cigarette smoke (CS) exposure.

Club (Clara) Cell Secretory Protein (CCSP, or CC16) is produced mainly by non-ciliated airway epithelial cells including bronchiolar club cells and the change of its expression has been shown to associate with the progress and severity of Chronic Obstructive Pulmonary Disease (COPD). In an animal model, the lack of CC16 renders the animal susceptible to the tumorigenic effect of a major CS carcinogen. A recent population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD) has indicated that the low serum CC16 concentration is closely linked with the smoke-related mortality, particularly that driven by the lung cancer. However, the study of CC16 expression in well-defined smoke exposure models has been lacking, and there is no experimental support for the potential causal link between CC16 and CS-induced pathophysiological changes in the lung. In the present study, we have found that airway CC16 expression was significantly repressed in COPD patients, in monkey CS exposure model, and in CS-induced mouse model of COPD. Additionally, the lack of CC16 exacerbated airway inflammation and alveolar loss in the mouse model. Therefore, CC16 may play an important protective role in CS-related diseases.

Protective phenotypes of club cells and alveolar macrophages are favored as part of endotoxin-mediated prevention of asthma.

Atopic asthma is a chronic allergic disease that involves T-helper type 2 (Th2)-inflammation and airway remodeling. Bronchiolar club cells (CC) and alveolar macrophages (AM) are sentinel cells of airway barrier against inhaled injuries, where allergy induces mucous metaplasia of CC and the alternative activation of AM, which compromise host defense mechanisms and amplify Th2-inflammation. As there is evidence that high levels of environmental endotoxin modulates asthma, the goal of this study was to evaluate if the activation of local host defenses by Lipopolysaccharide (LPS) previous to allergy development can contribute to preserving CC and AM protective phenotypes. Endotoxin stimulus before allergen exposition reduced hallmarks of allergic inflammation including eosinophil influx, Interleukin-4 and airway hyperreactivity, while the T-helper type 1 related cytokines IL-12 and Interferon-γ were enhanced. This response was accompanied by the preservation of the normal CC phenotype and the anti-allergic proteins Club Cell Secretory Protein (CCSP) and Surfactant-D, thereby leading to lower levels of CC metaplasia and preventing the increase of the pro-Th2 cytokine Thymic stromal lymphopoietin. In addition, classically activated alveolar macrophages expressing nitric oxide were promoted over the alternatively activated ones that expressed arginase-1. We verified that LPS induced a long-term overexpression of CCSP and the innate immune markers Toll-like receptor 4, and Tumor Necrosis Factor-α, changes that were preserved in spite of the allergen challenge. These results demonstrate that LPS pre-exposition modifies the local bronchioalveolar microenvironment by inducing natural anti-allergic mechanisms while reducing local factors that drive Th2 type responses, thus modulating allergic inflammation.

Serum concentrations of club cell secretory protein (Clara) and cancer mortality in adults: a population-based, prospective cohort study

Club cell secretory protein (Clara) (CC16) is produced mainly by bronchiolar club cells and has been shown to have protective effects against airway inflammation and oxidative stress from cigarette smoking and related carcinogens. The goal of this study was to establish whether serum CC16 concentrations predict all-cause and cancer-specific mortality in adults. We used data from the population-based Tucson Epidemiological Study of Airway Obstructive Diseases (TESAOD), a prospective cohort study of respiratory health initiated in Tucson, AZ, USA, in 1972, that recruited a multistage stratified cluster sample of non-Hispanic white households. We measured serum CC16 concentrations in cryopreserved serum samples and reviewed vital status up to Jan 1, 2011, through contact with next of kin, collection of death certificates, and searches of the National Death Index. Our primary analysis was the relation of baseline serum CC16 to all-cause mortality or cause-specific mortality risk, analysed by adjusted Cox proportional hazards models. 1086 TESAOD participants aged 21-70 years at enrolment were eligible for inclusion. Of these, 653 (60%) had died by 2011, and cause of death was ascertained for 649 (99%). When adjusted for sex, age, education, body-mass index, smoking and pack-years, and baseline levels of lung function, serum CC16 concentrations at baseline were inversely associated with mortality risk over the study follow-up. Mortality risk increased for each 1-SD decrease in CC16 (adjusted hazard ratio [HR] 1·16 [95% CI 1·06-1·26]; p=0·0007). For cause-specific mortality, each 1-SD decrease in serum CC16 was associated with an increased risk of dying of cancer (adjusted HR 1·41 [1·19-1·67]; p<0·0001). In the subset of smokers, the corresponding adjusted HR for mortality by lung cancer was 1·52 (1·14-2·03; p=0·004). Serum CC16 concentrations can predict mortality risk in the general adult population. The excess risk associated with lower CC16 concentrations is predominantly driven by cancer, particularly lung cancer. National Heart, Lung, and Blood Institute.

Club cell secretory protein improves survival in a murine obliterative bronchiolitis model

Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.