Pseudomonas aeruginosa senses and responds to epithelial potassium flux via Kdp operon to promote biofilm.

Mucosa-associated biofilms are associated with many human disease states, but the host mechanisms promoting biofilm remain unclear. In chronic respiratory diseases like cystic fibrosis (CF), Pseudomonas aeruginosa establishes chronic infection through biofilm formation. P. aeruginosa can be attracted to interspecies biofilms through potassium currents emanating from the biofilms. We hypothesized that P. aeruginosa could, similarly, sense and respond to the potassium efflux from human airway epithelial cells (AECs) to promote biofilm. Using respiratory epithelial co-culture biofilm imaging assays of P. aeruginosa grown in association with CF bronchial epithelial cells (CFBE41o-), we found that P. aeruginosa biofilm was increased by potassium efflux from AECs, as examined by potentiating large conductance potassium channel, BKCa (NS19504) potassium efflux. This phenotype is driven by increased bacterial attachment and increased coalescence of bacteria into aggregates. Conversely, biofilm formation was reduced when AECs were treated with a BKCa blocker (paxilline). Using an agar-based macroscopic chemotaxis assay, we determined that P. aeruginosa chemotaxes toward potassium and screened transposon mutants to discover that disruption of the high-sensitivity potassium transporter, KdpFABC, and the two-component potassium sensing system, KdpDE, reduces P. aeruginosa potassium chemotaxis. In respiratory epithelial co-culture biofilm imaging assays, a KdpFABCDE deficient P. aeruginosa strain demonstrated reduced biofilm growth in association with AECs while maintaining biofilm formation on abiotic surfaces. Furthermore, we determined that the Kdp operon is expressed in vivo in people with CF and the genes are conserved in CF isolates. Collectively, these data suggest that P. aeruginosa biofilm formation can be increased by attracting bacteria to the mucosal surface and enhancing coalescence into microcolonies through aberrant AEC potassium efflux sensed by the KdpFABCDE system. These findings suggest host electrochemical signaling can enhance biofilm, a novel host-pathogen interaction, and potassium flux could be a therapeutic target to prevent chronic infections in diseases with mucosa-associated biofilms, like CF.

Bacterial respiratory inhibition triggers dispersal of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa grows as a biofilm under many environmental conditions, and the bacterium can disperse from biofilms via highly regulated, dynamic processes. However, physiologic triggers of biofilm dispersal remain poorly understood. Based on prior literature describing dispersal triggered by forms of starvation, we tested bacterial respiratory inhibitors for biofilm dispersal in two models resembling chronic airway infections. Our underlying hypothesis was that respiratory inhibitors could serve as a model for the downstream effects of starvation. We used two experimental conditions. In the first condition, biofilms were grown and dispersed from the surface of airway epithelial cells, and the second condition was a model where biofilms were grown on glass in cell culture media supplemented with host-relevant iron sources. In both biofilm models, the respiratory inhibitors potassium cyanide and sodium azide each triggered biofilm dispersal. We hypothesized that cyanide-induced dispersal was due to respiratory inhibition rather than signaling via an alternative mechanism, and, indeed, if respiration was supported by overexpression of cyanide-insensitive oxidase, dispersal was prevented. Dispersal required the activity of the cyclic-di-GMP regulated protease LapG, reinforcing the role of matrix degradation in dispersal. Finally, we examined the roles of individual phosphodiesterases, previously implicated in dispersal to specific triggers, and found signaling to be highly redundant. Combined deletion of the phosphodiesterases dipA, bifA, and rbdA was required to attenuate the dispersal phenotype. In summary, this work adds insight into the physiology of biofilm dispersal under environmental conditions in which bacterial respiration is abruptly limited. IMPORTANCE The bacterium Pseudomonas aeruginosa grows in biofilm communities that are very difficult to treat in human infections. Growing as a biofilm can protect bacteria from antibiotics and the immune system. Bacteria can leave a biofilm through a process called "dispersal." Dispersed bacteria seed new growth areas and are more susceptible to killing by antibiotics. The triggers for biofilm dispersal are not well understood, and if we understood dispersal better it might lead to the development of new treatments for infection. In this paper, we find that inhibiting P. aeurginosa's ability to respire (generate energy) can trigger dispersal from a biofilm grown in association with human respiratory epithelial cells in culture. The dispersal process requires a protease which is previously known to degrade the biofilm matrix. These findings give us a better understanding of how the biofilm dispersal process works so that future research can discover better ways of clearing bacteria growing in biofilms.

Sodium Nitrite Inhibits Killing of Pseudomonas aeruginosa Biofilms by Ciprofloxacin.

Sodium nitrite inhibits bacterial respiration and is in development as an antimicrobial for chronic bacterial infections associated with cystic fibrosis. The goal of the current study was to investigate the interaction between nitrite and ciprofloxacin. Using liquid culture killing assays and a biotic biofilm model, we observed that nitrite induces tolerance of ciprofloxacin.

Sodium nitrite blocks the activity of aminoglycosides against Pseudomonas aeruginosa biofilms.

Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 µg/ml to >1,024 µg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm.

Persistence and evolution of Pseudomonas aeruginosa following initiation of highly effective modulator therapy in cystic fibrosis.

Today, more than 90% of people with cystic fibrosis (pwCF) are eligible for the highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy called elexacaftor/tezacaftor/ivacaftor (ETI) and its use is widespread. Given the drastic respiratory symptom improvement experienced by many post-ETI, clinical studies are already underway to reduce the number of respiratory therapies, including antibiotic regimens, that pwCF historically relied on to combat lung disease progression. Early studies suggest that bacterial burden in the lungs is reduced post-ETI, yet it is unknown how chronic Pseudomonas aeruginosa populations are impacted by ETI. We found that pwCF remain infected throughout their upper and lower respiratory tract with their same strain of P. aeruginosa post-ETI, and these strains continue to evolve in response to the newly CFTR-corrected airway. Our work underscores the continued importance of CF airway microbiology in the new era of highly effective CFTR modulator therapy. IMPORTANCE: The highly effective cystic fibrosis transmembrane conductance regulator modulator therapy Elexakaftor/Tezacaftor/Ivacaftor (ETI) has changed cystic fibrosis (CF) disease for many people with cystic fibrosis. While respiratory symptoms are improved by ETI, we found that people with CF remain infected with Pseudomonas aeruginosa. How these persistent and evolving bacterial populations will impact the clinical manifestations of CF in the coming years remains to be seen, but the role and potentially changing face of infection in CF should not be discounted in the era of highly effective modulator therapy.

Effects of highly effective modulator therapy on the dynamics of the respiratory mucosal environment and inflammatory response in cystic fibrosis.

BACKGROUND: While the widespread initiation of elexacaftor/tezacaftor/ivacaftor (ETI) has led to dramatic clinical improvements among persons with cystic fibrosis (pwCF), little is known about how ETI affects the respiratory mucosal inflammatory and physiochemical environment, or how these changes relate to lung function. METHODS: We performed a prospective, longitudinal study of adults with CF and chronic rhinosinusitis (CF-CRS) followed at our CF center (n = 18). Endoscopic upper respiratory tract (paranasal sinus) aspirates from multiple visit dates, both pre- and post-ETI initiation, were collected and tested for cytokines, metals, pH, and lactate levels. Generalized estimating equations were used to identify relationships between ETI and upper respiratory tract (URT) biomarker levels, and between URT biomarkers and lung function or clinical sinus parameters. RESULTS: ETI was associated with decreased upper respiratory mucosal cytokines B-cell activating factor (BAFF), IL-12p40, IL-32, IL-8, IL-22 and soluble tumor necrosis factor-1 (sTNFR1), and an increase in a proliferation-inducing ligand (APRIL) and IL-19. ETI was also associated with decreased URT levels of copper, manganese, and zinc. In turn, lower URT levels of BAFF, IL-8, lactate, and potassium were each associated with ~1.5% to 4.3% improved forced expiratory volume in 1 s (FEV1), while higher levels of IFNγ, iron, and selenium were associated with ~2% to 10% higher FEV1. CONCLUSIONS: Our observations suggest a dampening of inflammatory signals and restriction in microbial nutrients in the upper respiratory tract with ETI. These findings improve our understanding of how ETI impacts the mucosal environment in the respiratory tract, and may give insight into the improved infectious and inflammatory status and the resulting clinical improvements seen in pwCF.

Predictors of Sinonasal Improvement After Highly Effective Modulator Therapy in Adults with Cystic Fibrosis.

OBJECTIVES: The 22-question SinoNasal Outcome Test (SNOT-22) assesses chronic rhinosinusitis (CRS) severity. We aimed to identify predictors of SNOT-22 score improvement following highly effective modulator therapy (HEMT) initiation and to corroborate the SNOT-22 minimal clinically important difference (MCID) in adults with cystic fibrosis (CF). METHODS: Prospective observational data was pooled from four studies across 10 US centers investigating people with CF (PwCF) and CRS. Three studies evaluated HEMT's impact on CRS. For participants enrolled prior to HEMT initiation, SNOT-22 scores were obtained at baseline and after 3-6 months of HEMT. Multivariate regression identified predictors of improvement. Cronbach's alpha and four distribution-based methods were used to assess internal consistency and calculate the MCID of the SNOT-22. RESULTS: A total of 184 PwCF participated with mean baseline SNOT-22 scores ranging from 18.1 to 56.7. Cronbach's alpha was ≥0.90 across sites. Participants at sites with pre- and post-HEMT data reported improvement in SNOT-22 scores after initiating HEMT (all p < 0.05). Worse baseline SNOT-22 score (odds ratio (OR): 1.05, p < 0.001, 95% CI: 1.02-1.08), F508del homozygosity (OR: 4.30, p = 0.040, 95% CI: 1.14-18.99), and absence of prior modulator therapy (OR: 4.99, p = 0.017, 95% CI: 1.39-20.11) were associated with greater SNOT-22 improvement. The mean MCID calculated via distribution-based methods was 8.5. CONCLUSION: Worse baseline sinonasal symptoms, F508del homozygosity, and absence of prior modulator therapy predicted greater improvement after HEMT initiation. The mean MCID for SNOT-22 in PwCF is 8.5 points, similar to non-CF individuals with CRS, and provides a threshold specifically for PwCF. The SNOT-22 has strong internal consistency in PwCF. LEVEL OF EVIDENCE: 3 Laryngoscope, 2024.

Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression.

The cystic fibrosis (CF) respiratory tract harbors pathogenic bacteria that cause life-threatening chronic infections. Of these, Pseudomonas aeruginosa becomes increasingly dominant with age and is associated with worsening lung function and declining microbial diversity. We aimed to understand why P. aeruginosa dominates over other pathogens to cause worsening disease. Here, we show that P. aeruginosa responds to dynamic changes in iron concentration, often associated with viral infection and pulmonary exacerbations, to become more competitive via expression of the TseT toxic effector. However, this behavior can be therapeutically targeted using the iron chelator deferiprone to block TseT expression and competition. Overall, we find that iron concentration and TseT expression significantly correlate with microbial diversity in the respiratory tract of people with CF. These findings improve our understanding of how P. aeruginosa becomes increasingly dominant with age in people with CF and provide a therapeutically targetable pathway to help prevent this shift.

Elexacaftor-tezacaftor-ivacaftor decreases pseudomonas abundance in the sinonasal microbiome in cystic fibrosis.

BACKGROUND: Chronic rhinosinusitis (CRS) is common in individuals with cystic fibrosis (CF) and is marked by chronic inflammation and episodes of infection that negatively impact quality of life. Several studies have shown that elexacaftor-tezacaftor-ivacaftor (ETI) improves symptoms and examination findings in CF-CRS. The current study determines the effect of ETI on the sinonasal microbiota in CF. METHODS: Sinonasal samples were collected under endoscopic visualization before and after starting ETI. Samples were subjected to 16S amplicon sequencing and sequences were processed with the QIIME2 pipeline with subsequent analysis using the vegan R-package. RESULTS: Twenty-nine individual baseline samples and 23 sample pairs pre-/post-ETI were available. At baseline, the cohort had samples dominated by Staphylococcus, and alpha diversity was lower than that of a published reference set of individuals without sinonasal disease. Individuals with prior sinus surgery had lower alpha diversity as measured by Shannon Index, Observed Richness, and Faith's phylogenetic diversity Index. Beta diversity differed between individuals with and without allergic rhinitis, with higher Staphylococcus abundance in those with allergic rhinitis. No change in alpha or beta diversity was seen after a median of 9 months on ETI. With ETI, the Pseudomonas genus and the genus containing Burkholderia decreased in samples containing these taxa at baseline. Pseudomonas abundance decreased with treatment as measured by qPCR. Core sinonasal microbiome members Staphylococcus, Corynebacterium, and Streptococcus were unchanged, while Moraxella increased with ETI. CONCLUSIONS: Treatment with ETI leads to a reduction in Pseudomonas abundance within the sinonasal microbiome of individuals with Pseudomonas at baseline.

Successful treatment of refractory hypertension with bilateral nephrectomy in a patient with chronic kidney disease stage 3.

We present a case of life-threatening refractory hypertension (rHTN) in a patient with stage 3b chronic kidney disease that was unresponsive to open surgical renal denervation (RDN) but responded to bilateral nephrectomy (BLN). Both RDN and BLN reduce the increased sympathetic activation in rHTN. However, RDN has yet to show reductions in blood pressure adequate for the average patient with rHTN, and BLN has thus far been reserved for patients with preexisting end-stage kidney disease (ESKD). Our case suggests that there are patients with rHTN that warrant consideration of BLN prior to developing ESKD.

Elexacaftor-Tezacaftor- Ivacaftor improves sinonasal outcomes in cystic fibrosis.

BACKGROUND: Many individuals with cystic fibrosis (CF) have chronic rhinosinusitis resulting in nasal obstruction, sinus infections, and repeated surgeries. Elexacaftor-tezacaftor-ivacaftor is a highly effective modulator therapy approved for individuals aged 6 years or older with CF who have at least one F508del allele or other responsive mutation. The current study tests the hypothesis that ELX/TEZ/IVA improves sinonasal disease in CF. METHODS: The study was a pre/post, observational cohort study conducted at two sites. Participants underwent a study visit prior to starting ELX/TEZ/IVA and a second visit at a median of 9 months on therapy. Each visit included sinus CT scan, rigid nasal endoscopy, and sweat chloride measurement. Symptoms were measured with the 22 item Sinonasal Outcome Test at scheduled intervals during the study. Regression models were used to test for improvement in symptoms, endoscopy, and CT scales. RESULTS: The study enrolled 34 individuals, with a median age of 27 years (range 12-60). Symptoms improved within 7 days of therapy and plateaued by day 28. Endoscopic crusting resolved and nasal polyposis improved, with a decrease in size or resolution of polyps. Sinus opacification and mucosal thickening improved on CT radiographs with treatment. CONCLUSIONS: Sinonasal symptoms improved rapidly and durably for at least 180 days on ELX/TEZ/IVA therapy. Objective measures of disease including endoscopic and CT findings improved with ELX/TEZ/IVA.

Low Diversity and Instability of the Sinus Microbiota over Time in Adults with Cystic Fibrosis.

Chronic rhinosinusitis (CRS) is a common, yet underreported and understudied manifestation of upper respiratory disease in people with cystic fibrosis (CF). Recently developed standard of care guidelines for the management of CF CRS suggest treatment of upper airway disease may ameliorate lower airway disease. We sought to determine whether changes to sinus microbial community diversity and specific taxa known to cause CF lung disease are associated with increased respiratory disease and inflammation. We performed 16S rRNA gene sequencing, supplemented with cytokine analyses, microscopy, and bacterial culturing, on samples from the sinuses of 27 adults with CF CRS. At each study visit, participants underwent endoscopic paranasal sinus sampling and clinical evaluation. We identified key drivers of microbial community composition and evaluated relationships between diversity and taxa with disease outcomes and inflammation. Sinus community diversity was low, and the composition was unstable, with many participants exhibiting alternating dominance between Pseudomonas aeruginosa and staphylococci over time. Despite a tendency for dominance by these two taxa, communities were highly individualized and shifted composition during exacerbation of sinus disease symptoms. Exacerbations were also associated with communities dominated by Staphylococcus spp. Reduced microbial community diversity was linked to worse sinus disease and the inflammatory status of the sinuses (including increased interleukin-1ß [IL-1ß]). Increased IL-1ß was also linked to worse sinus endoscopic appearance, and other cytokines were linked to microbial community dynamics. Our work revealed previously unknown instability of sinus microbial communities and a link between inflammation, lack of microbial community diversity, and worse sinus disease. IMPORTANCE Together with prior sinus microbiota studies of adults with CF chronic rhinosinusitis, our study underscores similarities between sinus and lower respiratory tract microbial community structures in CF. We show how community structure tracks with inflammation and several disease measures. This work strongly suggests that clinical management of CRS could be leveraged to improve overall respiratory health in CF. Our work implicates elevated IL-1ß in reduced microbiota diversity and worse sinus disease in CF CRS, suggesting applications for existing therapies targeting IL-1ß. Finally, the widespread use of highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy has led to less frequent availability of spontaneous expectorated sputum for microbiological surveillance of lung infections. A better understanding of CF sinus microbiology could provide a much-needed alternative site for monitoring respiratory infection status by important CF pathogens.

Adaptation and genomic erosion in fragmented Pseudomonas aeruginosa populations in the sinuses of people with cystic fibrosis.

Pseudomonas aeruginosa notoriously adapts to the airways of people with cystic fibrosis (CF), yet how infection-site biogeography and associated evolutionary processes vary as lifelong infections progress remains unclear. Here we test the hypothesis that early adaptations promoting aggregation influence evolutionary-genetic trajectories by examining longitudinal P. aeruginosa from the sinuses of six adults with CF. Highly host-adapted lineages harbored mutator genotypes displaying signatures of early genome degradation associated with recent host restriction. Using an advanced imaging technique (MiPACT-HCR [microbial identification after passive clarity technique]), we find population structure tracks with genome degradation, with the most host-adapted, genome-degraded P. aeruginosa (the mutators) residing in small, sparse aggregates. We propose that following initial adaptive evolution in larger populations under strong selection for aggregation, P. aeruginosa persists in small, fragmented populations that experience stronger effects of genetic drift. These conditions enrich for mutators and promote degenerative genome evolution. Our findings underscore the importance of infection-site biogeography to pathogen evolution.

Antimicrobial Synergism Toward Pseudomonas aeruginosa by Gallium(III) and Inorganic Nitrite.

The ubiquitous involvement of key iron-containing metalloenzymes in metabolism is reflected in the dependence of virtually all bacteria on iron for growth and, thereby, potentially provides multiple biomolecular targets for antimicrobial killing. We hypothesized that nitrosative stress, which induces damage to iron metalloproteins, would sensitize bacteria to the ferric iron mimic gallium(III) (Ga3+), potentially providing a novel therapeutic combination. Using both laboratory and clinical isolates of Pseudomonas aeruginosa, we herein demonstrate that Ga3+ and sodium nitrite synergistically inhibit bacterial growth under both aerobic and anaerobic conditions. Nitric oxide also potentiated the antimicrobial effect of Ga3+. Because many chronic pulmonary infections are found as biofilms and biofilms have very high antibiotic tolerance, we then tested the combination against biofilms grown on plastic surfaces, as well as the apical surface of airway epithelial cells. Ga3+ and sodium nitrite had synergistic antimicrobial activity against both biofilms grown on plastic and on airway epithelial cell. Both Ga3+ and various NO donors are (independently) in clinical development as potential antimicrobials, however, we now propose the combination to have some particular advantages, while anticipating it should ultimately prove similarly safe for translation to treatment of human disease.

Dispersal of Epithelium-Associated Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa grows in highly antibiotic-tolerant biofilms during chronic airway infections. Dispersal of bacteria from biofilms may restore antibiotic susceptibility or improve host clearance. We describe models to study biofilm dispersal in the nutritionally complex environment of the human airway. P. aeruginosa was cocultured in the apical surface of airway epithelial cells (AECs) in a perfusion chamber. Dispersal, triggered by sodium nitrite, a nitric oxide (NO) donor, was tracked by live cell microscopy. Next, a static model was developed in which biofilms were grown on polarized AECs without flow. We observed that NO-triggered biofilm dispersal was an energy-dependent process. From the existing literature, NO-mediated biofilm dispersal is regulated by DipA, NbdA, RbdA, and MucR. Interestingly, altered signaling pathways appear to be used in this model, as deletion of these genes failed to block NO-induced biofilm dispersal. Similar results were observed using biofilms grown in an abiotic model on glass with iron-supplemented cell culture medium. In cystic fibrosis, airway mucus contributes to the growth environment, and a wide range of bacterial phenotypes are observed; therefore, we tested biofilm dispersal in a panel of late cystic fibrosis clinical isolates cocultured in the mucus overlying primary human AECs. Finally, we examined dispersal in combination with the clinically used antibiotics ciprofloxacin, aztreonam and tobramycin. In summary, we have validated models to study biofilm dispersal in environments that recapitulate key features of the airway and identified combinations of currently used antibiotics that may enhance the therapeutic effect of biofilm dispersal.IMPORTANCE During chronic lung infections, Pseudomonas aeruginosa grows in highly antibiotic-tolerant communities called biofilms that are difficult for the host to clear. We have developed models for studying P. aeruginosa biofilm dispersal in environments that replicate key features of the airway. We found that mechanisms of biofilm dispersal in these models may employ alternative or additional signaling mechanisms, highlighting the importance of the growth environment in dispersal events. We have adapted the models to accommodate apical fluid flow, bacterial clinical isolates, antibiotics, and primary human airway epithelial cells, all of which are relevant to understanding bacterial behaviors in the context of human disease. We also examined dispersal agents in combination with commonly used antipseudomonal antibiotics and saw improved clearance when nitrite was combined with the antibiotic aztreonam.

Reparative capacity of airway epithelium impacts deposition and remodeling of extracellular matrix.

Defective epithelial repair in the setting of chronic lung disease has been suggested to contribute to uncontrolled extracellular matrix (ECM) deposition and development of fibrosis. We sought to directly test this hypothesis through gene expression profiling of total lung RNA isolated from mouse models of selective epithelial cell injury that are associated with either productive or abortive repair. Analysis of gene expression in repairing lungs of naphthalene-exposed mice revealed prominent clusters of up-regulated genes with putative roles in regulation of the extracellular matrix and cellular proliferation. Further analysis of tenascin C (Tnc), a representative matrix protein, in total lung RNA revealed a transient 4.5-fold increase in mRNA abundance 1 day after injury and a return to steady-state levels by Recovery Day 3. Tnc was deposited by the peribronchiolar mesenchyme immediately after injury and was remodeled to basement membrane subtending the bronchiolar epithelium during epithelial repair. Epithelial restitution was accompanied by a decrease in Tnc mRNA and protein expression to steady-state levels. In contrast, abortive repair using a transgenic model allowing ablation of all reparative cells led to a progressive increase in Tnc mRNA within lung tissue and accumulation of its gene product within the subepithelial mesenchyme of both conducting airways and alveoli. These data demonstrate that the ECM is dynamically remodeled in response to selective epithelial cell injury and that this process is activated without resolution in the setting of defective airway epithelial repair.

Molecular staging of epithelial maturation using secretory cell-specific genes as markers.

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.

beta-Catenin is not necessary for maintenance or repair of the bronchiolar epithelium.

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.

Conditional stabilization of beta-catenin expands the pool of lung stem cells.

Maintenance of classic stem cell hierarchies is dependent upon stem cell self-renewal mediated in part by Wnt/beta-catenin regulation of the cell cycle. This function is critical in rapidly renewing tissues due to the obligate role played by the tissue stem cell. However, the stem cell hierarchy responsible for maintenance of the conducting airway epithelium is distinct from classic stem cell hierarchies. The epithelium of conducting airways is maintained by transit-amplifying cells in the steady state; rare bronchiolar stem cells are activated to participate in epithelial repair only following depletion of transit-amplifying cells. Here, we investigate how signaling through beta-catenin affects establishment and maintenance of the stem cell hierarchy within the slowly renewing epithelium of the lung. Conditional potentiation of beta-catenin signaling in the embryonic lung results in amplification of airway stem cells through attenuated differentiation rather than augmented proliferation. Our data demonstrate that the differentiation-modulating activities of stabilized beta-catenin account for expansion of tissue stem cells.