Mouse Tracheal Epithelial Cell Research Articles

P53 regulation by MDM2 contributes to self-renewal and differentiation of basal stem cells in mouse and human airway epithelium.

Proper physiological function of mammalian airways requires the differentiation of basal stem cells into secretory or multiciliated cells, among others. In addition, the self-renewal ability of these basal stem cells is crucial for developing a quick response to toxic agents in order to re-establish the epithelial barrier function of the airways. Although these epithelial missions are vital, little is known about those mechanism controlling airway epithelial regeneration in health and disease. p53 has been recently proposed as the guardian of homeostasis, promoting differentiation programs, and antagonizing a de-differentiation program. Here, we exploit mouse and human tracheal epithelial cell culture models to study the role of MDM2-p53 signaling in self-renewal and differentiation in the airway epithelium. We show that p53 protein regulation by MDM2 is crucial for basal stem cell differentiation and to keep proper cell proliferation. Therefore, we suggest that MDM2/p53 interaction modulation is a potential target to control regeneration of the mammalian airway epithelia without massively affecting the epithelium integrity and differentiation potential.

IL-33/ST2 signaling modulates Afghanistan particulate matter induced airway hyperresponsiveness in mice

Increased symptoms of asthma-like respiratory illnesses have been reported in soldiers returning from tours of duty in Afghanistan. Inhalation of desert particulate matter (PM) may contribute to this deployment-related lung disease (DRLD), but little is known about disease mechanisms. The IL-33 signaling pathway, including its receptor ST2, has been implicated in the pathogenesis of lung diseases including asthma, but its role in PM-mediated airway dysfunction has not been studied. The goal of this study was to investigate whether IL-33/ST2 signaling contributes to airway dysfunction in preclinical models of lung exposure to Afghanistan PM (APM). Wild-type (WT) and ST2 knockout (KO) mice on the BALB/C background were oropharyngeally instilled with a single dose of saline or 50 μg of APM in saline. Airway hyperresponsiveness (AHR) and inflammation were assessed after 24 h. In WT mice, a single APM exposure induced AHR and neutrophilic inflammation. Unlike the WT mice, ST2 KO mice that lack the receptor for IL-33 did not demonstrate AHR although airway neutrophilic inflammation was comparable to the WT mice. Oropharyngeal delivery of a soluble ST2 decoy receptor in APM-exposed WT mice significantly blocked AHR. Additional data in mouse tracheal epithelial cell and lung macrophage cultures demonstrated a role of APM-induced IL-33/ST2 signaling in suppression of regulator of G protein signaling 2 (RGS2), a gene known to protect against bronchoconstriction. We present for the first time that APM may increase AHR, one of the features of asthma, in part through the IL-33/ST2/RGS2 pathway.

Lack of GAS2L2 Causes PCD by Impairing Cilia Orientation and Mucociliary Clearance

Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8Hz versus control = 15.8Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X.laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results inPCD.

Inflammation Increases the Opening State of Channels in the Epithelium, Releasing High Levels of ATP that Resulted in a Decrease on Mucociliary Transport Velocity in the Airways

ATP is one of the chemical signals locally released by the airway epithelium associated with an autocrine/paracrine mechanism to control basal ciliary beat frequency (CBF). A recent study has reported that ATP has a dual effect on CBF, since ATP (μM) increases CBF, but high levels of ATP (mM) significantly decreases CBF in cultures of mouse tracheal epithelial cell. This nucleotide can be released by different mechanisms that include hemichannels of connexins (Cx43) and pannexins (Panx1), and more recently the Calhm1 channel. In several cell types it has recently reported that in pathophysiological conditions, connexins and pannexins channels could modify their opening state. Furthermore, high levels of ATP have been measured in the bronchoalveolar fluid of asthmatic patients and ovoalbumin‐induced asthmatic mouse model (OVA). The asthmatic patients also have infiltration of inflammatory cells in the pulmonary epithelium, reduced CBF and mucociliary transport effectiveness. Mucociliary transport is the first defense mechanism in the airways, where particles are trapped in the mucus and removed by the ciliary beating to the pharynx. We proposed the following objective: Determine the relation between levels of ATP of an asthmatic OVA mouse model, the opening state of the channels that control the release of this nucleotide and the effectiveness of mucociliary transport in the airways. The protocols used in this study were approved, by the ethics committee of the university that follow international guidelines for animal care. We measured extracellular ([ATP]e) with a luminometric assay and evaluated the infiltration of inflammatory cells in the lung parenchyma by histochemistry (H&E). The ethidium bromide (5μM) capture was recorded with a camera mounted on an Olympus upright microscope. The microspheres (6μm) velocity on the surface of the trachea epithelium was registered with a videomicroscopy system and analyzed with the ImageJ program. We observed greater infiltration of inflammatory cells in the pulmonary parenchyma of the asthmatic mouse, compared with the control mouse. Also, we established that high levels of ATP were present in asthmatic mice trachea lavage compared to control animals. The microspheres velocity was diminished in OVA mouse model compare to control mice and addition of 1mM of ATP in control or asthmatic mice transiently stopped the microspheres movement. Furthermore, we observed a significant increase in the percentage of ethidium bromide capture by the epithelium of asthmatic mice compare to controls. These results demonstrated that in an inflammatory condition, channels involved in the ATP release increased their opening state in the airways epithelium. This effect could be associated to high levels of ATP measured in asthma, condition that resulted in a deleterious effect on mucociliary transport effectiveness.Support or Funding InformationCONICYT 21160416, LL.A. Millennium Institute on Immunology and Immunotherapy (P09/016‐F) MV.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influences of influenza virus on cystic fibrosis transmembrane conductance regulator (CFTR)

Objective To evaluate the differentiation of mouse tracheal epithelial cells (MTEC) at an air-liquid interface and to investigate the influences of influenza virus on the cystic fibrosis transmembrane conductance regulator (CFTR) in primary cultured MTEC for further elucidating the possible mechanism of imbalanced fluid and salt transportation in respiratory system caused by influenza virus infection. Methods The morphology of primarily cultured MTEC was observed under inverted microscope. Trans epithelial electrical resistance (TEER) was measured by a resistance meter to evaluate the integrity of cultured MTEC. An Ussing chamber apparatus was used to record the short-circuit current of primary cultured MTEC. Results The primarily cultured MTEC clustered together and had a tight pavement-like appearance under light microscope. The TEER was greater than 1 000 Ω after 6 days of culture. Influenza virus could reduce the short-circuit current of CFTR to (52.77±10.30)% in intact cell membrane and to (41.50±1.09)% in monolayer MTEC after increasing the permeability of basement membrane. It had been proved that CFTR was essential to maintaining the balance of fluid and salt transportation in respiratory system. Conclusion Mouse MTEC are efficiently cultured at a air-liquid interface and the primarily cultured cells are highly similar to those in a normal physiologic state. Influenza virus may block the secretion of anions through inhibiting the function of CFTR, which may induce the development of chronic obstructive pulmonary disease and the incidence of asthma. Key words: Influenza virus; Mouse tracheal epithelial cell; Cystic fibrosis transmembrane conductance regulator; Short-circuit current

An innate defense peptide BPIFA1/SPLUNC1 restricts influenza A virus infection

The airway epithelium secretes proteins that function in innate defense against infection. Bactericidal/permeability-increasing fold-containing family member A1 (BPIFA1) is secreted into airways and has a protective role during bacterial infections, but it is not known whether it also has an antiviral role. To determine a role in host defense against influenza A virus (IAV) infection and to find the underlying defense mechanism, we developed transgenic mouse models that are deficient in BPIFA1 and used these, in combination with in vitro three-dimensional mouse tracheal epithelial cell (mTEC) cultures, to investigate its antiviral properties. We show that BPIFA1 has a significant role in mucosal defense against IAV infection. BPIFA1 secretion was highly modulated after IAV infection. Mice deficient in BPIFA1 lost more weight after infection, supported a higher viral load and virus reached the peripheral lung earlier, indicative of a defect in the control of infection. Further analysis using mTEC cultures showed that BPIFA1-deficient cells bound more virus particles, displayed increased nuclear import of IAV ribonucleoprotein complexes, and supported higher levels of viral replication. Our results identify a critical role of BPIFA1 in the initial phase of infection by inhibiting the binding and entry of IAV into airway epithelial cells.

Abstract 62: Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model

Abstract Background: Cancer cells may undergo mutations beyond their original causal changes, resulting in clonal expansion, and potentially driving tumor heterogeneity and evolution. Our hypothesis was that additional, “third hit” genetic changes in clonally derived tumors, originally generated by the same oncogenic stimulus, would have different phenotypes. Methods: Epithelial cells were isolated from LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- and C57BL/6 control mouse tracheas and were cultured on plastic. On day 3, cells were treated with an adenovirus expressing Cre recombinase. After 7 days, single EPCAM+, 7-aminoactinomycin D (7-AAD)-, and tdTomato+ cells from the LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse, and EPCAM+/ 7-AAD- cells from the control mouse were FACS sorted, and individually seeded in 96-well plates. Clones developing in each well were passaged separately and characterized using growth curves on plastic, staining for lineage markers, and colony forming efficiency (CFE) and colony morphology in 3D culture. Selected clones were further investigated in vivo after subcutaneous injection into the flanks of NOD/SCID mice. Agilent SureSelect targeted exome sequencing was used to evaluate genetic variations in cells derived from each cell clone. Results: Thirty-five LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- transformed clones and 2 control mouse clones were serially passaged, and 4 transformed cell lines representing distinct phenotypes (named MT1, 2, 3, and 4), were identified based on growth rates, lineage marker staining, CFE, and 3D colony morphology. All 4 clones were pancytokeratin+, 3 were thyroid trasnscription factor-1 (TTF-1)+ and one was p63+. All 4 cell lines generated tumors in NOD/SCID mice, which exhibited variable histology and marker expression. Exome sequencing revealed unique copy number profiles. Specifically, MT1 had gains and losses in chromosome 3, MT2 had losses in chromosome 9, MT3 had losses in chromosomes 7 and 14, and MT4 had losses in chromosome 7. Mutations potentially representing third hits were identified in each cell line, particularly Ss18l1 (synovial sarcoma translocation gene on chromosome 18-like 1) and Gas7 (growth arrest specific 7) in MT1, Runx1 (runt related transcription factor 1) and Dicer1 (dicer 1, ribonuclease type III) in MT2, Apc (adenomatous polyposis coli) in MT3, and Csmd1 (CUB and Sushi multiple domains 1) in MT4. Conclusions: Multiple single cell derived mouse tracheal epithelial tumor cell clones were generated from a single LSL-KrasG12D+/-/Trp53fl/fl/Rosa-CAG-LSL-tdTomato+/- mouse. Selected clones exhibited unique growth rates, lineage marker expression patterns, CFE, 3D cell morphology and variable in vivo tumor histology and marker expression. Unique copy number aberrations and third hit mutations likely generate distinct clone phenotypes, driving tumor heterogeneity and evolution. Citation Format: Sang-Won Um, Ping-Jie Xiao, David Neil Hayes, William Y. Kim, Scott H. Randell. Third hit genetic changes and clonal heterogeneity in a genetically engineered mouse lung cancer model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 62. doi:10.1158/1538-7445.AM2014-62

Arsenic Compromises Conducting Airway Epithelial Barrier Properties in Primary Mouse and Immortalized Human Cell Cultures

Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb)] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE) cell model we found that both micromolar (3.9 μM) and submicromolar (0.8 μM) arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl) Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-). We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

Direct and indirect roles for β-catenin in facultative basal progenitor cell differentiation.

The conducting airway epithelium is maintained and repaired by endogenous progenitor cells. Dysregulated progenitor cell proliferation and differentiation is thought to contribute to epithelial dysplasia in chronic lung disease. Thus modification of progenitor cell function is an attractive therapeutic goal and one that would be facilitated by knowledge of the molecular pathways that regulate their behavior. We modeled the human tracheobronchial epithelium using primary mouse tracheal epithelial cell cultures that were differentiated by exposure to the air-liquid-interface (ALI). A basal cell subset, termed facultative basal cell progenitors (FBP), initiate these cultures and are the progenitor for tracheal-specific secretory cells, the Clara-like cell, and ciliated cells. To test the hypothesis that β-catenin is necessary for FBP function, ALI cultures were generated from mice homozygous for the Ctnb(flox(E2-6)) allele. In this model, exons 2-6 of the β-catenin gene are flanked by LoxP sites, allowing conditional knockout of β-catenin. The β-catenin locus was modified through transduction with Adenovirus-5-encoding Cre recombinase. This approach generated a mosaic epithelium, comprised of β-catenin wild-type and β-catenin knockout cells. Dual immunostaining and quantitative histomorphometric analyses demonstrated that β-catenin played a direct role in FBP-to-ciliated cell differentiation and that it regulated cell-cell interactions that were necessary for FBP-to-Clara-like cell differentiation. β-catenin was also necessary for FBP proliferation and long-term FBP viability. We conclude that β-catenin is a critical determinant of FBP function and suggest that dysregulation of the β-catenin signaling pathway may contribute to disease pathology.

Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells

BackgroundAsthma causes significant morbidity worldwide in adults and children alike, and incurs large healthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases, have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, anti-inflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergic mouse model of asthma, we previously demonstrated a benefit of statins in reducing peribronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, and lung IL-4 and IL-13 production.ObjectivesIn this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatory gene expression of asthma-related cytokines in well-differentiated primary mouse tracheal epithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression of IL-13-inducible genes in MTE cells.MethodsWe harvested tracheal epithelial cells from naïve BALB/c mice, grew them under air-liquid interface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTE cells using a quantitative real-time PCR mouse gene array kit.ResultsWe found that simvastatin had differential effects on IL-13-mediated gene expression (inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 and CCL20 (MIP-3α)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10, CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible or statin effects on gene expression.ConclusionsSimvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatory cytokines and chemokines in primary mouse tracheal epithelial cells. The airway epithelium may be a viable target tissue for the statin drugs. Further research is needed to assess the mechanisms of how statins modulate epithelial gene expression.

Cystic fibrosis transmembrane conductance regulator regulates epithelial cell response to Aspergillus and resultant pulmonary inflammation.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) alter epithelial cell (EC) interactions with multiple microbes, such that dysregulated inflammation and injury occur with airway colonization in people with cystic fibrosis (CF). Aspergillus fumigatus frequently colonizes CF airways, but it has been assumed to be an innocent saprophyte; its potential role as a cause of lung disease is controversial. To study the interactions between Aspergillus and EC, and the role of the fungus in evoking inflammatory responses. A. fumigatus expressing green fluorescent protein was developed for in vitro and in vivo models, which used cell lines and mouse tracheal EC. Fungal spores (conidia) are rapidly ingested by ECs derived from bronchial cell lines and murine tracheas, supporting a role for EC in early airway clearance. Bronchial ECs harboring CFTR mutations (ΔF508) or deletion demonstrate impaired uptake and killing of conidia, and ECs with CFTR mutation undergo more conidial-induced apoptosis. Germinated (hyphal) forms of the fungus evoke secretion of inflammatory mediators, with CFTR mutation resulting in increased airway levels of macrophage inflammatory protein 2 and KC, and higher lung monocyte chemotactic protein-1. After A. fumigatus inhalation, CFTR(-/-) mice develop exaggerated lymphocytic inflammation, mucin accumulation, and lung injury. Data demonstrate a critical role for CFTR in mediating EC responses to A. fumigatus. Results suggest that the fungus elicits aberrant pulmonary inflammation in the setting of CFTR mutation, supporting the potential role of antifungals to halt progressive CF lung disease.

Nitric oxide and airway epithelial barrier function: Regulation of tight junction proteins and epithelial permeability

Acute airway inflammation is associated with enhanced production of nitric oxide (NO ) and altered airway epithelial barrier function, suggesting a role of NO or its metabolites in epithelial permeability. While high concentrations of S-nitrosothiols disrupted transepithelial resistance (TER) and increased permeability in 16HBE14o− cells, no significant barrier disruption was observed by NONOates, in spite of altered distribution and expression of some TJ proteins. Barrier disruption of mouse tracheal epithelial (MTE) cell monolayers in response to inflammatory cytokines was independent of NOS2, based on similar effects in MTE cells from NOS2−/− mice and a lack of effect of the NOS2-inhibitor 1400W. Cell pre-incubation with LPS protected MTE cells from TER loss and increased permeability by H 2O 2, which was independent of NOS2. However, NOS2 was found to contribute to epithelial wound repair and TER recovery after mechanical injury. Overall, our results demonstrate that epithelial NOS2 is not responsible for epithelial barrier dysfunction during inflammation, but may contribute to restoration of epithelial integrity.

Mammalian cilia function is independent of the polymeric state of tubulin glycylation

Polyglycylation is a polymeric post-translational modification of tubulin that is ubiquitous and widely present in cilia and flagella. It consists of the addition of highly variable numbers of glycyl residues as side chains onto the gamma carboxyl group of specific glutamyl residues at the C-termini of alpha- and beta-tubulin. The function of polyglycylation is poorly understood, however, studies in Tetrahymena have shown that the mutation of polyglycylation sites in beta-tubulin resulted in axonemal abnormality or lethality. This suggests that polyglycylation is functionally essential in protists. We hypothesize that polyglycylation is also essential in mammalian cilia and that the extent of polyglycylation has functional significance. In this study, we examined polyglycylation states in ciliated tissues and in mouse tracheal epithelial cell cultures. We utilized two antibodies, TAP 952 and AXO 49, which recognize glutamyl sites possessing monomeric glycylation sites and glutamyl sites possessing polymeric glycylation sites, respectively. Monomeric glycylation sites were observed in cilia of all the ciliated tissues examined but were invariably excluded from the distal tips. In contrast, polymeric glycylation sites were rare, but when observed, they were localized at the bases of cilia. During ciliogenesis, in epithelial cell cultures, monomeric glycylation sites were observed, but the extent of polymeric glycylation sites were variable and were only observed during the early stages of the cultures. Our observations suggest that while monomeric glycylation sites are universal and likely essential in mammalian cilia, polymeric glycylation sites are not required for ciliary beating. Rather, our observations suggest that the number of added glycyl residues increases progressively from the tips of cilia toward their bases.

Expression of nucleotide-regulated Cl(-) currents in CF and normal mouse tracheal epithelial cell lines.

The dominant route for Cl(-) secretion in mouse tracheal epithelium is via Cl(-) channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(-/-)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/-)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10(-4) M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I(sc)) and resistance of both cell lines, with larger increases in I(sc) being elicited by ionomycin (10(-6) M). Both cell lines expressed P(2)Y(2) receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10(-4) M) with an increase in I(sc). This response could be inhibited by DIDS and was abolished in the presence of Cl(-)-free Ringer solution. Reducing the mucosal Cl(-) concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(-/-) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I(sc) and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl(-) currents and may prove valuable tools in studying the properties of this pathway.

Mycoplasma pulmonis inhibits electrogenic ion transport across murine tracheal epithelial cell monolayers.

Murine chronic respiratory disease is characterized by persistent colonization of tracheal and bronchial epithelial cell surfaces by Mycoplasma pulmonis, submucosal and intraluminal immune and inflammatory cells, and altered airway activity. To determine the direct effect of M. pulmonis upon transepithelial ion transport in the absence of immune and inflammatory cell responses, primary mouse tracheal epithelial cell monolayers (MTEs) were apically infected and assayed in Ussing chambers. M. pulmonis-infected MTEs, but not those infected with a nonmurine mycoplasma, demonstrated reductions in amiloride-sensitive Na+ absorption, cyclic AMP, and cholinergic-stimulated Cl- secretion and transepithelial resistance. These effects were shown to require interaction of viable organisms with the apical surface of the monolayer and to be dependent upon organism number and duration of infection. Altered transport due to M. pulmonis was not merely a result of epithelial cell death as evidenced by the following: (i) active transport of Na+ and Cl-, albeit at reduced rates; (ii) normal cell morphology, including intact tight junctions, as demonstrated by electron microscopy; (iii) maintenance of a mean transepithelial resistance of 440 omega/cm2; and (iv) lack of leakage of fluid from the basolateral to the apical surface of the monolayer. Alteration in epithelial ion transport in vitro is consistent with impaired pulmonary clearance and altered airway function in M. pulmonis-infected animals. Furthermore, the ability of M. pulmonis to alter transport without killing the host cell may explain its successful parasitism and long-term persistence in the host. Further study of the MTE-M. pulmonis model should elucidate the molecular mechanisms which mediate this reduction in transepithelial ion transport.