Bile Acid Aspiration Research Articles

(703) - Bile Acids Aspiration Induces Fibrosis and Disrupts Surfactant Homeostasis in Human Lungs

(703) - Bile Acids Aspiration Induces Fibrosis and Disrupts Surfactant Homeostasis in Human Lungs

Large Airway Bronchial Wash Lipidomics as Novel Biomarkers for Chronic Lung Allograft Dysfunction

<h3>Purpose</h3> Chronic lung allograft dysfunction (CLAD) is the primary cause of long-term mortality after lung transplantation. Aspiration of bile acids has been associated with CLAD and lipidomic dysregulation. This study evaluates the role of large airway bronchial wash (LABW) lipidomics and diagnosis of CLAD. <h3>Methods</h3> LABW (n=611) samples were obtained from 305 lung transplant recipients at different timepoints. Concentrations of 26 specific lipid subfamilies were obtained using liquid chromatography mass spectrometry. Lipid percentages where stratified by highest tertiles. The primary outcome of interest was development of CLAD from sampling time. Time-to-event analysis was done using Kaplan-Meier functions and a multivariable Cox proportional hazards model adjusting for baseline comorbidities. <h3>Results</h3> Samples with CLAD at the time of collection showed higher percentages of hexosylceramides (HEXCER), CE, sphingomyelin (SM) and lactosylceramide (LACCER). A high percentage of bismethyl-phosphatidic acid (BMP), CE, phosphatidylethanolamine (PEP), dehydrosphingosine (DHSM), lysophosphatidylethanolamine (LPE), ganglioside (GM3), phosphatidyl-serine (PS) were associated with an increased hazards of CLAD by univariable analysis. A high percentage of CE (HR: 1.48, 95% CI: 1.09-2.03, <i>P</i>=0.013) were independent predictors of CLAD by multivariable analysis. High percentages of CER (HR: 0.49, 95% CI: 0.36-0.66, <i>P</i><0.001) and a high LACCER:CER ratio (HR: 0.45, 95% CI: 0.34-0.62, <i>P</i><0.001) were independently associated with decreased hazards of CLAD. <h3>Conclusion</h3> Airway lipidomic qualititative changes show a distinct phenotype that associate with the presence and risk of development of CLAD. Percentages of CE, CER and LACCER:CER ratios correlate independently with increased risk of development of CLAD. Changes in LABW lipids may serve as biomarkers for CLAD and elucidate pathophysiologic changes after transplant.

Aspiration of conjugated bile acids predicts adverse lung transplant outcomes and correlates with airway lipid and cytokine dysregulation

Duodeno-gastroesophageal reflux aspiration is associated with chronic lung allograft dysfunction (CLAD). Reflux aspirate can contain bile acids (BA), functional molecules in the gastro-intestinal tract with emulsifying properties. We sought to determine and quantify the various BA species in airways of the lung transplant recipients to better understand the various effects of aspirated BA that contribute to post-transplantation outcomes. Bronchial washings (BW) were prospectively collected from lung transplant recipients and subsequently assayed by liquid chromatography-mass spectrometry for 13 BA and 25 lipid families. Patients were monitored for CLAD, rejection, inflammation and airway infections. Detectable BA were present in 45/50 patients (90%) at 3 months after transplant. Elevated BA and predominance of conjugated species were independent predictors of CLAD (hazard ratio 7.9; 95% confidence interval 2.7-23.6; p < 0.001 and 7.3; 2.4-22; p < 0.001, respectively) and mortality (hazard ratio 4.4; 1.5-12.7; p=0.007 and 4.8; 1.4-15.8; p=0.01, respectively). High BA associated with increased positive bacterial cultures (60% vs 25%, p=0.02). Primary conjugated species independently correlated with the rate of bacterial cultures during the first-year post-transplant (Beta coefficient: 0.77; 0.28-1.26; p=0.003) and changes in airway lipidome and cytokines. Higher BA levels and predominance of conjugated BA are independent predictors of chronic lung allograft dysfunction, mortality and bacterial infections. Primary conjugated BA are related to distinct changes in airway lipidome and inflammatory cytokines. This elucidates novel evidence into the mechanism following BA aspiration and proposes novel markers for prediction of adverse post-transplant outcomes.

Assessment of Donor Lung Aspiration on the Ex Vivo Lung Perfusion (EVLP) Platform

Donor aspiration of gastric contents represents a significant risk for lung injury and severe primary graft dysfunction after lung transplantation. Concern about possible aspiration is a common reason for clinicians to decline donor lungs for transplantation. The presence of bile acid in donor bronchial wash (BW) or bronchoalveolar lavage (BAL) fluid may indicate that a donor has aspirated. Previous reports have also shown that aspiration of bile acids cause increased inflammation in the lung. Ex vivo lung perfusion (EVLP) has become an important platform to assess questionable or "marginal" donors. In this study, we examined the relationship between possible donor aspiration and physiologic and biologic assessment of inflammation on EVLP. Large airway BW samples were collected from 270 donors (n=111 non-EVLP, n=159 EVLP) prior to transplantation. Bile acid levels were measured in BW samples using a commercially available colorimetric assay and clinically approved platform. Protein and biochemical measurements were made in perfusate samples collected hourly after the start of EVLP. The majority of donor lungs with clinically suspected aspiration were successfully transplanted after EVLP assessment (58% [23/40]). Donor bile acid levels were significantly higher in donor lungs that were ultimately declined for transplantation at the end of EVLP (0.28 vs. 0.12 μM, p<0.05, n=143). During EVLP there was a positive correlation between bile acid in the donor BW and inflammation (IL-8 levels (r=0.19, p<0.05, n=159)). Similarly, there was an increase in biochemical markers of cell death ([Ca2+]) in the EVLP perfusate of donors with high bile acid levels in BW (r=0.27, p<0.001, n=142). Following EVLP and transplantation, we observed no significant effects of donor bile acid levels on recipient ICU length-of-stay (r=0.09, p=0.35, n=117). Suspected donor aspiration can be assessed using the EVLP platform. Bile acid levels measured in the donor lung are associated with increased inflammation and lung injury observed during EVLP, but not recipient outcomes post-EVLP. Therefore, EVLP may be an ideal platform to identify marginal donor lungs suspected of aspiration but are ultimately suitable for clinical transplantation.

Bile Acid Aspiration is Associated with Airway Infections: A Targeted Metabolomic Approach

Gastroesophageal reflux and bile acid aspiration are risk factors for chronic lung allograft dysfunction. Bile acids may compromise the broncho-alveolar innate immunity by deranging the lipid surface barrier and/or by directly modulating macrophage activity. Targeted bile acid metabolomics and bacterial, fungal and viral cultures were investigated in post lung transplant surveillance bronchial washings.

Bile Acids Aspiration Modulates Cholinergic and Serotonergic Responses of the Distal Airways

Gastro-esophageal reflux and bile acids (BAs) aspiration is associated to chronic lung allograft dysfunction (CLAD). We investigated for the first time the reactivity of small airways to a comprehensive panel of BAs as detected in post lung transplant bronchial washings.

Effects of bile acids on human airway epithelial cells: implications for aerodigestive diseases.

Gastro-oesophageal reflux and aspiration have been associated with chronic and end-stage lung disease and with allograft injury following lung transplantation. This raises the possibility that bile acids may cause lung injury by damaging airway epithelium. The aim of this study was to investigate the effect of bile acid challenge using the immortalised human bronchial epithelial cell line (BEAS-2B).The immortalised human bronchial epithelial cell line (BEAS-2B) was cultured. A 48-h challenge evaluated the effect of individual primary and secondary bile acids. Post-challenge concentrations of interleukin (IL)-8, IL-6 and granulocyte−macrophage colony-stimulating factor were measured using commercial ELISA kits. The viability of the BEAS-2B cells was measured using CellTiter-Blue and MTT assays.Lithocholic acid, deoxycholic acid, chenodeoxycholic acid and cholic acid were successfully used to stimulate cultured BEAS-2B cells at different concentrations. A concentration of lithocholic acid above 10 μmol·L−1 causes cell death, whereas deoxycholic acid, chenodeoxycholic acid and cholic acid above 30 μmol·L−1 was required for cell death. Challenge with bile acids at physiological levels also led to a significant increase in the release of IL-8 and IL6 from BEAS-2B.Aspiration of bile acids could potentially cause cell damage, cell death and inflammation in vivo. This is relevant to an integrated gastrointestinal and lung physiological paradigm of chronic lung disease, where reflux and aspiration are described in both chronic lung diseases and allograft injury.

Bile signalling promotes chronic respiratory infections and antibiotic tolerance.

Despite aggressive antimicrobial therapy, many respiratory pathogens persist in the lung, underpinning the chronic inflammation and eventual lung decline that are characteristic of respiratory disease. Recently, bile acid aspiration has emerged as a major comorbidity associated with a range of lung diseases, shaping the lung microbiome and promoting colonisation by Pseudomonas aeruginosa in Cystic Fibrosis (CF) patients. In order to uncover the molecular mechanism through which bile modulates the respiratory microbiome, a combination of global transcriptomic and phenotypic analyses of the P. aeruginosa response to bile was undertaken. Bile responsive pathways responsible for virulence, adaptive metabolism, and redox control were identified, with macrolide and polymyxin antibiotic tolerance increased significantly in the presence of bile. Bile acids, and chenodeoxycholic acid (CDCA) in particular, elicited chronic biofilm behaviour in P. aeruginosa, while induction of the pro-inflammatory cytokine Interleukin-6 (IL-6) in lung epithelial cells by CDCA was Farnesoid X Receptor (FXR) dependent. Microbiome analysis of paediatric CF sputum samples demonstrated increased colonisation by P. aeruginosa and other Proteobacterial pathogens in bile aspirating compared to non-aspirating patients. Together, these data suggest that bile acid signalling is a leading trigger for the development of chronic phenotypes underlying the pathophysiology of chronic respiratory disease.

(599) - Bile Acid Aspiration Associates with CLAD and Affects the Bronchial District Lipid Profile: Targeted Bile Acid Metabolomics and Lipidomics in Bronchial Washing

Lung transplant is the ultimate option for end stage lung disease pts. Overall survival is halted by rejection and chronic lung allograft dysfunction (CLAD). Gastro-esophageal reflux and aspiration has been considered a risk factor for CLAD. Bile acid aspiration was investigated by targeted metabolomics in the bronchial washings from lung transplant patients. Given the detergent properties of bile acids, their effect of the bronchial district lipids was studied using a lipidomics approach. Bronchial washings (BW) (56 samples) were prospectively collected from 51 patients at routine surveillance post-transplant bronchoscopies. Liquid chromatography-mass spectrometry retrospectively assayed BW for 13 bile acids and 25 lipid families inclusive of 250 lipids. Patients were monitored for CLAD and transbronchial biopsies for rejection and inflammation. Bile acids were detected in all BW. BW from pts with CLAD (22/51) had overall greater levels of bile acids (Mann Whitney p=0.03). Different levels (Kruscal Wallis p<0.05) were noted in BW from pts with early (onset <24 mo) CLAD (19.4 nM/L, IQR 15.2 - 35.8), late CLAD (26, 19.3-55.9), and no-CLAD (17.1, 13.5-26.4). Late CLAD vrs no-CLAD group (p=0.02); late CLAD vrs early CLAD (p=0.05); no difference for early CLAD vrs. no CLAD. Total Bile acids correlated (p<0.0005) with: cholesteryl-esters (CE) (Spearman r=0.6); Sphingomyelin (SM) (r=0.5); dihydrosphingomyelin (dhSM) (r=0.55); lactosyl-ceramide (Lac-Cer) (r=0.6); Lysophosphatidylcholine ether (LPCe) (r=0.5). Bile acids levels, high (upper quartile >27.5 nM/L) vrs. low, showed greater levels (p<0.05) of: CE, SM, dhSM, hexosyl-Cer, Lac-Cer, ganglioside GM3, phosphatidic acid, dipalmitoylphosphatidylcholine phosphatidylcholin ether, lysophosphatidylcholine, LPCe, lysophosphatidylinositol, phosphatidylethanolamine-p, lysophosphatidylethanolamine, lysophosphatidylethanolamine-p, phosphatidylinositol, and acylphosphatidylglycerol. Bile acids assayed by mass spec are detected in all lung transplant bronchial washings. High level of bile acids associate with late CLAD onset and have a significant dose related effect on the bronchial district lipidomic profile.

Bile Acid Aspiration Associated With Lung Chemical Profile Linked to Other Biomarkers of Injury After Lung Transplantation

Aspiration of gastrointestinal contents has been linked to worse outcomes following lung transplantation but uncertainty exists about underlying mechanisms. We applied high-resolution metabolomics of bronchoalveolar lavage fluid (BALF) in patients with episodic aspiration (defined by bile acids in the BALF) to identify potential metabolic changes associated with aspiration. Paired samples, one with bile acids and another without, from 29 stable lung transplant patients were studied. Liquid chromatography coupled to high-resolution mass spectroscopy was used to interrogate metabolomic contents of these samples. Data were obtained for 7068 ions representing intermediary metabolites, environmental agents and chemicals associated with microbial colonization. A substantial number (2302) differed between bile acid positive and negative samples when analyzed by false discovery rate at q = 0.01. These included pathways associated with microbial metabolism. Hierarchical cluster analysis defined clusters of chemicals associated with bile acid aspiration that were correlated to previously reported biomarkers of lung injury including T cell granzyme B level and the chemoattractants CXCL9 and CXCL10. These data specifically link bile acids presence in lung allografts to inflammatory pathways known to segregate with worsening allograft outcome, and provide additional mechanistic insight into the association between reflux and lung allograft injury.

Bile acids increase alveolar epithelial permeability via mitogen‐activated protein kinase, cytosolic phospholipase A2, cyclooxygenase‐2, prostaglandin E2 and junctional proteins

Bile acid (BA) aspiration is associated with various lung diseases. It was hypothesized that BA may induce changes in alveolar epithelium permeability and contribute to the pathogenesis of lung injury. Human alveolar epithelial cells were grown in monolayer and stimulated with a major component of BA, chenodeoxycholic acid (CDCA). Transepithelial electrical resistance (TER) and paracellular fluxes were measured to assess permeability alteration. Prostaglandin E2 ( PGE2 ) production was measured, and its effect on TER and junctional proteins (JP) was also examined. Reverse transcription polymerase chain reaction and Western blots were used to investigate the expression of messenger RNA and JP. CDCA induced significant p38 and c-Jun N-terminal kinase (JNK) phosphorylation, cytosolic phospholipase A2 (cPLA2 ) and cyclooxygenase-2 (COX-2) messenger RNA expression, PGE2 production, TER reduction and decay of JP (including occludin, zonula occludens-1 (ZO-1) and E-cadherin, in which ZO-1 had maximal change). CDCA also increased paracellular fluxes, which was abolished by dexamethasone. Both CDCA and PGE2 contributed to TER reduction in an identical trend and a dose-response manner. PGE2 also reduced ZO-1 expression, which was similar to that observed by CDCA stimulation. Pretreatment with inhibitors of p38 (SB203580), JNK (SP600125), cPLA2 (mepacrine) and COX-2 (NS398) as well as dexamethasone reversed the CDCA-induced PGE2 production, TER reduction and decay of ZO-1. The increase in alveolar permeability was associated with decay of JP. BA may induce permeability alteration through the upregulation of mitogen-activated protein kinase, cPLA2 , COX-2, PGE2 and JP, which may contribute to the pathogenesis of BA-associated lung injury.

Integrative Omics of Aspiration in Lung Transplantation

<h3>Purpose</h3> Prior studies have defined lung biomarkers associated with graft injury in lung transplant. We have developed a lung metabolomic profile of bile acid aspiration. We correlated the metabolites of aspiration to other omic measures of lung injury. <h3>Methods and Materials</h3> We performed metabolomic profiling on 29 recipients who had episodic bile detected in lung lavages. Each patient contributed a lung lavage specimen that was positive and negative for bile acid resulting in 58 total samples. Lavages were tested using C18 liquid chromatography coupled to mass spectroscopy. We used three approaches: Principle component analysis, Principle component regression, and Hierarchical clustering analysis. 252 Bile acid associated features were found at the intersection of these approaches. Cluster analysis developed 6 clusters positively or negatively associated with aspiration. Clusters were evaluated against biomarkers CXCL9, CXCL10, MCP1, FoxP3 and GranzymeB. <h3>Results</h3> Bile acid aspiration was associated with a distinct metabolomic profile resolved using 3 bioinformatics approaches with a bias of increased small species in the bile positive samples, suggesting an increase in pulmonary permeability with aspiration. Presence of bile only weakly correlated with the other omic markers of injury (r .17-.26, p .09-.2). Individual bile acid associated clusters showed significant positive and negative correlation with known other markers of injury. [figure 1] <h3>Conclusions</h3> In this discovery phase study there is a strong association between metabolomic features of bile acid aspiration and known biomarkers of injury from other omic platforms. This strengthens the claim that aspiration of gastric contents is dangerous and also provides an avenue for risk stratification in a prospective cohort of patients.

Bile Acids in Sputum and Increased Airway Inflammation in Patients With Cystic Fibrosis

Up to 80% of patients with cystic fibrosis (CF) may have increased gastroesophageal reflux and aspiration of duodenogastric contents into the lungs. We aimed to assess aspiration in patients with CF by measuring duodenogastric components in induced sputum and to investigate whether the presence of bile acids (BAs) in sputum was correlated with disease severity and markers of inflammation. In 41 patients with CF, 15 healthy volunteers, 29 patients with asthma, and 28 patients with chronic cough, sputum was obtained after inhalation of hypertonic saline. Sputum supernatant was tested for BA and neutrophil elastase. Spirometry and BMI were assessed on the day of sputum collection. Two of 15 healthy patients (13%), eight of 29 patients (28%) with asthma, four of 28 patients (14%) with chronic cough, and 23 of 41 patients (56%) with CF had BA in sputum. BA concentrations were similar in patients who are positive for BA with genotype F508del homozygote, F508del heterozygote, and other CF mutations and were not related with BMI and age. Patients with CF with BA in sputum had a higher concentration of neutrophil elastase compared with patients without BA in sputum (31.25 [20.33-54.78] μg/mL vs 14.45 [7.11-27.88] μg/mL, P < .05). There was a significant correlation between BA concentrations and dynamic lung volumes (FEV(1) % predicted [r = -0.53, P < .01], FVC% [r = -0.59, P < .01]) as well as with number of days of antibiotic IV treatment (r = 0.58, P < .01). BAs are present in the sputum of more than one-half of patients with CF, suggesting aspiration of duodenogastric contents. Aspiration of BA was associated with increased airway inflammation. In patients with BA aspiration, the levels of BA were clearly associated with the degree of lung function impairment as well as the need for IV antibiotic treatment.

Bile Acids Aspiration Reduces Survival in Lung Transplant Recipients with BOS Despite Azithromycin

Azithromycin (AZM) improved bronchiolitis obliterans syndrome (BOS) and reduced aspiration in lung transplant (LTx) recipients. We hypothesize that AZM could improve graft and overall survival more efficiently in LTx patients with BOS who have bile acid (BA) aspiration by protecting against the aspiration-induced progression of BOS. The goal was to compare FEV(1) (% baseline), BOS progression and overall survival in LTx recipients treated with AZM for BOS, both with versus without BA aspiration. Therefore, LTx recipients treated with AZM for BOS were recruited and broncho-alveolar lavage (BAL) samples were analyzed for the presence of BA and neutrophilia before the start of AZM treatment. Short-term effect of AZM on FEV(1) and BAL neutrophilia was assessed, progression of BOS and survival were followed-up for 3 years and results were compared between patients with/without BA aspiration. 19/37 LTx patients had BA in BAL. BA aspiration predisposed to a significantly worse outcome, in terms of decline in FEV(1) , progression of BOS ≥ 1 and survival. AZM does not seem to protect against the long-term allograft dysfunction caused by gastroesophageal reflux (GER) and aspiration and an additional treatment targeting aspiration may be indicated in those LTx patients.

W1064 Bile Acids Aspiration Reduces Survival in Lung Transplant Recipients Despite Azithromycin Therapy

W1064 Bile Acids Aspiration Reduces Survival in Lung Transplant Recipients Despite Azithromycin Therapy

427: Reduced Survival in Lung Transplant Recipients with Bile Acids Aspiration Despite Azithromycin Therapy

427: Reduced Survival in Lung Transplant Recipients with Bile Acids Aspiration Despite Azithromycin Therapy

Bile Acid Aspiration in Suspected Ventilator-Associated Pneumonia

The aims of this study were to measure the levels of bile acids in patients with suspected ventilator-associated pneumonia (VAP) and provide a possible pathway for neutrophilic inflammation to explain its proinflammatory effect on the airway. Bile acid levels were measured by spectrophotometric enzymatic assay, and liquid chromatography mass spectrometry was used to quantify the major bile acids. Alveolar cells were grown on modified air-liquid interface culture inserts, and bile acids were then employed to stimulate the cells. Reverse transcriptase polymerase chain reaction and Western blots were used to determine the involved gene expression and protein levels. The mean (+/- SE) concentration of total bile acids in tracheal aspirates was 6.2 +/- 2.1 and 1.1 +/- 0.4 mumol/L/g sputum, respectively, for patients with and without VAP (p < 0.05). The interleukin (IL)-8 level was significantly higher in the VAP group (p < 0.05). The major bile acid, chenodeoxycholic acid, stimulated alveolar epithelial cells to increase IL-8 production at both the messenger RNA and protein level through p38 and c-Jun N-terminal kinase (JNK) activation. The selective p38 and JNK inhibitors, as well as dexamethasone, successfully inhibited IL-8 production. These data suggest that early intervention to prevent bile acid aspiration may reduce the intensity of neutrophilic inflammation in intubated and mechanically ventilated patients in the ICU.

Azithromycin Reduces Gastroesophageal Reflux and Aspiration in Lung Transplant Recipients

Azithromycin (AZI) is a macrolide antibiotic that improves lung function in lung transplant recipients (LTx). Gastroesophageal reflux (GER) has been implicated in the pathogenesis of chronic rejection after LTx. Macrolide antibiotics may affect GER by modifying esophageal and gastric motility. The purpose of this study was to evaluate the effect of AZI on GER and gastric aspiration after LTx. Acid and weakly acidic GER was measured with 24-h pH-impedance monitoring in 47 LTx patients (12 patients "on" AZI). Gastric aspiration was assessed in a separate group of 30 LTx patients before and after AZI by measurements of pepsin and bile acid in bronchoalveolar lavage fluid (BALF). Patients "on" AZI had a significant lower total number of reflux events [41 (30-61) vs. 22.5 (7-37.5)], number of acid reflux events [24 (16-41) vs. 8 (4-18)], esophageal acid exposure [2.9% (0.7-7.3) vs. 0.2% (0.1-2.0)], bolus exposure [0.73% (0.5-1.4) vs. 0.21% (0.12-0.92)], and proximal extent of reflux [14 (9-24) vs. 5 (2-7)]. AZI reduced the concentration of bile acids in BALF without affecting levels of pepsin. LTx patients "on" AZI have less GER and bile acids aspiration. This effect might be due to enhanced esophageal motility and accelerated gastric emptying.

Nocturnal Weakly Acidic Reflux Promotes Aspiration of Bile Acids in Lung Transplant Recipients

Gastroesophageal reflux (GER) and aspiration of bile acids have been implicated as non-alloimmune risk factors for the development of bronchiolitis obliterans syndrome (BOS) after lung transplantation. The aim of our study was to investigate the association between GER and gastric aspiration of bile acids and to establish which reflux characteristics may promote aspiration of bile acids into the lungs and may feature as a potential diagnostic tool in identifying lung transplantation (LTx) patients at risk for aspiration. Twenty-four stable LTx recipients were studied 1 year after transplantation. All patients underwent 24-hour ambulatory impedance-pH recording for the detection of acid (pH <4) and weakly acidic (pH 4 to 7) reflux. On the same day, bronchoalveolar lavage fluid (BALF) was collected and then analyzed for the presence of bile acids (Bioquant enzymatic assay). Increased GER was detected in 13 patients, of whom 9 had increased acid reflux and 4 had exclusively increased weakly acidic reflux. Sixteen patients had detectable bile acids in the BALF (0.6 [0.4 to 1.5] micromol/liter). The 24-hour esophageal volume exposure was significantly increased in patients with bile acids compared to patients without bile acids in the BALF. Acid exposure and the number of reflux events (total, acid and weakly acidic) were unrelated to the presence of bile acids in the BALF. However, both nocturnal volume exposure and the number of nocturnal weakly acidic reflux events were significantly higher in patients with bile acids in the BALF. Weakly acidic reflux events, especially during the night, are associated with the aspiration of bile acids in LTx recipients and may therefore feature as a potential risk factor for the development of BOS.

Bile acids induce CCN2 production through p38 MAP kinase activation in human bronchial epithelial cells: A factor contributing to airway fibrosis

Bile acid aspiration occurs in a variety of acute and chronic airway disorders. The consequence of bile acid aspiration and lung disease remains unclear. It was hypothesized that airway epithelium exposure to bile acids would induce fibrosis via production of connective tissue growth factor (CCN2), CCN2 is essential for transforming growth factor-beta (TGFbeta)-induced fibrogenesis and functions as a downstream mediator of TGF-beta action on fibroblasts. Human bronchial epithelial cells were grown on air-liquid interface culture inserts. Cells were stimulated with the major components of bile acids, chenodeoxycholic acid (CD) and glycochenodeoxycholic acid (GCD). RT-PCR and real-time quantitative PCR were used to detect mRNA expression. ELISA and western blotting were used to measure protein. CD-stimulated airway epithelial cells produce CCN2 at both mRNA and protein levels in a dose-dependent manner. GCD failed to increase CCN2 production. CCN2 expression occurred via activation of p38 mitogen-activated protein (MAP) kinase and the downstream transcription factor ATF-2. Dexamethasone and the selective p38 MAP kinase inhibitor SB203580 successfully inhibited p38 MAP kinase, ATF-2 phosphorylation and subsequent CCN2 production. CD induced TGF-beta1 release from airway epithelium via the same signalling pathway. TGF-beta1 therefore enhanced CCN2production in an autocrine manner. Early intervention to stop these processes may be useful in preventing fibrogenesis in chronic airway diseases associated with bile acid exposure.