Myeloid Heterogeneity Mediates Acute Exacerbations of Pulmonary Fibrosis.

Epidemiological evidence indicates that exposure to particulate matter is linked to the development of idiopathic pulmonary fibrosis (IPF) and increases the incidence of acute exacerbations of IPF. In addition to accelerating the rate of lung function decline, exposure to fine particulate matter (particulate matter smaller than 2.5 µm [PM2.5]) is a risk factor for increased mortality in subjects with IPF. In this article, we show that exposure to PM2.5 mediates monocyte recruitment and fibrotic progression in mice with established fibrosis. In mice with established fibrosis, bronchoalveolar lavage cells showed monocyte/macrophage heterogeneity after exposure to PM2.5. These cells had a significant inflammatory and anti-inflammatory signature. The mixed heterogeneity of cells contributed to the proinflammatory and anti-inflammatory response. Although monocyte-derived macrophages were recruited to the lung in bleomycin-injured mice treated with PM2.5, recruitment of monocytes expressing Ly6Chi to the lung promoted progression of fibrosis, reduced lung aeration on computed tomography, and impacted lung compliance. Ly6Chi monocytes isolated from PM2.5-exposed fibrotic mice showed enhanced expression of proinflammatory markers compared with fibrotic mice exposed to vehicle. Moreover, IPF bronchoalveolar lavage cells treated ex vivo with PM2.5 showed an exaggerated inflammatory response. Targeting Ly6Chi monocyte recruitment inhibited fibrotic progression in mice. Moreover, the adoptive transfer of Ly6Chi monocytes exacerbated established fibrosis. These observations suggest that enhanced recruitment of Ly6Chi monocytes with a proinflammatory phenotype mediates acute exacerbations of pulmonary fibrosis, and targeting these cells may provide a potential novel therapeutic target to protect against acute exacerbations of IPF.

Use of a pulmosphere model to evaluate drug antifibrotic responses in interstitial lung diseases.

BACKGROUND: Interstitial lung diseases (ILD) encompass a heterogenous group of diffuse parenchymal lung disorders characterized by variable degrees of inflammation and fibrosis. Pretherapeutic clinical testing models for such diseases can serve as a platform to test and develop effective therapeutic strategies. In this study, we developed patient derived 3D organoid model to recapitulate the disease process of ILDs. We characterized the inherent property of invasiveness in this model and tested for antifibrotic responses with an aim to develop a potential platform for personalized medicine in ILDs. METHODS: In this prospective study, 23 patients with ILD were recruited and underwent lung biopsy. 3D organoid-based models (pulmospheres) were developed from the lung biopsy tissues. Pulmonary functioning testing and other relevant clinical parameters were collected at the time of enrollment and follow up visits. The patient derived pulmospheres were compared to normal control pulmospheres obtained from 9 explant lung donor samples. These pulmospheres were characterized by their invasive capabilities and responsiveness to the antifibrotic drugs, pirfenidone and nintedanib. RESULTS: Invasiveness of the pulmospheres was measured by the zone of invasiveness percentage (ZOI%). The ILD pulmospheres (n = 23) had a higher ZOI% as compared to control pulmospheres (n = 9) (516.2 ± 115.6 versus 54.63 ± 19.6 respectively. ILD pulmospheres were responsive to pirfenidone in 12 of the 23 patients (52%) and responsive to nintedanib in all 23 patients (100%). Pirfenidone was noted to be selectively responsive in patients with connective tissue disease related ILD (CTD-ILD) at low doses. There was no correlation between the basal pulmosphere invasiveness, response to antifibrotics, and FVC change (Δ FVC). CONCLUSIONS: The 3D pulmosphere model demonstrates invasiveness which is unique to each individual subject and is greater in ILD pulmospheres as compared to controls. This property can be utilized to test responses to drugs such as antifibrotics. The 3D pulmosphere model could serve as a platform for the development of personalized approaches to therapeutics and drug development in ILDs and potentially other chronic lung diseases.

Cutaneous Exposure to Arsenicals Is Associated with Development of Constrictive Bronchiolitis in Mice.

Organoarsenicals, such as lewisite and related chloroarsine, diphenylchloroarsine (DPCA), are chemical warfare agents developed during World War I. Stockpiles in Eastern Europe remain a threat to humans. The well-documented effects of cutaneous exposure to these organoarsenicals include skin blisters, painful burns, and life-threatening conditions such as acute respiratory distress syndrome. In survivors, long-term effects such as the development of respiratory ailments are reported for the organoarsenical sulfur mustard; however, no long-term pulmonary effects are documented for lewisite and DPCA. No animal models exist to explore the relationship between skin exposure to vesicants and constrictive bronchiolitis. We developed and characterized a mouse model to study the long-term effects of cutaneous exposure on the lungs after exposure to a sublethal dose of organoarsenicals. We exposed mice to lewisite, DPCA, or a less toxic surrogate organoarsenic chemical, phenyl arsine oxide, on the skin. The surviving mice were followed for 20 weeks after skin exposure to arsenicals. Lung microcomputed tomography, lung function, and histology demonstrated increased airway resistance, increased thickness of the smooth muscle layer, increased collagen deposition in the subepithelium, and peribronchial lymphocyte infiltration in mice exposed to arsenical on skin.

Pathophysiological Role of Vimentin Intermediate Filaments in Lung Diseases.

Vimentin intermediate filaments, a type III intermediate filament, are among the most widely studied IFs and are found abundantly in mesenchymal cells. Vimentin intermediate filaments localize primarily in the cytoplasm but can also be found on the cell surface and extracellular space. The cytoplasmic vimentin is well-recognized for its role in providing mechanical strength and regulating cell migration, adhesion, and division. The post-translationally modified forms of Vimentin intermediate filaments have several implications in host-pathogen interactions, cancers, and non-malignant lung diseases. This review will analyze the role of vimentin beyond just the epithelial to mesenchymal transition (EMT) marker highlighting its role as a regulator of host-pathogen interactions and signaling pathways for the pathophysiology of various lung diseases. In addition, we will also examine the clinically relevant anti-vimentin compounds and antibodies that could potentially interfere with the pathogenic role of Vimentin intermediate filaments in lung disease.

Fibrinogen mediates cadmium-induced macrophage activation and serves as a predictor of cadmium exposure in chronic obstructive pulmonary disease.

The etiologies of chronic obstructive pulmonary disease (COPD) remain unclear. Cadmium (Cd) causes both pulmonary fibrosis and emphysema; however, the predictors for Cd exposure and the mechanisms by which Cd causes COPD remain unknown. We demonstrated that Cd burden was increased in lung tissue from subjects with COPD and this was associated with cigarette smoking. Fibrinogen levels increased markedly in lung tissue of patients with smoked COPD compared with never-smokers and control subjects. Fibrinogen concentration also correlated positively with lung Cd load, but inversely with the predicted % of FEV1 and FEV1/FVC. Cd enhanced the secretion of fibrinogen in a cdc2-dependent manner, whereas fibrinogen further mediated Cd-induced peptidylarginine deiminase 2 (PAD2)-dependent macrophage activation. Using lung fibroblasts from CdCl2-treated Toll-like receptor 4 (TLR4) wild-type and mutant mice, we demonstrated that fibrinogen enhanced Cd-induced TLR4-dependent collagen synthesis and cytokine/chemokine production. We further showed that fibrinogen complexed with connective tissue growth factor (CTGF), which in turn promoted the synthesis of plasminogen activator inhibitor-2 (PAI-2) and fibrinogen and inhibited fibrinolysis in Cd-treated mice. The amounts of fibrinogen were increased in the bronchoalveolar lavage fluid (BALF) of Cd-exposed mice. Positive correlations were observed between fibrinogen with hydroxyproline. Our data suggest that fibrinogen is involved in Cd-induced macrophage activation and increases in fibrinogen in patients with COPD may be used as a marker of Cd exposure and predict disease progression.

Targeting Cpt1a-Bcl-2 interaction modulates apoptosis resistance and fibrotic remodeling.

The mitochondrial calcium uniporter (MCU) regulates metabolic reprogramming in lung macrophages and the progression of pulmonary fibrosis. Fibrosis progression is associated with apoptosis resistance in lung macrophages; however, the mechanism(s) by which apoptosis resistance occurs is poorly understood. Here, we found a marked increase in mitochondrial B-cell lymphoma-2 (Bcl-2) in lung macrophages from subjects with idiopathic pulmonary fibrosis (IPF). Similar findings were seen in bleomycin-injured wild-type (WT) mice, whereas Bcl-2 was markedly decreased in mice expressing a dominant-negative mitochondrial calcium uniporter (DN-MCU). Carnitine palmitoyltransferase 1a (Cpt1a), the rate-limiting enzyme for fatty acid ß-oxidation, directly interacted with Bcl-2 by binding to its BH3 domain, which anchored Bcl-2 in the mitochondria to attenuate apoptosis. This interaction was dependent on Cpt1a activity. Lung macrophages from IPF subjects had a direct correlation between CPT1A and Bcl-2, whereas the absence of binding induced apoptosis. The deletion of Bcl-2 in macrophages protected mice from developing pulmonary fibrosis. Moreover, mice had resolution when Bcl-2 was deleted or was inhibited with ABT-199 after fibrosis was established. These observations implicate an interplay between macrophage fatty acid ß-oxidation, apoptosis resistance, and dysregulated fibrotic remodeling.

NETosis in the pathogenesis of acute lung injury following cutaneous chemical burns.

Despite the high morbidity and mortality among patients with extensive cutaneous burns in the intensive care unit due to the development of acute respiratory distress syndrome, effective therapeutics remain to be determined. This is primarily because the mechanisms leading to acute lung injury (ALI) in these patients remain unknown. We test the hypothesis that cutaneous chemical burns promote lung injury due to systemic activation of neutrophils, in particular, toxicity mediated by the deployment of neutrophil extracellular traps (NETs). We also demonstrate the potential benefit of a peptidyl arginine deiminase 4 (PAD4) inhibitor to prevent NETosis and to preserve microvascular endothelial barrier function, thus reducing the severity of ALI in mice. Our data demonstrated that phenylarsine oxide (PAO) treatment of neutrophils caused increased intracellular Ca2+-associated PAD4 activity. A dermal chemical burn by lewisite or PAO resulted in PAD4 activation, NETosis, and ALI. NETs disrupted the barrier function of endothelial cells in human lung microvascular endothelial cell spheroids. Citrullinated histone 3 alone caused ALI in mice. Pharmacologic or genetic abrogation of PAD4 inhibited lung injury following cutaneous chemical burns. Cutaneous burns by lewisite and PAO caused ALI by PAD4-mediated NETosis. PAD4 inhibitors may have potential as countermeasures to suppress detrimental lung injury after chemical burns.

Citrullinated vimentin mediates development and progression of lung fibrosis.

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)-dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-ß1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2-/- and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

Cutaneous lewisite exposure causes acute lung injury.

Lewisite is a strong vesicating and chemical warfare agent. Because of the rapid transdermal absorption, cutaneous exposure to lewisite can also elicit severe systemic injury. Lewisite (2.5, 5.0, and 7.5 mg/kg) was applied to the skin of Ptch1+/- /SKH-1 mice and acute lung injury (ALI) was assessed after 24 hours. Arterial blood gas measurements showed hypercapnia and hypoxemia in the lewisite-exposed group. Histological evaluation of lung tissue revealed increased levels of proinflammatory neutrophils and a dose-dependent increase in structural changes indicative of injury. Increased inflammation was also confirmed by altered expression of cytokines, including increased IL-33, and a dose-dependent elevation of CXCL1, CXCL5, and GCSF was observed in the lung tissue. In the bronchoalveolar lavage fluid of lewisite-exposed animals, there was a significant increase in HMGB1, a damage-associated molecular pattern molecule, as well as elevated CXCL1 and CXCL5, which coincided with an influx of neutrophils to the lungs. Complete blood cell analysis revealed eosinophilia and altered neutrophil-lymphocyte ratios as a consequence of lewisite exposure. Mean platelet volume and RBC distribution width, which are predictors of lung injury, were also increased in the lewisite group. These data demonstrate that cutaneous lewisite exposure causes ALI and may contribute to mortality in exposed populations.

Increased flux through the mevalonate pathway mediates fibrotic repair without injury.

Macrophages are important in mounting an innate immune response to injury as well as in repair of injury. Gene expression of Rho proteins is known to be increased in fibrotic models; however, the role of these proteins in idiopathic pulmonary fibrosis (IPF) is not known. Here, we show that BAL cells from patients with IPF have a profibrotic phenotype secondary to increased activation of the small GTPase Rac1. Rac1 activation requires a posttranslational modification, geranylgeranylation, of the C-terminal cysteine residue. We found that by supplying more substrate for geranylgeranylation, Rac1 activation was substantially increased, resulting in profibrotic polarization by increasing flux through the mevalonate pathway. The increased flux was secondary to greater levels of acetyl-CoA from metabolic reprogramming to ß oxidation. The polarization mediated fibrotic repair in the absence of injury by enhancing macrophage/fibroblast signaling. These observations suggest that targeting the mevalonate pathway may abrogate the role of macrophages in dysregulated fibrotic repair.

Vimentin intermediate filament assembly regulates fibroblast invasion in fibrogenic lung injury.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease, with a median survival of 3-5 years following diagnosis. Lung remodeling by invasive fibroblasts is a hallmark of IPF. In this study, we demonstrate that inhibition of vimentin intermediate filaments (VimIFs) decreases the invasiveness of IPF fibroblasts and confers protection against fibrosis in a murine model of experimental lung injury. Increased expression and organization of VimIFs contribute to the invasive property of IPF fibroblasts in connection with deficient cellular autophagy. Blocking VimIF assembly by pharmacologic and genetic means also increases autophagic clearance of collagen type I. Furthermore, inhibition of expression of collagen type I by siRNA decreased invasiveness of fibroblasts. In a bleomycin injury model, enhancing autophagy in fibroblasts by an inhibitor of VimIF assembly, withaferin A (WFA), protected from fibrotic lung injury. Additionally, in 3D lung organoids, or pulmospheres, from patients with IPF, WFA reduced the invasiveness of lung fibroblasts in the majority of subjects tested. These studies provide insights into the functional role of vimentin, which regulates autophagy and restricts the invasiveness of lung fibroblasts.

Autoimmunity to Vimentin Is Associated with Outcomes of Patients with Idiopathic Pulmonary Fibrosis.

Autoimmunity has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF); however, the repertoire of autoantigens involved in this disease and the clinical relevance of these autoimmune responses are still being explored. Our initial discovery assays demonstrated that circulating and intrapulmonary vimentin levels are increased in IPF patients. Subsequent studies showed native vimentin induced HLA-DR-dependent in vitro proliferation of CD4 T cells from IPF patients and enhanced the production of IL-4, IL-17, and TGF-ß1 by these lymphocytes in contrast to normal control specimens. Vimentin supplementation of IPF PBMC cultures also resulted in HLA-DR-dependent production of IgG with anti-vimentin specificities. Circulating anti-vimentin IgG autoantibody levels were much greater in IPF subjects from the University of Alabama at Birmingham (n = 102) and the University of Pittsburgh (U. Pitt., n = 70) than in normal controls. Anti-vimentin autoantibody levels in IPF patients were HLA biased and inversely correlated with physiological measurements of lung function (i.e., forced expiratory volumes and diffusing capacities). Despite considerable intergroup differences in transplant-free survival between these two independent IPF cohorts, serious adverse outcomes were most frequent among the patients within each population that had the highest anti-vimentin autoantibody levels (University of Alabama at Birmingham: hazard ratio 2.5, 95% confidence interval 1.2-5.3, p = 0.012; University of Pittsburgh: hazard ratio 2.7, 95% confidence interval 1.3-5.5, p = 0.006). These data show that anti-vimentin autoreactivity is prevalent in IPF patients and is strongly associated with disease manifestations. These findings have implications with regard to the pathogenesis of this enigmatic disease and raise the possibility that therapies specifically directed at these autoimmune processes could have therapeutic efficacy.

Low-dose cadmium exposure induces peribronchiolar fibrosis through site-specific phosphorylation of vimentin.

Exposure to cadmium (Cd) has been associated with development of chronic obstructive lung disease (COPD). The mechanisms and signaling pathways whereby Cd causes pathological peribronchiolar fibrosis, airway remodeling, and subsequent airflow obstruction remain unclear. We aimed to evaluate whether low-dose Cd exposure induces vimentin phosphorylation and Yes-associated protein 1 (YAP1) activation leading to peribronchiolar fibrosis and subsequent airway remodeling. Our data demonstrate that Cd induces myofibroblast differentiation and extracellular matrix (ECM) deposition around small (<2 mm in diameter) airways. Upon Cd exposure, α-smooth muscle actin (α-SMA) expression and the production of ECM proteins, including fibronectin and collagen-1, are markedly induced in primary human lung fibroblasts. Cd induces Smad2/3 activation and the translocation of both Smad2/3 and Yes-associated protein 1 (YAP1) into the nucleus. In parallel, Cd induces AKT and cdc2 phosphorylation and downstream vimentin phosphorylation at Ser39 and Ser55, respectively. AKT and cdc2 inhibitors block Cd-induced vimentin fragmentation and secretion in association with inhibition of α-SMA expression, ECM deposition, and collagen secretion. Furthermore, vimentin silencing abrogates Cd-induced α-SMA expression and decreases ECM production. Vimentin-deficient mice are protected from Cd-induced peribronchiolar fibrosis and remodeling. These findings identify two specific sites on vimentin that are phosphorylated by Cd and highlight the functional significance of vimentin phosphorylation in YAP1/Smad3 signaling that mediates Cd-induced peribronchiolar fibrosis and airway remodeling.

3D pulmospheres serve as a personalized and predictive multicellular model for assessment of antifibrotic drugs.

Idiopathic pulmonary fibrosis (IPF) is a fatal progressive fibrotic lung disease characterized by the presence of invasive myofibroblasts in the lung. Currently, there are only two FDA-approved drugs (pirfenidone and nintedanib) for the treatment of IPF. There are no defined criteria to guide specific drug therapy. New methodologies are needed not only to predict personalized drug therapy, but also to screen novel molecules that are on the horizon for treatment of IPF. We have developed a model system that exploits the invasive phenotype of IPF lung tissue. This ex vivo 3D model uses lung tissue from patients to develop pulmospheres. Pulmospheres are 3D spheroids composed of cells derived exclusively from primary lung biopsies and inclusive of lung cell types reflective of those in situ, in the patient. We tested the pulmospheres of 20 subjects with IPF and 9 control subjects to evaluate the responsiveness of individual patients to antifibrotic drugs. Clinical parameters and outcomes were also followed in the same patients. Our results suggest that pulmospheres simulate the microenvironment in the lung and serve as a personalized and predictive model for assessing responsiveness to antifibrotic drugs in patients with IPF.

Attenuated heme oxygenase-1 responses predispose the elderly to pulmonary nontuberculous mycobacterial infections.

Pulmonary infections with nontuberculous mycobacteria (P-NTM), such as by Mycobacterium avium complex (M. avium), are increasingly found in the elderly, but the underlying mechanisms are unclear. Recent studies suggest that adaptive immunity is necessary, but not sufficient, for host defense against mycobacteria. Heme oxygenase-1 (HO-1) has been recognized as a critical modulator of granuloma formation and programmed cell death in mycobacterial infections. Old mice (18-21 mo) infected with M. avium had attenuated HO-1 response with diffuse inflammation, high burden of mycobacteria, poor granuloma formation, and decreased survival (45%), while young mice (4-6 mo) showed tight, well-defined granuloma, increased HO-1 expression, and increased survival (95%). To further test the role of HO-1 in increased susceptibility to P-NTM infections in the elderly, we used old and young HO-1+/+ and HO-1-/- mice. The transcriptional modulation of the JAK/STAT signaling pathway in HO-1-/- mice due to M. avium infection demonstrated similarities to infected wild-type old mice with upregulation of SOCS3 and inhibition of Bcl2. Higher expression of SOCS3 with downregulation of Bcl2 resulted in higher macrophage death via cellular necrosis. Finally, peripheral blood monocytes (PBMCs) from elderly patients with P-NTM also demonstrated attenuated HO-1 responses after M. avium stimulation and increased cell death due to cellular necrosis (9.69% ± 2.02) compared with apoptosis (4.75% ± 0.98). The augmented risk for P-NTM in the elderly is due, in part, to attenuated HO-1 responses, subsequent upregulation of SOCS3, and inhibition of Bcl2, leading to programmed cell death of macrophages, and sustained infection.

Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice.

Pulmonary exposure to cadmium, a major component of cigarette smoke, has a dramatic impact on lung function and the development of emphysema. Cigarette smoke exposure induces heme oxygenase-1 (HO-1), a cytoprotective enzyme. In this study, we employed a truncated mouse model of emphysema by intratracheal instillation of cadmium (CdCl2) solution (0.025% per 1 mg/kg body wt) in HO-1(+/+), HO-1(-/-), and overexpressing humanized HO-1 bacterial artificial chromosome (hHO-1BAC) mice. We evaluated the role of HO-1 in cadmium-induced emphysema in mice by analyzing histopathology, micro-computed tomography scans, and lung function tests. CdCl2-exposed HO-1(-/-) mice exhibited more severe emphysema compared with HO-1(+/+) or hHO-1BAC mice. Loss of pulmonary endothelial cells (PECs) from the alveolar capillary membrane is recognized to be a target in emphysema. PECs from HO-1(+/+), HO-1(-/-), and hHO-1BAC were employed to define the underlying molecular mechanism for the protection from emphysema by HO-1. Electron microscopy, expression of autophagic markers (microtubule-associated protein 1B-light chain 3 II, autophagy protein 5, and Beclin1) and apoptotic marker (cleaved caspase 3) suggested induction of autophagy and apoptosis in PECs after CdCl2 treatment. CdCl2-treated HO-1(-/-) PECs exhibited downregulation of autophagic markers and significantly increased cleaved caspase 3 expression and activity (∼4-fold higher). Moreover, hHO-1BAC PECs demonstrated upregulated autophagy and absence of cleaved caspase 3 expression or activity. Pretreatment of HO-1(+/+) PECs with rapamycin induced autophagy and resulted in reduced cell death upon cadmium treatment. Induction of autophagy following CdCl2 treatment was found to be protective from apoptotic cell death. HO-1 induced protective autophagy in PECs and mitigated cadmium-induced emphysema.

Heme oxygenase-1 protects corexit 9500A-induced respiratory epithelial injury across species.

The effects of Corexit 9500A (CE) on respiratory epithelial surfaces of terrestrial mammals and marine animals are largely unknown. This study investigated the role of CE-induced heme oxygenase-1 (HO-1), a cytoprotective enzyme with anti-apoptotic and antioxidant activity, in human bronchial airway epithelium and the gills of exposed aquatic animals. We evaluated CE-mediated alterations in human airway epithelial cells, mice lungs and gills from zebrafish and blue crabs. Our results demonstrated that CE induced an increase in gill epithelial edema and human epithelial monolayer permeability, suggesting an acute injury caused by CE exposure. CE induced the expression of HO-1 as well as C-reactive protein (CRP) and NADPH oxidase 4 (NOX4), which are associated with ROS production. Importantly, CE induced caspase-3 activation and subsequent apoptosis of epithelial cells. The expression of the intercellular junctional proteins, such as tight junction proteins occludin, zonula occludens (ZO-1), ZO-2 and adherens junctional proteins E-cadherin and Focal Adhesion Kinase (FAK), were remarkably inhibited by CE, suggesting that these proteins are involved in CE-induced increased permeability and subsequent apoptosis. The cytoskeletal protein F-actin was also disrupted by CE. Treatment with carbon monoxide releasing molecule-2 (CORM-2) significantly inhibited CE-induced ROS production, while the addition of HO-1 inhibitor, significantly increased CE-induced ROS production and apoptosis, suggesting a protective role of HO-1 or its reaction product, CO, in CE-induced apoptosis. Using HO-1 knockout mice, we further demonstrated that HO-1 protected against CE-induced inflammation and cellular apoptosis and corrected CE-mediated inhibition of E-cadherin and FAK. These observations suggest that CE activates CRP and NOX4-mediated ROS production, alters permeability by inhibition of junctional proteins, and leads to caspase-3 dependent apoptosis of epithelial cells, while HO-1 and its reaction products protect against oxidative stress and apoptosis.