Correction to: Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls.

After publication of our article [1], we have been notified that an extra alpha symbol (α) was mistakenly added at the beginning of the title.

Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls [corrected].

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects. METHODS: Primary normal human lung and IPF-FB were exposed to TGF-ß (5 ng/ml), Azithromycin (50 µM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry. RESULTS: AZT significantly reduced collagen secretion in TGF-ß treated IPF-FB compared to TGF-ß treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-ß treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-ß treated IPF-FB. CONCLUSION: We report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

Surfactant replacement therapy reduces acute lung injury and collapse induration-related lung remodeling in the bleomycin model.

Bleomycin-induced lung injury leads to surfactant dysfunction and permanent loss of alveoli due to a remodeling process called collapse induration. Collapse induration also occurs in acute interstitial lung disease and idiopathic pulmonary fibrosis in humans. We hypothesized that surfactant dysfunction aggravates lung injury and early remodeling resulting in collapse induration within 7 days after lung injury. Rats received bleomycin to induce lung injury and either repetitive surfactant replacement therapy (SRT: 100 mg Curosurf/kg BW = surf group) or saline (0.9% NaCl = saline group). After 3 (D3) or 7 (D7) days, invasive pulmonary function tests were performed to determine tissue elastance (H) and static compliance (Cst). Bronchoalveolar lavage (BAL) was taken for surfactant function, inflammatory markers, and protein measurements. Lungs were fixed by vascular perfusion for design-based stereology and electron microscopic analyses. SRT significantly improved minimum surface tension of alveolar surfactant as well as H and Cst at D3 and D7. At D3 decreased inflammatory markers including neutrophilic granulocytes, IL-1ß, and IL-6 correlated with reduced BAL-protein levels after SRT. Numbers of open alveoli were significantly increased at D3 and D7 in SRT groups whereas at D7 there was also a significant reduction in septal wall thickness and parenchymal tissue volume. Septal wall thickness and numbers of open alveoli highly correlated with improved lung mechanics after SRT. In conclusion, reduction in surface tension was effective to stabilize alveoli linked with an attenuation of parameters of acute lung injury at D3 and collapse induration at D7. Hence, SRT modifies disease progression to collapse induration.

Toll-like receptor 4 activation attenuates profibrotic response in control lung fibroblasts but not in fibroblasts from patients with IPF.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a median survival of 3 yr. IPF deteriorates upon viral or bacterial lung infection although pulmonary infection (pneumonia) in healthy lungs rarely induces fibrosis. Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), initiating proinflammatory pathways. As TLR4 has already been linked to hepatic fibrosis and scleroderma, we now investigated the role of TLR4 in IPF fibroblasts. Lung tissue sections from patients with IPF were analyzed for TLR4 expression. Isolated normal human lung fibroblasts (NL-FB) and IPF fibroblasts (IPF-FB) were exposed to LPS and transforming growth factor-ß (TGF-ß) before expression analysis of receptors, profibrotic mediators, and cytokines. TLR4 is expressed in fibroblast foci of IPF lungs as well as in primary NL-FB and IPF-FB. As a model for a gram-negative pneumonia in the nonfibrotic lung, NL-FB and IPF-FB were coexposed to LPS and TGF-ß. Whereas NL-FB produced significantly less connective tissue growth factor upon costimulation compared with TGF-ß stimulation alone, IPF-FB showed significantly increased profibrotic markers compared with control fibroblasts after costimulation. Although levels of antifibrotic prostaglandin E2 were elevated after costimulation, they were not responsible for this effect. However, significant downregulation of TGF-ß receptor type 1 in control fibroblasts seems to contribute to the reduced profibrotic response in our in vitro model. Normal and IPF fibroblasts thus differ in their profibrotic response upon LPS-induced TLR4 stimulation.

Lysophosphatidic Acid Signaling through the Lysophosphatidic Acid-1 Receptor Is Required for Alveolarization.

Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.

Comparison of innate immune responses towards rhinovirus infection of primary nasal and bronchial epithelial cells.

BACKGROUND AND OBJECTIVE: Rhinoviruses (RV) replicate in both upper and lower airway epithelial cells. We evaluated the possibility of using nasal epithelial cells (NEC) as surrogate of bronchial epithelial cells (BEC) for RV pathogenesis cell culture studies. METHODS: We used primary paired NEC and BEC cultures established from healthy subjects and compared the replication of RV belonging to the major (RV16) and minor (RV1B) group, and the cellular antiviral and proinflammatory cytokine responses towards these viruses. We related antiviral and pro-inflammatory responses of NEC isolated from CF and COPD patients with those of BEC. RESULTS: RV16 replication and major group surface receptor (ICAM-1) expression were higher in healthy NEC compared with BEC (P < 0.05); RV1B replication and minor group surface receptor (LDLR) expression were similar. Healthy NEC and BEC produced similar levels of IFN-ß and IFN-λ2/3 upon RV infection or after simulation with poly(IC). IL-8 production was similar between healthy NEC and BEC. IL-6 release at baseline (P < 0.01) and upon infection with RV16 (P < 0.05) and poly(IC) stimulation (P < 0.05) was higher in NEC. RV1B viral load in NEC was related to RV1B viral load in BEC (r = 0.49, P = 0.01). There was a good correlation of IFN levels between NEC and BEC (r = 0.66, P = 0.0004 after RV1B infection). IL-8 production in NEC was related to IL-8 production in BEC (r = 0.48, P = 0.02 after RV1B infection). CONCLUSION: NEC are a suitable alternative cellular system to BEC to study the pathophysiology of RV infections and particularly to investigate IFN responses induced by RV infection.