Alveolar epithelium plays a pivotal role in protecting the lungs from inhaled infectious agents. Therefore, the regenerative capacity of the alveolar epithelium is critical for recovery from these insults in order to rebuild the epithelial barrier and restore pulmonary functions. Here, we show that sublethal infection of mice with Streptococcus pneumoniae, the most common pathogen of community-acquired pneumonia, led to exclusive damage in lung alveoli, followed by alveolar epithelial regeneration and resolution of lung inflammation. We show that surfactant protein C–expressing (SPC-expressing) alveolar epithelial type II cells (AECIIs) underwent proliferation and differentiation after infection, which contributed to the newly formed alveolar epithelium. This increase in AECII activities was correlated with increased nuclear expression of Yap and Taz, the mediators of the Hippo pathway. Mice that lacked Yap/Taz in AECIIs exhibited prolonged inflammatory responses in the lung and were delayed in alveolar epithelial regeneration during bacterial pneumonia. This impaired alveolar epithelial regeneration was paralleled by a failure to upregulate IκBa, the molecule that terminates NF-κB–mediated inflammatory responses. These results demonstrate that signals governing resolution of lung inflammation were altered in Yap/Taz mutant mice, which prevented the development of a proper regenerative niche, delaying repair and regeneration of alveolar epithelium during bacterial pneumonia.
Ryan LaCanna, Daniela Liccardo, Peggy Zhang, Lauren Tragesser, Yan Wang, Tongtong Cao, Harold A. Chapman, Edward E. Morrisey, Hao Shen, Walter J. Koch, Beata Kosmider, Marla R. Wolfson, Ying Tian
The lung is a specialized barrier organ that must tightly regulate interstitial fluid clearance and prevent infection in order to maintain effective gas exchange. Lymphatic vessels are important for these functions in other organs, but their roles in the lung have not been fully defined. In the present study, we addressed how the lymphatic vasculature participates in lung homeostasis. Studies using mice carrying a lymphatic reporter allele revealeded that, in contrast to other organs, lung lymphatic collecting vessels lack smooth muscle cells entirely, suggesting that forward lymph flow is highly dependent on movement and changes in pressure associated with respiration. Functional studies using CLEC2-deficient mice in which lymph flow is impaired due to loss of lympho-venous hemostasis or using inducible lung-specific ablation of lymphatic endothelial cells in a lung transplant model revealeded that loss of lymphatic function leads to an inflammatory state characterized by the formation of tertiary lymphoid organs (TLOs). In addition, impaired lymphatic flow in mice resulteds in hypoxia and features of lung injury that resemble emphysema. These findings reveal both a lung-specific mechanism of lymphatic physiology and a lung-specific consequence of lymphatic dysfunction that may contribute to chronic lung diseases that arise in association with TLO formation.
Hasina Outtz Reed, Liqing Wang, Jarrod Sonett, Mei Chen, Jisheng Yang, Larry Li, Petra Aradi, Zoltán Jakus, Jeanine M. D'Armiento, Wayne W. Hancock, Mark L. Kahn
Idiopathic pulmonary fibrosis (IPF) is a chronic and deadly disease with a poor prognosis and few treatment options. Pathological remodeling of the extracellular matrix (ECM) by myofibroblasts is a key factor that drives disease pathogenesis, although the underlying mechanisms remain unknown. Alternative polyadenylation (APA) has recently been shown to play a major role in cellular responses to stress by driving the expression of fibrotic factors and ECMs through altering microRNA sensitivity, but a connection to IPF has not been established. Here, we demonstrate that CFIm25, a global regulator of APA, is down-regulated in the lungs of patients with IPF and mice with pulmonary fibrosis, with its expression selectively reduced in alpha-smooth muscle actin (α-SMA) positive fibroblasts. Following the knockdown of CFIm25 in normal human lung fibroblasts, we identified 808 genes with shortened 3′UTRs, including those involved in the transforming growth factor-β signaling pathway, the Wnt signaling pathway, and cancer pathways. The expression of key pro-fibrotic factors can be suppressed by CFIm25 overexpression in IPF fibroblasts. Finally, we demonstrate that deletion of CFIm25 in fibroblasts or myofibroblast precursors using either the Col1a1 or the Foxd1 promoter enhances pulmonary fibrosis after bleomycin exposure in mice. Taken together, our results identified CFIm25 down-regulation as a novel mechanism to elevate pro-fibrotic gene expression in pulmonary fibrosis.
Tingting Weng, Junsuk Ko, Chioniso P. Masamha, Zheng Xia, Yu Xiang, Ning-yuan Chen, Jose G. Molina, Scott Collum, Tinne C. Mertens, Fayong Luo, Kemly Philip, Jonathan Davies, Jingjing Huang, Cory Wilson, Rajarajan A. Thandavarayan, Brian A. Bruckner, Soma S.K. Jyothula, Kelly A. Volcik, Lei Li, Leng Han, Wei Li, Shervin Assassi, Harry Karmouty-Quintana, Eric J. Wagner, Michael R. Blackburn
Goblet cell metaplasia, a disabling hallmark of chronic lung disease, lacks curative treatments at present. To identify novel therapeutic targets for goblet cell metaplasia, we studied the transcriptional response profile of IL-13–exposed primary human airway epithelia in vitro and asthmatic airway epithelia in vivo. A perturbation-response profile connectivity approach identified geldanamycin, an inhibitor of heat shock protein 90 (HSP90) as a candidate therapeutic target. Our experiments confirmed that geldanamycin and other HSP90 inhibitors prevented IL-13–induced goblet cell metaplasia in vitro and in vivo. Geldanamycin also reverted established goblet cell metaplasia. Geldanamycin did not induce goblet cell death, nor did it solely block mucin synthesis or IL-13 receptor–proximal signaling. Geldanamycin affected the transcriptome of airway cells when exposed to IL-13, but not when exposed to vehicle. We hypothesized that the mechanism of action probably involves TGF-β, ERBB, or EHF, which would predict that geldanamycin would also revert IL-17–induced goblet cell metaplasia, a prediction confirmed by our experiments. Our findings suggest that persistent airway goblet cell metaplasia requires HSP90 activity and that HSP90 inhibitors will revert goblet cell metaplasia, despite active upstream inflammatory signaling. Moreover, HSP90 inhibitors may be a therapeutic option for airway diseases with goblet cell metaplasia of an unknown mechanism of action.
Alejandro A. Pezzulo, Rosarie A. Tudas, Carley G. Stewart, Luis G. Vargas Buonfiglio, Brian D. Lindsay, Peter J. Taft, Nicholas D. Gansemer, Joseph Zabner
BACKGROUND. Chronic Obstructive Pulmonary Disease (COPD) is a heterogeneous smoking-related disease characterized by airway obstruction and inflammation. This inflammation may persist even after smoking cessation and responds variably to corticosteroids. Personalizing treatment to biologically similar “molecular phenotypes” may improve therapeutic efficacy in COPD. IL-17A is involved in neutrophilic inflammation and corticosteroid resistance, and thus may be particularly important in a COPD molecular phenotype. METHODS. We generated a gene expression signature of IL-17A response in bronchial airway epithelial brushings (“BAE”) from smokers with and without COPD (n = 238), and validated it using data from two randomized trials of IL-17 blockade in psoriasis. This IL-17 signature was related to clinical and pathologic characteristics in two additional human studies of COPD: (1) SPIROMICS (n = 47), which included former and current smokers with COPD, and (2) GLUCOLD (n = 79), in which COPD participants were randomized to placebo or corticosteroids. RESULTS. The IL-17 signature was associated with an inflammatory profile characteristic of an IL-17 response, including increased airway neutrophils and macrophages. In SPIROMICS the signature was associated with increased airway obstruction and functional small airway disease on quantitative chest CT. In GLUCOLD the signature was associated with decreased response to corticosteroids, irrespective of airway eosinophilic or Type 2 inflammation. CONCLUSION. These data suggest that a gene signature of IL-17 airway epithelial response distinguishes a biologically, radiographically, and clinically distinct COPD subgroup that may benefit from personalized therapy. TRIAL REGISTRATION. ClinicalTrials.gov NCT01969344. FUNDING. Primary support from NIH/NHLBI. For others see below.
Stephanie A. Christenson, Maarten van den Berge, Alen Faiz, Kai Imkamp, Nirav Bhakta, Luke R. Bonser, Lorna T. Zlock, Igor Z. Barjaktarevic, R. Graham Barr, Eugene R. Bleecker, Richard C. Boucher, Russell P. Bowler, Alejandro P. Comellas, Jeffrey L. Curtis, MeiLan K. Han, Nadia N. Hansel, Pieter S. Hiemstra, Robert J. Kaner, Jerry A. Krishnan, Fernando J. Martinez, Wanda K. O'Neal, Robert Paine III, Wim Timens, J. Michael Wells, Avrum Spira, David J. Erle, Prescott G. Woodruff
In the era of combined antiretroviral therapy (cART), lung diseases such as chronic bronchitis (CB) and COPD are common among persons living with HIV (PLWH), particularly smokers. Although smoking is highly prevalent among PLWH, HIV may be an independent risk factor for lung diseases; however, the role of HIV and cigarette smoke (CS) and their potential interaction in the development of chronic lung diseases among PLWH has not been delineated. To investigate this interaction, cynomolgus macaques were exposed to CS and/or simian-adapted human immunodeficiency virus (SHIV) and treated with cART. The development of CB and the lung functions were evaluated following CS±SHIV treatment. The results showed that in the lung, SHIV was a strong independent risk factor for goblet cell metaplasia/hyperplasia and mucus formation, MUC5AC synthesis, loss of tight junction proteins, and increased expression of Th2 cytokines/transcription factors. In addition, SHIV and CS synergistically reduced the lung function and increased the extrathoracic tracheal ring thickness. Interestingly, SHIV-infection generated significant numbers of HIV-gp120+ epithelial cells (HGECs) in small airways and alveoli and their numbers doubled in CS+SHIV-infected lungs. We conclude that even with cART, SHIV independently induces CB and pro-COPD changes in the lung and the effects are exacerbated by CS.
Hitendra S. Chand, Rodrigo Vazquez-Guillamet, Christopher M. Royer, Karin Rudolph, Neerad C. Mishra, Shashi P. Singh, Shah S. Hussain, Edward G. Barrett, Shannon Callen, Siddappa N. Byrareddy, Maria Cristina Vazquez Guillamet, Jawad Abukhalaf, Aryaz Sheybani, Vernat Exil, Veena Raizada, Hemant Agarwal, Madhavan Nair, Francois Villinger, Shilpa Buch, Mohan Sopori
Allergic asthma is characterized by airway hyperresponsiveness, inflammation, and a cellular infiltrate dominated by eosinophils. Numerous epidemiological studies have related the exacerbation of allergic asthma with an increase in ambient inhalable particulate matter from air pollutants. This is because inhalable particles efficiently deliver airborne allergens deep into the airways, where they can aggravate allergic asthma symptoms. However, the cellular mechanisms by which inhalable particulate allergens (pAgs) potentiate asthmatic symptoms remain unknown, in part because most in vivo and in vitro studies exploring the pathogenesis of allergic asthma use soluble allergens (sAgs). Using a mouse model of allergic asthma, we found that, compared with their sAg counterparts, pAgs triggered markedly heightened pulmonary eosinophilia in allergen-sensitized mice. Mast cells (MCs) were implicated in this divergent response, as the differences in airway inflammatory responses provoked by the physical nature of the allergens were attenuated in MC-deficient mice. The pAgs were found to mediate MC-dependent responses by enhancing retention of pAg/IgE/FcεRI complexes within lipid raft–enriched, CD63+ endocytic compartments, which prolonged IgE/FcεRI-initiated signaling and resulted in heightened cytokine responses. These results reveal how the physical attributes of allergens can co-opt MC endocytic circuitry and signaling responses to aggravate pathological responses of allergic asthma in mice.
Cong Jin, Christopher P. Shelburne, Guojie Li, Kristina J. Riebe, Gregory D. Sempowski, W. Michael Foster, Soman N. Abraham
BACKGROUND. Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling. Characterization of airway changes on computed tomography has been challenging due to the complexity of the recurring branching patterns, and this can be better measured using fractal dimensions. METHODS. We analyzed segmented airway trees of 8135 participants enrolled in the COPDGene cohort. The fractal complexity of the segmented airway tree was measured by the Airway Fractal Dimension (AFD) using the Minkowski-Bouligand box-counting dimension. We examined associations between AFD and lung function and respiratory morbidity using multivariable regression analyses. We further estimated the extent of peribronchial emphysema (%) within 5mm of the airway tree as this is likely to affect AFD. We classified participants into 4 groups based on median AFD and %peribronchial emphysema, and estimated survival. RESULTS. AFD was significantly associated with FEV1 (p<0.001) and FEV1/FVC (p<0.001) after adjusting for age, race, gender, smoking status, pack-years of smoking, body-mass-index, CT emphysema, air trapping, airway thickness, and CT scanner type. On multivariable analysis, AFD was also associated with respiratory-quality of life and six-minute walk distance, as well as exacerbations, lung function decline and mortality on longitudinal follow-up. We identified a subset of participants with AFDmedian who had worse survival compared with participants with high AFD and low peribronchial emphysema (adjusted HR = 2.72, 95%CI 2.20 to 3.35; p<0.001), a substantial number of whom were not identified by traditional spirometry severity grades. CONCLUSIONS. Airway fractal dimension as a measure of airway branching complexity and remodeling in smokers is associated with respiratory morbidity and lung function change, offers prognostic information additional to traditional CT measures of airway wall thickness, and can be used to estimate mortality risk.
Sandeep Bodduluri, Abhilash S. Kizhakke Puliyakote, Sarah E. Gerard, Joseph M. Reinhardt, Eric A. Hoffman, John D. Newell Jr., Hrudaya P. Nath, MeiLan K. Han, George R. Washko, Raúl San José Estépar, Mark T. Dransfield, Surya P. Bhatt
Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.
Chaoqun Wang, Nabora S. Reyes de Mochel, Stephanie A. Christenson, Monica Cassandras, Rebecca Moon, Alexis N. Brumwell, Lauren E. Byrnes, Alfred Li, Yasuyuki Yokosaki, Peiying Shan, Julie B. Sneddon, David Jablons, Patty J. Lee, Michael A. Matthay, Harold A. Chapman, Tien Peng
Epithelial cell dysfunction is postulated as an important component in the pathogenesis of Idiopathic Pulmonary Fibrosis (IPF). Mutations in the Surfactant Protein C [SP-C] gene [SFTPC], an alveolar type 2 (AT2) cell restricted protein, have been found in sporadic and familial IPF. To causally link these events, we developed a knock-in mouse model capable of regulated expression of an IPF-associated Isoleucine to Threonine substitution at codon 73 [I73T] in Sftpc (SP-CI73T). Tamoxifen treated SP-CI73T cohorts developed rapid increases in SftpcI73T mRNA and misprocessed proSP-CI73T protein accompanied by increased early mortality (days 7-14). This acute phase was marked by diffuse parenchymal lung injury, tissue infiltration by monocytes, polycellular alveolitis, and elevations in bronchoalveolar lavage and AT2 mRNA contents of select inflammatory cytokines. Resolution of alveolitis (2-4 weeks), commensurate with a rise in TGFB1, was followed by aberrant remodeling marked by collagen deposition, AT2 cell hyperplasia, a-SMA positive cells, and restrictive lung physiology. The translational relevance of the model was supported by detection of multiple IPF biomarkers previously reported in human cohorts. These data provide proof of principle that mutant SP-C expression in vivo causes spontaneous lung fibrosis strengthening the role of AT2 dysfunction as a key upstream driver of IPF pathogenesis.
Shin-Ichi Nureki, Yaniv Tomer, Alessandro Venosa, Jeremy Katzen, Scott J. Russo, Sarita Jamil, Matthew Barrett, Vivian Nguyen, Meghan Kopp, Surafel Mulugeta, Michael F. Beers