Pulmonologies
Abstract
Cholesterol-25-hydroxylase (CH25H), the biosynthetic enzyme for 25-hydroxycholesterol (25HC), is most highly expressed in the lung, but its role in lung biology is poorly defined. Recently, we reported that Ch25h is induced in monocyte-derived macrophages recruited to the airspace during resolution of lung inflammation and that 25HC promotes Liver X Receptor (LXR)-dependent clearance of apoptotic neutrophils by these cells. Ch25h and 25HC are, however, also robustly induced by lung-resident cells during the early hours of lung inflammation, suggesting additional cellular sources and targets. Here, using Ch25h-/- mice and exogenous 25HC in lung injury models, we provide evidence that 25HC sustains pro-inflammatory cytokines in the airspace and augments lung injury, at least in part, by inducing LXR-independent endoplasmic reticulum stress and endothelial leak. Suggesting an autocrine effect in endothelium, inhaled LPS upregulates pulmonary endothelial Ch25h and non-hematopoietic Ch25h deletion is sufficient to confer lung protection. In acute respiratory distress syndrome patients, airspace 25HC and alveolar macrophage CH25H were associated with markers of microvascular leak, endothelial activation, endoplasmic reticulum stress, inflammation, and clinical severity. Taken together, our findings suggest that 25HC deriving from and acting upon different cell types in the lung communicates distinct, temporal LXR-independent and -dependent signals to regulate inflammatory homeostasis.
Authors
Jennifer H. Madenspacher, Eric D. Morrell, Jeffrey G. McDonald, Bonne M. Thompson, Yue Li, Konstantin G. Birukov, Anna A. Birukova, Renee D. Stapleton, Aidin Alejo, Peer W. Karmaus, Julie M. Meacham, Prashant Rai, Carmen Mikacenic, Mark M. Wurfel, Michael B. Fessler
Abstract
Anti-CD36 antibodies have been suggested to induce transfusion-related acute lung injury (TRALI) upon blood transfusion, particularly in Asian populations. However, little is known about the pathological mechanism of anti-CD36-mediated TRALI, and potential therapies haven’t yet been identified. Here, we developed a murine model of anti-CD36-mediated TRALI to address these questions. We found that administration of mouse monoclonal antibody against CD36 (mAb GZ1) or human anti-CD36 IgG, but not GZ1 F(ab’)2 fragments, induced severe TRALI in Cd36+/+ male mice. Pre-depletion of recipient monocytes or complement, but not neutrophils or platelets, prevented the development of murine TRALI. Moreover, plasma C5a levels after TRALI induction by anti-CD36 were increased more than 3-fold, implying a critical role of complement C5 activation in the mechanism of Fc-dependent anti-CD36-mediated TRALI. Administration of GZ1 F(ab’)2, antioxidant (NAC) or C5-blocker (mAb BB5.1) before TRALI induction completely protected mice from anti-CD36-mediated TRALI. Although no significant amelioration in TRALI was observed when mice were injected with GZ1 F(ab’)2 after TRALI induction, significant improvement was achieved when mice were treated post-induction with NAC or anti-C5. Importantly, anti-C5 treatment completely rescued mice from TRALI, suggesting the potential role of existing anti-C5 drugs in the treatment of patients with TRALI caused by anti-CD36.
Authors
Da-Wei Chen, Tian Kang, Xiu-Zhang Xu, Wen-Jie Xia, Xin Ye, Yong-Bin Wu, Yao-Ri Xu, Jing Liu, Hui Ren, Jing Deng, Yang-Kai Chen, Hao-Qiang Ding, Muhammad Aslam, Wioleta M. Zelek, B. Paul Morgan, Rick Kapur, Sentot Santoso, Yong-Shui Fu
Abstract
Patients with progressive fibrosing interstitial lung diseases (PF-ILDs) carry a poor prognosis and have limited therapeutic options. A hallmark feature is fibroblast resistance to apoptosis, leading to their persistence, accumulation, and excessive deposition of extracellular matrix. A complex balance of the B cell lymphoma 2 (BCL-2) protein family controlling the intrinsic pathway of apoptosis and fibroblast reliance on antiapoptotic proteins has been hypothesized to contribute to this resistant phenotype. Examination of lung tissue from patients with PF-ILD (idiopathic pulmonary fibrosis and silicosis) and mice with PF-ILD (repetitive bleomycin and silicosis) showed increased expression of antiapoptotic BCL-2 family members in α–smooth muscle actin–positive fibroblasts, suggesting that fibroblasts from fibrotic lungs may exhibit increased susceptibility to inhibition of antiapoptotic BCL-2 family members BCL-2, BCL-XL, and BCL-W with the BH3 mimetic ABT-263. We used 2 murine models of PF-ILD to test the efficacy of ABT-263 in reversing established persistent pulmonary fibrosis. Treatment with ABT-263 induced fibroblast apoptosis, decreased fibroblast numbers, and reduced lung collagen levels, radiographic disease, and histologically evident fibrosis. Our studies provide insight into how fibroblasts gain resistance to apoptosis and become sensitive to the therapeutic inhibition of antiapoptotic proteins. By targeting profibrotic fibroblasts, ABT-263 offers a promising therapeutic option for PF-ILDs.
Authors
Joseph C. Cooley, Nomin Javkhlan, Jasmine A. Wilson, Daniel G. Foster, Benjamin L. Edelman, Luis A. Ortiz, David A. Schwartz, David W.H. Riches, Elizabeth F. Redente
Abstract
Type II alveolar epithelial cell (AECII) redox imbalance contributes to the pathogenesis of idiopathic pulmonary fibrosis (IPF) – a deadly disease with restricted and limited treatment options. Here, we show that expression of membrane-bound cytochrome B5 reductase 3 (CYB5R3), an enzyme critical for maintaining cellular redox homeostasis and soluble guanylate cyclase (sGC) heme iron redox state, is diminished in IPF AECII. Deficiency of CYB5R3 in AECII leads to sustained activation of the profibrotic factor TGF-β1 and increased susceptibility to lung fibrosis. We further show that CYB5R3 is a critical regulator of ERK1/2 phosphorylation and sGC-cGMP-protein kinase G axis that modulates activation of TGF-β1 signaling pathway. We demonstrate that sGC agonists (BAY 41-8543 and BAY 54-6544) are effective in reducing the pulmonary fibrotic outcomes of in vivo deficiency of CYB5R3 in AECII. Taken together, these results establish that CYB5R3 in AECII is required to maintain resilience against lung injury and fibrosis, and that therapeutic manipulation of sGC redox state could provide a basis for treating fibrotic conditions in the lung and beyond.
Authors
Marta Bueno, Jazmin Calyeca, Timur Khaliullin, Megan Miller, Diana Álvarez, Lorena Rosas, Judith Brands, Christian M. Baker, Amro Nasser, Stephanie Shulkowski, August Mathien, Nneoma O, Uzoukwu, John Sembrat, Brenton G. Mays, Kaitlin Fiedler, Scott A. Hahn, Sonia R. Salvatore, Francisco J. Schopfer, Mauricio Rojas, Peter Sandner, Adam Straub, Ana L. Mora
Abstract
Hypoxia is a sentinel feature of IPF. The IPF microenvironment contains high lactate levels and hypoxia enhances cellular lactate production. Lactate, acting through the GPR81 lactate receptor, serves as a signal molecule regulating cellular processes. We previously identified intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the lungs of IPF patients that drive fibrosis. However, whether hypoxia enhances IPF MPC fibrogenicity is unclear. We hypothesized that hypoxia increases IPF MPC fibrogenicity via lactate and its cognate receptor GPR81. Here we show that hypoxia promotes IPF MPC self-renewal. The mechanism involves hypoxia-mediated enhancement of LDHA function and lactate production and release. Hypoxia also increases HIF1α levels, which in turn augments the expression of GPR81. Exogenous lactate operating through GPR81 promotes IPF MPC self-renewal. IHC analysis of IPF lung tissue demonstrate IPF MPCs expressing GPR81 and hypoxic markers on the periphery of the fibroblastic focus. We show that hypoxia enhances IPF MPC fibrogenicity in vivo. We demonstrate that knock-down of GPR81 inhibits hypoxia-induced IPF MPC self-renewal in vitro and attenuates hypoxia-induced IPF MPC fibrogenicity in vivo. Our data demonstrate that hypoxia creates a feed-forward loop that augments IPF MPC fibrogenicity via the lactate/GPR81/HIF1α pathway.
Authors
Libang Yang, Adam Gilbertsen, Hong Xia, Alexey Benyumov, Karen A. Smith, Jeremy A. Herrera, Emilian Racila, Peter B. Bitterman, Craig A. Henke
Abstract
Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with ARDS secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here, using a mouse model of hypercapnia exposure, cell lineage-tracing, spatial transcriptomics and 3D-cultures, we show that hypercapnia limits β-catenin signaling in AT2 cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+-fibroblasts from those maintaining AT2 progenitor activity towards those that antagonize β-catenin signaling thereby limiting progenitor function. Constitutive activation of β-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in hypercapnic patients may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier, increasing lung flooding, ventilator dependency and mortality.
Authors
Laura A. Dada, Lynn C. Welch, Natalia D. Magnani, Ziyou Ren, Hyebin Han, Patricia L. Brazee, Diego Celli, Annette S. Flozak, Anthea Weng, Mariana Maciel Herrerias, Vitalii Kryvenko, István Vadász, Constance E. Runyan, Hiam Abdala-Valencia, Masahiko Shigemura, S. Marina Casalino-Matsuda, Alexander V. Misharin, G.R. Scott Budinger, Cara J. Gottardi, Jacob I. Sznajder
Abstract
In pulmonary arterial hypertension (PAH), inflammation promotes a fibroproliferative pulmonary vasculopathy. Reductionist studies emphasizing single biochemical reactions suggest a shift toward glycolytic metabolism in PAH; however, key questions remain regarding the metabolic profile of specific cell types within PAH vascular lesions in vivo. We used RNA-seq to profile the transcriptome of pulmonary artery endothelial cells (PAECs) freshly isolated from an inflammatory vascular injury model of PAH ex vivo, and these data were integrated with information from human gene ontology pathways. Network medicine was then used to map all amino acid and glucose pathways to the consolidated human interactome, which includes data on 233,957 physical protein-protein interactions. Glucose and proline pathways were significantly close to the human PAH disease module, suggesting that these pathways are functionally relevant to PAH pathobiology. To test this observation in vivo, we used multi-isotope imaging mass spectrometry (MIMS) to map and quantify utilization of glucose and proline in the PAH pulmonary vasculature at subcellular resolution. Our findings suggest suggest that elevated glucose and proline avidity underlies increased biomass in PAECs and the media of fibrosed PAH pulmonary arterioles. Overall, these data show that anabolic utilization of glucose and proline are fundamental to the vascular pathology of PAH.
Authors
Bradley M. Wertheim, Rui-Sheng Wang, Christelle Guillermier, Christiane V.R. Hütter, William M. Oldham, Jörg Menche, Matthew L. Steinhauser, Bradley A. Maron
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease. Recent findings have shown a marked metabolic reprogramming associated with changes in mitochondrial homeostasis and autophagy during pulmonary fibrosis. The microRNA-33 (miR-33) family of microRNAs (miRNAs) encoded within the introns of SREBP (sterol regulatory element binding protein) genes are master regulators of sterol and fatty acid (FA) metabolism. miR-33 controls macrophage immuno-metabolic response and enhances mitochondrial biogenesis, FA oxidation, and cholesterol efflux. Here, we show that miR-33 levels are increased in Broncho Alveolar Lavage (BAL) cells isolated from IPF patients compared to healthy controls. We demonstrate that specific genetic ablation of miR-33 in macrophages protects against bleomycin-induced pulmonary fibrosis. The absence of miR-33 in macrophages improves mitochondrial homeostasis and increases autophagy while decreasing inflammatory response after bleomycin injury. Notably, pharmacological inhibition of miR-33 in macrophages via administration of anti-miR-33 Peptide Nucleic Acids (PNA-33) attenuates fibrosis in different in vivo and ex vivo mice and human models of pulmonary fibrosis. Together, these studies elucidate a major role of miR-33 in macrophages in the regulation of pulmonary fibrosis and uncover a novel therapeutic approach to treat this disease.
Authors
Farida Ahangari, Nathan L. Price, Shipra Malik, Maurizio Chioccioli, Thomas Bärnthaler, Taylor S. Adams, Jooyoung Kim, Sai Pallavi Pradeep, Shuizi Ding, Carlos Cosme Jr, Kadi-Ann S. Rose, John E. McDonough, Nachelle R. Aurelien, Gabriel Ibarra, Norihito Omote, Jonas C. Schupp, Giuseppe DeIuliis, Julian A. Villalba Nunez, Lokesh Sharma, Changwan Ryu, Charles S. Dela Cruz, Xinran Liu, Antje Prasse, Ivan Rosas, Raman Bahal, Carlos Fernandez-Hernando, Naftali Kaminski
Abstract
In the progression phase of idiopathic pulmonary fibrosis (IPF) the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a co–culture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single–cell RNA sequencing (sc–RNA–seq) revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB cell populations adopted distinct pro–fibrotic cell differentiation states upon co-cultivation, resembling specific cell populations that were previously identified in lungs of IPF patients. Transcriptomic analysis revealed active nuclear factor NF–kappa–B (NF–κB) signaling early in the co–cultured EC and FB cells and the identified NF–κB expression signatures were also found in “HAS1 High FB” and “PLIN2+ FB” populations from IPF patient lungs. Pharmacological blockade of NF–κB signaling attenuated specific phenotypic changes of EC and prevented FB–mediated interleukin–6 (IL6), interleukin–8 (IL–8) and C–X–C motif chemokine ligand 6 (CXCL6) cytokine secretion, as well as collagen alpha–1(I) chain (COL1A1) and alpha–smooth muscle actin (α–SMA) accumulation. Thus, we identified NF–κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.
Authors
Patrick Sieber, Anny Schäfer, Raphael Lieberherr, Silvia L Caimi, Urs Lüthi, Jesper Ryge, Jan H. Bergmann, Francois Le Goff, Manuel Stritt, Peter Blattmann, Bérengère Renault, Patrick Rammelt, Bruno Sempere, Diego Freti, Rolf Studer, Eric S. White, Magdalena Birker-Robaczewska, Maxime Boucher, Oliver Nayler
Abstract
Keratin expression dynamically changes in airway basal cells (BCs) following acute and chronic injury, yet the functional consequences of these changes on BC behavior remain unknown. In Bronchiolitis Obliterans (BO) following lung transplantation, BC clonogenicity declines which is associated with a switch from Keratin15 (Krt15) to Keratin14 (Krt14). We investigated the roles of these keratins using Crispr-KO in vitro and in vivo and found that Krt14-KO and Krt15-KO produce contrasting phenotypes in terms of differentiation and clonogenicity. Primary mouse Krt14-KO BCs failed to differentiate into club and ciliated cells, but had enhanced clonogenicity. By contrast, Krt15-KO did not alter BC differentiation, but impaired clonogenicity in vitro and reduced the number of label-retaining BCs in vivo following injury. Krt14, but not Krt15, bound the tumor suppressor Stratifin (Sfn). Disruption of Krt14, but not of Krt15, reduced Sfn protein abundance and increased expression of the oncogene dNp63a during BC differentiation, while dNp63a levels were reduced in Krt15-KO BCs. Overall, the phenotype of Krt15-KO BCs contrasts that of Krt14-KO and resembles the phenotype in BO with decreased clonogenicity, increased Krt14 and decreased dNp63a expression. This work demonstrates that Krt14 and Krt15 functionally regulate BC behavior which is relevant in chronic disease states like BO.
Authors
Vitaly Ievlev, Thomas J. Lynch, Kyle W. Freischlag, Caitlyn B. Gries, Anit Shah, Albert C. Pai, Bethany A. Ahlers, Soo Yeun Park, John F. Engelhardt, Kalpaj R. Parekh
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