Hypoxia enhances IPF mesenchymal progenitor cell fibrogenicity via the lactate/GPR81/HIF1α pathway

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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

Hypercapnia alters stromal-derived Wnt production limiting β-catenin signaling and proliferation in alveolar type 2 cells

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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

Proline and glucose metabolic reprogramming supports vascular endothelial and medial biomass in pulmonary arterial hypertension

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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

microRNA-33 deficiency in macrophages enhances autophagy, improves mitochondrial homeostasis, and protects against lung fibrosis

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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

NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model

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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

Krt14 and Krt15 differentially regulate regenerative properties and differentiation potential of airway basal cells

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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

Culture impact on the transcriptomic programs of primary and iPSC-derived human alveolar type 2 cells

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Abstract

Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single cell resolution. Here, we perform head-to-head comparisons between the transcriptomes of fresh primary (1o) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1o and iAEC2s) exhibiting similarities to and differences from freshly purified 1o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires a “transitional cell state” described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1o and engineered AEC2s, two in vitro models that can be harnessed to study human lung health and disease.

Authors

Konstantinos-Dionysios Alysandratos, Carolina Garcia-de-Alba, Changfu Yao, Patrizia Pessina, Jessie Huang, Carlos Villacorta-Martin, Olivia T. Hix, Kasey Minakin, Claire L. Burgess, Pushpinder Bawa, Aditi Murthy, Bindu Konda, Michael F. Beers, Barry R. Stripp, Carla F. Kim, Darrell N. Kotton

Mitochondrial fission and bioenergetics mediate human lung fibroblast durotaxis

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Abstract

Mitochondria are dynamic organelles responsible for energy production and many processes central to cellular function. Alterations in mitochondrial function is associated with human fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF). Pulmonary fibrosis is characterized by stiffening of the extracellular matrix (ECM). Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we identified mitochondria as an important mechanotransducer at the intersection between extracellular mechanical signals and durotactic lung fibroblast migration. Primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness-regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a DRP1/MFF-dependent pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. Our findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of IPF.

Authors

Ting Guo, Chun-sun Jiang, Shan-Zhong Yang, Yi Zhu, Chao He, A. Brent Carter, Veena B. Antony, Hong Peng, Yong Zhou

NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes

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Abstract

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first post-transplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. Natural killer (NK) cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. Together, these data support an aberrant NK cell-neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

Authors

Daniel R. Calabrese, Tasha Tsao, Mélia Magnen, Colin Valet, Ying Gao, Beñat Mallavia, Jennifer J. Tian, Emily A. Aminian, Kristin M. Wang, Avishai Shemesh, Elman B. Punzalan, Aartik Sarma, Carolyn S. Calfee, Stephanie A. Christenson, Charles R. Langelier, Steven R. Hays, Jeff A. Golden, Lorriana E. Leard, Mary E. Kleinhenz, Nicholas A. Kolaitis, Rupal J. Shah, Aida Venado, Lewis L. Lanier, John R. Greenland, David M. Sayah, Abbas Ardehali, Jasleen Kukreja, S. Sam Weigt, John A. Belperio, Jonathan P Singer, Mark R. Looney

HIF1A-dependent induction of alveolar epithelial PFKFB3 dampens acute lung injury

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Abstract

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us towards a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxp/loxp SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid installation. Studies in genetic models linked Pfkfb3 expression and function to hypoxia-inducible factor Hif1a. Intra-tracheal pyruvate instillation not only reconstituted Pfkfb3loxp/loxp or Hif1aloxp/loxp SPC ER Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies confirmed increased PFKFB3 staining in injured lungs and co-localized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counter-balancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI.

Authors

Christine U. Vohwinkel, Nana Burns, Ethan Coit, Xiaoyi Yuan, Eszter K. Vladar, Christina Sul, Eric P. Schmidt, Peter Carmeliet, Kurt Stenmark, Eva S. Nozik, Rubin M. Tuder, Holger K. Eltzschig