Glycolytic reprogramming is involved in tissue remodeling on chronic rhinosinusitis

PLOS ONE RESEARCH ARTICLE Glycolytic reprogramming is involved in tissue remodeling on chronic rhinosinusitis Min-Sik Jo1‡, Hyun-Woo Yang1‡, Joo-Hoo Park ID1, Jae-Min Shin ID1,2,3‡*, Il-Ho Park1,2,3‡* 1 Upper Airway Chronic Inflammatory Diseases Laboratory, Korea University College of Medicine, Seoul, South Korea, 2 Medical Device Usability Test Center, Guro Hospital, Korea University College of Medicine, Seoul, South Korea, 3 Department of Otorhinolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 ‡ MSJ and HWY are co-first authors and JMS and IHP are co-corresponding authors to this work. * (JMS); (IHP) Abstract Background OPEN ACCESS Citation: Jo M-S, Yang H-W, Park J-H, Shin J-M, Park I-H (2023) Glycolytic reprogramming is involved in tissue remodeling on chronic rhinosinusitis. PLoS ONE 18(2): e0281640. https:// doi.org/10.1371/journal.pone.0281640 Editor: Kishor Pant, The Hormel Institute (University of Minnesota), UNITED STATES Glycolytic reprogramming is a key feature of chronic inflammatory disease. Extracellular matrix (ECM) produced by myofibroblasts plays an important role in tissue remodeling of nasal mucosa in chronic rhinosinusitis (CRS). This study aimed to determine whether glycolytic reprogramming contributes to myofibroblast differentiation and ECM production in nasal fibroblasts. Methods Received: December 2, 2022 Accepted: January 27, 2023 Published: February 16, 2023 Peer Review History: PLOS recognizes the benefits of transparency in the peer review process; therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. The editorial history of this article is available here: https://doi.org/10.1371/journal.pone.0281640 Copyright: © 2023 Jo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting information files. Primary nasal fibroblasts were isolated from the nasal mucosa of patients with CRS. Glycolytic reprogramming was assessed by measuring the extracellular acidification and oxygen consumption rates in nasal fibroblast, with and without transforming growth factor beta 1 (TGF-β1) treatment. Expression of glycolytic enzymes and ECM components was measured by real-time polymerase chain reaction, western blotting, and immunocytochemical staining. Gene set enrichment analysis was performed using whole RNA-sequencing data of nasal mucosa of healthy donors and patients with CRS. Result Glycolysis of nasal fibroblasts stimulated with TGF-B1 was upregulated along with glycolytic enzymes. Hypoxia-inducing factor (HIF)-1α was a high-level regulator of glycolysis, and increased HIF-1α expression promoted glycolysis of nasal fibroblasts, and inhibition of HIF-1α down-regulated myofibroblasts differentiation and ECM production. Funding: This research was supported by the Bio & Medical Technology Development Program of PLOS ONE | https://doi.org/10.1371/journal.pone.0281640 February 16, 2023 1 / 16 PLOS ONE the National Research Foundation (NRF), funded by the Korean government (MSIT) (2019M3E5D1A01068992, 2020R1C1C1004572). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Glycolytic reprogramming in CRS Conclusion This study suggests that inhibition of the glycolytic enzyme and HIF-1α in nasal fibroblasts regulates myofibroblast differentiation and ECM generation associated with nasal mucosa remodeling. Competing interests: The authors have declared that no competing interests exist. Introduction Chronic rhinosinusitis (CRS) is a chronic inflammatory disease that affects 5.5–10% of the population [1]. It is characterized by symptoms, including tenderness, headache, and nasal discharge, which impairs the quality of life of patients. Morphologically, CRS is classified as CRSsNP (without nasal polyps) and CRSwNP (with nasal polyps). However, it is currently classified into Th1, Th2 and Th17 type or eosinophilic, non-eosinophilic CRS based on cytokine expression and immune cells. Among them, some types of CRS strongly accompanied by tissue remodeling on mucosa tend to be recurrent and recalcitrant even after proper management, including medications and surgery [2]. Therefore, our research has focused on tissue remodeling of CRS. Tissue remodeling refers to the structural changes in damaged tissues due to chronic inflammation or injury, and this process involves continuous extracellular matrix (ECM) production and degradation [3]. Under pathological conditions, uncontrolled ECM production induces irreversible structural changes and leads to recalcitrant and recurrent CRS [4]. ECM accumulation that results from myofibroblasts differentiation and transforming growth factor (TGF)-β1 produced by immune cells and structural cells, is well known as a key activator of this process [5]. Glycolysis and mitochondrial respiration are the two major energy-yielding pathways. Metabolic activity in normal cells depends on mitochondrial oxidative phosphorylation (OXPHOS) to use glucose to generate ATP for energy [6]. However, cells undergoing phenotypic or functional changes have increased glycolysis used for biosynthesis and bioenergetic demand. This phenomenon is referred to as metabolic reprogramming because glycolysis is quicker than OXPHOS in ATP generation [7]. Glycolytic reprogramming in metabolic reprogramming is associated with chronic inflammatory and airway diseases, such as asthma and chronic obstructive pulmonary disease [8– 10]. Hypoxia inducible factor (HIF)-1α, a transcription factor, upregulates glycolytic enzymes (hexokinase 2 [HK2], phosphofructokinase-1 [PFK-1], and pyruvate kinase [PK]), which results in glycolytic reprogramming [11]. There is evident that HIF-1α is expressed in fibroblasts of nasal polyps, but it is not known whether this contributes to glycolytic reprogramming [12]. Based on the above background, we hypothesized that glycolytic reprogramming related to HIF-1α contributes to ECM accumulation in CRS. Thus, the purpose of this study was to determine whether glycolytic reprogramming by HIF-1α is involved in myofibroblast differentiation and ECM production in nasal fibroblasts. Materials and methods Reagents Human recombinant TGF-β1 was obtained from R&D systems (Minneapolis, MN, USA). The inhibitors, 2-Deoxy-D-glucose (2-DG) for HK2 inhibitor and 3-(3-pyridinyl)-1-(4-pyridinyl)2-propen-1-one (3-PO) for 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase (PFKFB) 3 PLOS ONE | https://doi.org/10.1371/journal.pone.0281640 February 16, 2023 2 / 16 PLOS ONE Glycolytic reprogramming in CRS Table 1. Clinic (...truncated)