Keeya Sunata ^1,2 Jun Miyata ^1,2* Yusuke Kawashima ³ Ryo Konno ³ Masaki Ishikawa ³ Yoshinori Hasegawa ³ Ryuta Onozato ¹ Yo Otsu ¹ Emiko Matsuyama ¹ Hisashi Sasaki ⁴ Shinichi Okuzumi ¹ Takao Mochimaru ^1,5 Katsunori Masaki ¹ Hiroki Kabata ¹ Shotaro Chubachi ¹ Makoto Arita ^2,6,7,8 Koichi Fukunaga ¹

• ¹ School of Medicine, Keio University, Tokyo, Japan
• ² RIKEN Yokohama, Yokohama, Kanagawa, Japan
• ³ Department of Applied genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan
• ⁴ National Defense Medical College (Japan), Tokorozawa, Saitama, Japan
• ⁵ Tokyo Medical Center (NHO), Meguro-ku, Tokyo, Japan
• ⁶ Yokohama City University, Yokohama, Kanagawa, Japan
• ⁷ Faculty of Pharmacy, Keio University, Minato, Tokyo, Japan
• ⁸ Human Biology-Microbiome-Quantum Research Center (WPI-Bio2Q), Keio University, Tokyo, Japan

Elevated blood eosinophil levels in patients with chronic obstructive pulmonary disease (COPD) with or without asthma are linked to increased exacerbations and the effectiveness of inhaled corticosteroid treatment. This study aimed to delineate the inflammatory cellular properties of eosinophils in patients with asthma-COPD overlap (ACO) and eosinophilic COPD (eCOPD). Eosinophils were isolated from the peripheral blood of healthy volunteers, patients with non-eCOPD, and those with ACO/eCOPD. Multi-omics analysis involving transcriptomics, proteomics, and lipidomics was performed, followed by bioinformatic data analyses. In vitro experiments using eosinophils from healthy volunteers were conducted to investigate the molecular mechanisms underlying cellular alterations in eosinophils. Proteomics and transcriptomics analyses revealed cellular characteristics in overall COPD patients represented by viral infection (elevated expression of sterol regulatory element-binding protein-1) and inflammatory responses (elevated levels of IL1 receptor-like 1, Fc epsilon receptor Ig, and transmembrane protein 176B). Cholesterol metabolism enzymes were identified as ACO/eCOPD-related factors. Gene Ontology and pathway enrichment analyses demonstrated the key roles of antiviral responses, cholesterol metabolism, and inflammatory molecules-related signaling pathways in ACO/eCOPD. Lipidomics showed the impaired synthesis of cyclooxygenase-derived mediators including prostaglandin E2 (PGE2) in ACO/eCOPD. In vitro assessment confirmed that IL-33 or TNF-α stimulation combined with IL-5 and IFN-γ stimulation induced cellular signatures in eosinophils in ACO/eCOPD. Atorvastatin, dexamethasone, and PGE2 differentially modulated these inflammatory changes. ACO/eCOPD is associated with viral infection and an inflammatory milieu. Therapeutic strategies using statins and inhaled corticosteroids are recommended to control these pathogenic changes.