Dynamic Shifts in Inflammation and Fibrotic Remodeling in Post-HSCT Bronchiolitis Obliterans: A Spatial Transcriptomics Study

Bronchiolitis obliterans (BO) is a severe complication following hematopoietic stem cell transplantation (HSCT), significantly impacting patients' prognosis and quality of life. Despite its clinical importance, the underlying pathophysiological mechanisms remain poorly understood.

We analyzed lung specimens from three patients who developed BO post-HSCT and subsequently underwent lung transplantation. All patients had acute lymphoblastic leukemia as their primary disease and developed BO 1-2 years after HSCT. Healthy lung regions from two cancer patients served as controls. Using NanoString GeoMx Digital Spatial Profiling, we performed transcriptional profiling with the Human Whole Transcriptome Atlas on 94 regions of interest (ROIs). BO lesions were classified based on our previously established staging system (Kuroi T et al. Int J Hematol. 2021) and analyzed for both inner and outer bronchiolar regions.

Linear mixed model analysis identified significant differential gene expression between BO and control groups. GO enrichment analysis highlighted biological processes such as phagocytosis, positive regulation of leukocyte activation, and cell activation, indicating a close association with immune cells, particularly macrophages, which are known to obstruct bronchioles in BO. Processes such as regulation of cell-cell adhesion and post-Golgi vesicle-mediated transport were also enriched, suggesting involvement in extracellular matrix remodeling and fibroblast activation. GSEA further identified significant processes, including inflammatory response, defense response, positive regulation of response to stimulus, and vesicle-mediated transport. These results suggest an interplay between immune response and tissue remodeling. Notably, changes in the expression of genes such as HES1, IGF2BP2, PTPRF, and ITGB2, which are involved in cell adhesion and cytoskeletal reorganization, were closely associated with fibroblast activation and macrophage infiltration. These findings parallel the fibrosis process observed in BO following lung transplantation, where macrophage infiltration and myofibroblast activation are critical. Given these insights, our focus shifted to the dynamics of macrophages and myofibroblasts, aiming to understand their roles in the progression from inflammation to tissue remodeling.

CD68, a pan-macrophage marker, showed higher expression in Early and Middle-outside stages (Inflammatory Phase) of BO, but significantly decreased in Middle-inside and Late stages (Remodeling Phase) as the disease progressed. Unsupervised clustering analysis revealed distinct gene expression profiles between these phases, suggesting a temporal shift in disease mechanisms as BO progresses from inner bronchioles outwards. Correlation analysis revealed significant associations between CD68 and JAK1/STAT1 expression, as well as genes associated with myofibroblast activity and extracellular matrix remodeling (e.g., ACTA2, TGFB1). These findings suggest a coordinated process of inflammation and remodeling in BO, potentially involving the JAK-STAT pathway.

Linear mixed model analysis revealed distinct gene expression patterns between Inflammatory and Remodeling Phases, characterized by inflammatory/proliferation-related genes (e.g., CXCL6, HLA-family) and tissue remodeling/protein synthesis and genes (e.g., ITGB1, RPL and RPS families), respectively. This transition aligns with changes in macrophage-related gene expression. Pathway analysis revealed enrichment of translation, ribosomal activity, and metabolic processes in the Remodeling Phase, indicating a dynamic shift in biological activities as BO progresses.

In conclusion, this study reveals a transition in BO pathology from inflammation to remodeling, with macrophages playing a central role. The coordinated changes in gene expression provide a comprehensive picture of BO progression. These insights into post-HSCT BO pathophysiology highlight potential therapeutic targets in macrophage function and tissue remodeling pathways, suggesting possibilities for stage-specific interventions in BO treatment.

Fujii:MSD: Honoraria; Gilead Sciences, Inc.: Honoraria; Asahi Kasei Corporation: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Janssen Pharmaceuticals, Inc.: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Daiichi Sankyo Co., Ltd.: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria. Sugimoto:GlaxoSmithKline K.K.: Research Funding; Janssen Pharmaceutical K.K.: Research Funding. Kondo:ONO Pharmaceutical Co., Ltd.: Research Funding. Kobayashi:Chugai: Honoraria; Sanofi: Honoraria. Asada:Novartis: Research Funding, Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Abbvie: Speakers Bureau; Meiji: Speakers Bureau; Kyowa KIRIN: Speakers Bureau; Astellas: Speakers Bureau; Asahi KASEI: Speakers Bureau; Otsuka: Speakers Bureau. Ennishi:Kyowa-Kirin: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Nipponshinyaku Pharmaceutical Co., Ltd.: Honoraria, Research Funding; AbbVie: Honoraria; Astrazeneca: Honoraria; Bristol-Myers Squibb: Honoraria; Eisai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Janssen Pharmaceutical K.K.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; SymBio Pharmaceuticals: Honoraria; DAIICHI SANKYO COMPANY, LIMITED: Honoraria; Illumina, Inc: Honoraria, Research Funding. Toyooka:Chugai Pharma: Honoraria, Research Funding; Daiichi Sankyo: Honoraria; Astellas Pharma: Honoraria; Boehringer Ingelheim: Honoraria; Taiho Pharmaceutical: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria; Bayer: Honoraria; Medtronic: Honoraria; Johnson & Johnson: Honoraria; Kyorin: Honoraria; Eli Lilly: Honoraria, Research Funding; Novartis: Honoraria; AstraZeneca: Honoraria, Research Funding; Merck & Co: Honoraria; AstraZeneca: Consultancy; Guardant Health: Consultancy; Eli Lilly: Research Funding; Taiho Pharmaceutical: Research Funding. Maeda:Japan Blood Products Organization: Research Funding; TEIJIN PHARMA LIMITED.: Research Funding; NIPPON KAYAKU CO., LTD.: Research Funding; Mallinckrodt Pharma K.K.: Research Funding; REGiMMUNE Co, Ltd.: Research Funding; Asahi Kasei Pharma Corporation: Honoraria, Research Funding; Astellas Pharma Inc.: Honoraria; AstraZeneca K.K.: Honoraria; Amgen K.K.: Honoraria; AbbVie GK: Honoraria; Eisai Co., Ltd: Honoraria; Viatris Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; ONO Pharmaceutical Co., Ltd.: Honoraria; KYORIN Pharmaceutical Co., Ltd.: Honoraria, Research Funding; KISSEI Pharmaceutical Co., Ltd.: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Gilead Sciences, Inc.: Honoraria; KONICA MINOLTA, Inc.: Honoraria; Sanofi K.K.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; JCR Pharmaceuticals Co., Ltd.: Honoraria; Celgene Corporation: Honoraria; CSL Behring K.K.: Honoraria; DAIICHI SANKYO COMPANY, LIMITED: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; TERUMO Corporation: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria; Bayer Yakuhin, Ltd.: Honoraria; Bristol-Myers Squibb K.K.: Honoraria; Novartis Pharma K.K.: Honoraria; Pfizer Japan Inc.: Honoraria; Pharma Essentia Corp.: Honoraria; Mundipharma K.K.: Honoraria; Human Life CORD Japan Inc.: Honoraria; Meiji Seika Pharma Co., Ltd.: Honoraria; Medical Review Co.,Ltd: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Yakult Honsha Co., Ltd.: Honoraria; Asahi Kasei Pharma Corporation: Consultancy; Meiji Seika Pharma Co., Ltd.: Consultancy; Medical Review Co.,Ltd: Consultancy; TAIHO Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.