Unraveling the molecular architecture of autoimmune thyroid diseases at spatial resolution

Autoimmune thyroid diseases (AITD) such as Graves' disease (GD) or Hashimoto's thyroiditis (HT) are organ-specific diseases related to complex interactions between distinct components of the thyroid ecosystem. We applied spatial transcriptomics (ST) to explore the molecular architecture and heterogeneity of different cells present in the thyroid tissue, including thyroid follicular cells (TFCs) and stromal cells such as fibroblasts and endothelial cells. We identified damaged antigen-presenting TFCs that had an upregulated CD74/MIF axis in thyroids from AITD patients. Furthermore, we discerned two main fibroblast subpopulations in the connective tissue including ADIRF+ myofibroblasts, mainly upregulated in GD, and inflammatory-associated fibroblasts (IAFs), mainly overexpressed in HT patients. We also demonstrated an increase of fenestrated PLVAP+ vessels in AITD, especially in GD. Our data unveil novel stromal and thyroid epithelial cell subpopulations that could play an essential role in the pathogenesis of AITD. Overall design: We used thyroid tissue samples from AITD lesions and histologically normal parenchyma adjacent to other thyroid lesions from patients who underwent thyroidectomy at the Surgical Department of the Hospital Universitario la Princesa. Thyroid tissue samples were immediately snap-frozen in liquid nitrogen cooled in isopentane and transferred to a -80 °C freezer. RNA was isolated from 40 fresh-frozen thyroid tissues (21 controls, 11 HT and 8 GD samples). Total RNA was isolated using miRNeasy Mini Kit (Qiagen). The quality and quantity of RNA was assessed in a 2100 Bioanalyzer using a RNA 6000 Nano kit (Agilent Technologies). Eight samples with RIN>7 were selected for Spatial Transcriptomics using the Visium spatial Gene expression Kits from 10X Genomics. Briefly, the Visium Spatial Tissue Optimization Slide & Reagent kit (10X Genomics) was used to optimize permeabilization conditions for thyroid tissue. Then, they were cryosectioned at 10µm onto 6.5 × 6.5 mm capture areas from Visium spatial slides and processed according to the manufacturer's instructions (10x Genomics).