Single-Cell Imaging Maps Inflammatory Cell Subsets to Pulmonary Arterial Hypertension Vasculopathy

Abstract

Experimental and clinical observations are advancing immunotherapies to clinical trial in pulmonary arterial hypertension (PAH). However, comprehensive mapping of the immune landscape in and around pulmonary arteries (PAs) in lung tissue is necessary to predict how immune cells might induce pulmonary vascular pathology. While more severe vascular pathology is reported in BMPR2 mutant patients classified as hereditary (H)PAH, the relationship to specific immune cell subsets has not been established. Emerging high throughput approaches such as multiplexed ion beam imaging by time-of-flight (MIBI-TOF) that labels antibodies with mass elements enables the study of multiple cells and states of activation in the same tissue section. Methods: We applied MIBI-TOF to compare PAs and surrounding tissue from unused donor lungs with PAH lungs that were diagnosed as idiopathic (IPAH) or HPAH. Measurements: We measured the number and proximity of immune cells to vascular lesions and to each other. Immune cell subsets that tracked with vascular pathology were interrogated ex vivo for stimulation of pulmonary arterial smooth muscle cell (PASMC) proliferation. Results: Extensive immune infiltration in I/HPAH was observed with intramural infiltration linked to PA occlusive changes. The presence of CD11c+DCs expressing SAMHD1, TIM-3 and IDO-1 within immune enriched microenvironments and neutrophils were associated with greater immune activation in HPAH. CD11c-DC3s (mo-DC-like cells) were linked to iNOS+ endothelial cells and proliferating SMCs. Co-culture studies reinforced a causal relationship between neutrophil extracellular vesicles and mo-DCs in mediating PASMC proliferation. Conclusions: These findings merit consideration in predicting and assessing the efficacy of immunotherapies for PAH.