Differential expression of viral entry protein neuropilin 1 (NRP1) and neuropilin 2 (NRP2) in fatal COVID-19

Coronavirus disease 2019 (COVID-19) is associated with hyperinflammation, endothelialitis, hypoxemia, and hypercoagulation, contributing to thrombosis in acute severe and long COVID. While ACE2 is the primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, its low expression in certain infected cell types suggests alternative co-receptors. Neuropilins (NRP1 and NRP2), widely expressed, have been proposed as co-factors for viral entry. We analyzed NRP1 and NRP2 expression in autopsy tissues from heart, lung, and hematolymphoid organs using immunohistochemistry (<i>n</i> = 20) and compared findings with public single-cell RNA sequencing (scRNAseq) data. Selected cases were further examined by spatial multiplex immunofluorescence (CODEX). <i>In vitro</i> binding of NRP1/NRP2 to SARS-CoV-2 spike fragments S1 and S1' was assessed by immunofluorescence microscopy. NRP1 was abundantly expressed in myocardial capillary endothelial cells (ECs) and macrophages in the heart and lung; NRP2 was found in alveolar macrophages and mast cells. scRNAseq re-analysis confirmed these <i>in situ</i> patterns. <i>In vitro</i>, NRP1 exclusively bound S1, while NRP2 bound both S1 and S1'. SARS-CoV-2 RNA was detected in neuropilin-positive, <i>ACE2</i>/<i>TMPRSS2</i>-negative vascular EC and mast cells. The detection of SARS-CoV-2 RNA in neuropilin-positive but <i>ACE2</i>/<i>TMPRSS2</i>-negative cell clusters supports that neuropilins are involved in systemic viral dissemination. NRP1 on vascular EC may contribute to angiogenesis, vascular damage, and microangiopathy, while NRP2 represents a potential immunomodulatory target to regulate macrophage activity, resolve inflammation, and potentially prevent the progression of pulmonary fibrosis and limit excessive mast cell activation in long COVID.IMPORTANCEThe well-known severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), exhibits low expression in key cell types implicated in coronavirus disease 2019 (COVID-19) pathology, such as endothelial cells and B cells, macrophages, and mast cells. In contrast, neuropilins, identified as co-receptors for SARS-CoV-2, are abundantly expressed in these cells under physiological conditions and may be involved in virus-host interactions. This study presents a detailed <i>in situ</i> analysis of Neuropilin 1 (NRP1) and Neuropilin 2 (NRP2) expression in fatal COVID-19 cases using immunohistochemistry and spatial multiplex immunofluorescence phenotyping, complemented by single cell RNA sequencing. Additionally, it demonstrates differential binding affinities of NRP1 and NRP2 to SARS-CoV-2 spike protein fragments S1 and S1' <i>in vitro</i>, suggesting distinct roles for these neuropilins in viral recognition. This study highlights the impact of the unique furin cleavage site in SARS-CoV-2, which may contribute to increased pathogenicity through its interaction with NRP1.