Fig 2: Ligand-specific engagement of neuropilin-1 receptor (NRP-1) signaling induces nociceptor activity, promoting a pain-like phenotype. (A) Graph showing normalized NRP-1 binding to increasing concentrations of recombinant spike protein (n = 8 replicates per concentration). The data were fit assuming a one site binding mode and yielded a Kd of ∼166.2 nM. (B) Mean action potential firing rates (Hz, events per second) of cultured DRG sensory neurons incubated for 30 minutes with VEGF-B (1 nM), Sema3A (3 nM), VEGF-A (1 nM), VEGF-A plus spike (100 nM), or VEGF-A plus NRP-1 inhibitor EG00229 (30 μM).26 Of the ligands tested, only VEGF-A, acting on VEGFR2, is a ligand for NRP-1 that triggers an increase in spontaneous firing of nociceptors. Data are shown as mean ± SEM and were analyzed by nonparametric two-way analysis of variance (post hoc: Sidak). P values, vs control (PBS) or VEGF-A, are indicated. EG00229 is an NRP-1 inhibitor (PDB 3i97). (C) Top left: VEGF-A heparin binding domain (gray cartoon with R323 in sticks) in complex with the NRP-1 b1 domain (white surface with binding site in red; PDB 4deq43). Top right: Peptide from C-terminus of furin cleaved SARS-CoV-2 spike protein 681-PRRAR-685 (blue sticks) docked to NRP1-b1 domain (white surface with binding site in red; PDB 6fmc46) using Glide (Schrödinger). Additional spike residues 678-TNS-680 modeled for illustration purposes only (blue cartoon). Bottom: Compound EG00229 (cyan sticks) in complex with NRP-1 b1 domain (white surface with binding site in red; PDB 3i9726). (D) Schematic illustration of the hypothesis that SARS-CoV-2 spike protein binding to NRP-1 b1b2 domain triggers an intracellular cascade that increases sodium and calcium channel activity to increase nociceptor activity culminating in enhanced pain. DRG, dorsal root ganglia; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; VEGF, vascular endothelial growth factor. Panel D of this figure was created with BioRender.com.

Fig 3: Immune resolution is promoted by Sema3A via selective induction of a phenotypic switch in pro-inflammatory macrophages towards resolution-phase-macrophages. Bone marrow derived macrophages were isolated and polarized for 24 h towards M1 with and without the presence of Sema3A. a–c The expression of iNOS, IL-10 and YM1 increased significantly in response to stimulation with Sema3A (*p < 0.05, ***p < 0.001, n = 3). Interestingly, the addition of Sema3A resulted in a marked induction of COX2 transcript levels (d) and protein levels (e) (***p < 0.001, n = 3). Clearance of apoptotic cells by means of efferocytosis was assessed in vitro. Fluorescently labeled apoptotic Jurkat-cells were fed to M2 and M1 with and without the presence of Sema3A. As expected, M2 were significantly better at clearing apoptotic Jurkat-cells than M1 (f, g). Remarkably, addition of Sema3A to M1 resulted in a significant increase in the percentage of efferocytotic cells (f, g) (**p < 0.01, n = 3). All experiments were repeated at least twice

Fig 4: Sema3A regulates inflammation during infarct healing. a Coronary artery occlusion in Sema3A WT and HZ mice did not result in a difference in mortality. b, c Despite comparable infarct sizes between both groups, Sema3A HZ mice showed worse cardiac function with decreased fractional shortening (d, *p < 0.05, n ≥ 13). The number of recruited immune cells was determined by immuno-histochemical staining for both the infarct and the border zone, 3 and 14 days after MI. e Sema3A HZ had more leukocytes in the border zone 3 days after ischemic injury as compared to WT (**p < 0.01, n ≥ 4). f Fourteen days after infarction the increased presence of leukocytes in Sema3A HZ mice was also apparent in the infarct area (***p < 0.001, n ≥ 9). All experiments were repeated at least twice

Fig 5: Sema3A expression increases during infarct healing and is present on macrophages. a, b Increased expression of Sema3A and Cx3CR1 following myocardial infarction in circulating monocytes from patients at day 3 and significant increases at day 30 post admission (p = 0.001 and 0.04, n = 11). c Sema3A gene expression increased significantly in the border zone 3 days after myocardial infarction in the murine model (*p < 0.05, **p < 0.01, n ≥ 4). Fourteen days after ischemic injury, the expression also increased in the infarcted area (***p < 0.001, n ≥ 4). d The expression of Sema3A in both the infarct and border zone was confirmed by immunohistochemistry. Where Sema3A expression predominated in the border zone 3 days after MI, 14 days after MI the increased expression in the infarct became more apparent (e). RNA expression decreased to baseline levels in the infarct border zone at 14 days post-MI. Counterintuitively, immuno-histochemical staining still showed substantial Sema3A presence at this time. This apparent discrepancy might be indicative of the slower rate of protein degradation. Immunofluorescence further revealed Sema3A location on leukocytes and macrophages (f). Scale bar 50 µm