COVID-19 suppresses pain

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SARS-CoV-2, the virus that causes COVID-19, can relieve pain, according to a new study by University of Arizona Health Sciences researchers.

The finding may explain why nearly half of people who get COVID-19 experience few or no symptoms, even though they are able to spread the disease, according to the study’s corresponding author Rajesh Khanna, Ph.D., a professor in the College of Medicine—Tucson’s Department of Pharmacology.

“It made a lot of sense to me that perhaps the reason for the unrelenting spread of COVID-19 is that in the early stages, you’re walking around all fine as if nothing is wrong because your pain has been suppressed,” said Dr. Khanna.

“You have the virus, but you don’t feel bad because you pain is gone. If we can prove that this pain relief is what is causing COVID-19 to spread further, that’s of enormous value.”

The paper, “SARS-CoV-2 Spike protein co-opts VEGF-A/Neuropilin-1 receptor signalingto induce analgesia,” will be published in PAIN.

The U.S. Centers for Disease Control and Prevention released updated data Sept. 10 estimating 50% of COVID-19 transmission occurs prior to the onset of symptoms and 40% of COVID-19 infections are asymptomatic.

“This research raises the possibility that pain, as an early symptom of COVID-19, may be reduced by the SARS-CoV-2 spike protein as it silences the body’s pain signaling pathways,” said UArizona Health Sciences Senior Vice President Michael D. Dake, MD.

“University of Arizona Health Sciences researchers at the Comprehensive Pain and Addiction Center are leveraging this unique finding to explore a novel class of therapeutics for pain as we continue to seek new ways to address the opioid epidemic.”

Viruses infect host cells through protein receptors on cell membranes.

Early in the pandemic, scientists established that the SARS-CoV-2 spike protein uses the angiotensin-converting enzyme 2 (ACE2) receptor to enter the body.

But in June, two papers posted on the preprint server bioRxiv pointed to neuropilin-1 as a second receptor for SARS-CoV-2.

“That caught our eye because for the last 15 years my lab has been studying a complex of proteins and pathways that relate to pain processing that are downstream of neuropilin,” said Dr. Khanna, who is affiliated with the UArizona Health Sciences Comprehensive Pain and Addiction Center and is a member of the UArizona BIO5 Institute.

“So we stepped back and realized this could mean that maybe the spike protein is involved in some sort of pain processing.”

Many biological pathways signal the body to feel pain. One is through a protein named vascular endothelial growth factor-A (VEGF-A), which plays an essential role in blood vessel growth but also has been linked to diseases such as cancer, rheumatoid arthritis and, most recently, COVID-19.

Like a key in a lock, when VEGF-A binds to the receptor neuropilin, it initiates a cascade of events resulting in the hyperexcitability of neurons, which leads to pain. Dr. Khanna and his research team found that the SARS-CoV-2 spike protein binds to neuropilin in exactly the same location as VEGF-A.

With that knowledge, they performed a series of experiments in the laboratory and in rodent models to test their hypothesis that the SARS-CoV-2 spike protein acts on the VEGF-A/neuropilin pain pathway.

They used VEGF-A as a trigger to induce neuron excitability, which creates pain, then added the SARS-CoV-2 spike protein.

“The spike protein completely reversed the VEGF-induced pain signaling,” Dr. Khanna said. “It didn’t matter if we used very high doses of spike or extremely low doses—it reversed the pain completely.”

Dr. Khanna is teaming up with UArizona Health Sciences immunologists and virologists to continue research into the role of neuropilin in the spread of COVID-19.

In his lab, he will be examining neuropilin as a new target for non-opioid pain relief. During the study, Dr. Khanna tested existing small molecule neuropilin inhibitors developed to suppress tumor growth in certain cancers and found they provided the same pain relief as the SARS-CoV-2 spike protein when binding to neuropilin.

“We are moving forward with designing small molecules against neuropilin, particularly natural compounds, that could be important for pain relief,” Dr. Khanna said. “We have a pandemic, and we have an opioid epidemic. They’re colliding. Our findings have massive implications for both. SARS-CoV-2 is teaching us about viral spread, but COVID-19 has us also looking at neuropilin as a new non-opioid method to fight the opioid epidemic.”


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, a coronavirus disease that, as of August 24, has infected more than 23.5 million people and caused nearly 810,000 deaths worldwide [15].

Most patients infected with SARS-CoV-2 report mild to severe respiratory illness with symptoms such as fever, cough and shortness of breath [30]. On the other hand, a subset of patients who are diagnosed by a positive nuclei acids test but are either asymptomatic or minimally symptomatic [30].

Increasing evidence shows that asymptomatic individuals can spread the virus efficiently, and the emergence of these silent spreaders of SARS-CoV-2 has limited control of the pandemic [14; 41].

Pain is a rising concern in symptomatic patients, likely emanating from a direct attack of SARS-CoV-2 on cells and the “cytokine storm” unleashed by affected cells [51; 68]. Whether asymptomatic or minimally symptomatic individuals have reduced pain thresholds, or whether their pain is silenced is unknown, but either could contribute to increased disease transmission dynamics.

The surface expressed angiotensin converting enzyme 2 (ACE2) has been lionized as the main receptor for uptake of SARS-CoV-2 [22; 60; 64]. Emerging evidence points to a subset of ACE2 expressing sensory neurons [48] that synapse with spinal and brainstem CNS neurons to produce neurological effects, including headache and nerve pain [32; 34].

Curiously, ACE2 is not present in most neurons [48], despite increasing reports of neurological symptoms being common in COVID-19 patients [32]. Paradoxically, though the levels of ACE2 expression decline in aging [49], increased COVID-19 severity was noted in older patient populations, such as that of Italy’s [2], supporting the contention that ACE2 is not the sole gateway for entry of SARS-CoV-2 [1].

Two recent reports demonstrated that the SARS-CoV-2 Spike protein can bind to the b1b2 domain of the neuropilin-1 receptor (NRP-1). This interaction occurs through a polybasic amino acid sequence (682RRAR685), not conserved in SARS and MERS, termed the ‘C-end rule’ (CendR) motif, which significantly potentiates its entry into cells [6; 11].

Importantly, ‘omic’ analyses revealed a significant upregulation of NRP-1 in biological samples from COVID-19 patients compared to healthy controls [6]. Using vascular endothelial growth factor-A (VEGF-A), a physiological ligand for the b1b2 pocket in NRP-1, we interrogated whether the Spike protein, the major surface antigen of SARS-CoV-2, could block VEGF-A/NRP-1 signaling to affect pain behaviors.

Given parallels between the pro-nociceptive effects of VEGF-A in rodents [4; 58] and humans [23; 58] and clinical findings demonstrating increased VEGF-A levels in bronchial alveolar lavage fluid from COVID-19 patients [47] coupled with substantially lower levels in the sera of asymptomatic individuals compared to symptomatic patients [30], a secondary question was to test whether Spike protein could confer analgesia. We found that VEGF-A sensitizes nociceptor activity – a hallmark of neuropathic pain [59], which was blocked by the Spike protein and NRP-1 inhibitor EG00229 [26]. Furthermore, we identify a novel analgesic role for Spike protein, which is mirrored by NRP-1 inhibition.

Results

Ligand specific engagement of NRP-1 signaling induces nociceptor activity and pain

Initially, we assessed the involvement of Spike and NRP-1 in the VEGF-A/NRP-1 pathway. An interaction between Spike (S1 domain aa 16-685, containing the CendR motif 682RRAR685) and the extracellular portion of NRP-1 was confirmed by enzyme-linked immunosorbent assay (ELISA) (Fig. 1A).

We calculated an equilibrium constant of dissociation (Kd) for this interaction to be ∼166.2 nM (Fig. 1A). Next, we plated sensory neurons on multiwell microelectrode arrays (MEAs), an approach enabling multiplexed measurements of spontaneous, as well as stimulus-evoked extracellular action potentials from large populations of cells [9].

VEGF-A increased spontaneous firing of dorsal root ganglion (DRG) neurons, which was blocked by the S1 domain of the Spike protein and by the NRP-1 inhibitor EG00229 (Fig. 1A). In contrast, ligands VEGF-B (ligand for VEGFR1 – a co-receptor for NRP-1 [31]) and semaphorin 3A (Sema3A, ligand for plexin receptor – also a co-receptor for NRP-1) [13; 53]) did not affect the spontaneous firing of nociceptors (Fig. 1B, C).

The lack of effect of VEGF-B and Sema3A rule out a role for VEGF-R1 and plexin, respectively, thus implicating a novel ligand-, VEGF-A, and receptor-, NRP-1, specific pathway driving nociceptor firing (Fig. 1D).

Figure 1.Ligand specific engagement of 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). Data were normalized to wells with no antibody and background subtracted. The data was fit assuming a one site of binding mode and yielded a Kd of ∼166.2 nM. (B) Mean action potential firing rates (Hz, event per second) of cultured DRG sensory neurons incubated for 30 min with VEGF-B (3 nM), Sema3A (100 ng), 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 is shown as mean ± s.e.m. and was analyzed by non-parametric two-way analysis of variance (post hoc: Sidak). P values, versus 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 4deq [43]). 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 6fmc [46]) 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 3i97 [26]). (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.

As both VEGF-A and Spike protein share a common binding pocket on NRP-1 (Fig. 1C) [6; 11; 43], we asked if the Spike protein could block VEGF-A/NRP-1 signaling to affect pain behaviors. Consistent with previous reports [4; 58], we confirmed that VEGF-A is pro-nociceptive as intra-plantar injection of VEGF-A decreased both paw withdrawal thresholds (Fig. 2A, B and Table S1) and latencies to a thermal stimulus (Fig. 2C, D and Table S1) in male rats.

Similar results were obtained in female rats as well (Fig. 2E-H and Table S1). Preventing VEGF-A from binding to NRP-1 with the NRP-1 inhibitor EG00229 or Spike from activating VEGF-A/NRP-1 signaling, blunted the mechanical allodynia and thermal hyperalgesia induced by VEGF-A alone (Fig. 2 and Table S1). Neither Spike nor EG00229 alone had any effect on these behaviors (Fig. 2 and Table S1) in either sex. Together, these data provide functional evidence that VEGF-A/NRP-1 signaling promotes a pain-like phenotype by sensitizing nociceptor activity (Fig. 1D).

Figure 2.
VEGF-A promotes a pain-like phenotype that is blocked by Spike protein or NRP-1 inhibition in male and female rats.
Paw withdrawal thresholds (A, B – male and E, F – female) or latencies (C, D – male and G, H – female) for male and female naïve rats injected in the paw with VEGF-A (10 nM), Spike (100 nM), EG00229 (30 µM) or PBS (vehicle), alone or in combination (50 µl/rat; = 6-12). For clarity, statistical significance is not presented in the time course graphs, instead it is presented in Table S1. Panels B, F, D and H are the area under the curve for 0-24 hours. Data is shown as mean ± s.e.m. and was analyzed by non-parametric two-way analysis of variance where time was the within subject factor and treatment was the between subject factor (post hoc: Sidak), *p<0.05. Areas under the curve were compared by a one-way analysis of variance with Kruskal-Wallis post-hoc test. The experiments were analyzed by an investigator blinded to the treatment. For full statistical analyses see Table S1.

VEGF-A–mediated increases in DRG ion channel currents are normalized by disruption of VEGF-A/NRP-1 signaling
To gain insight into the mechanism by which VEGF-A contributed to increased nociceptor activity, we postulated that ion channels in DRGs may be affected, as these contribute to nociceptive plasticity [61].

Typical families of Na+ currents from small diameter DRG neurons are shown in Figure 3A. VEGF-A facilitated a 1.9–fold increase in total Na+ currents compared to vehicle (PBS)-treated DRGs, which was completely blocked by Spike protein (Fig. 3B, C). Spike protein alone did not affect Na+ currents (Fig. 3B, C and Table S1).

Since this decreased current could arise from changes in channel gating, we determined if activation and inactivation kinetics of DRG Na+ currents were affected. Half-maximal activation and inactivation (V1/2), as well as slope values (k) for activation and inactivation, were no different between the conditions tested (Fig. 3D, E and Tables S1, S2), except for an ∼8 mV hyperpolarizing shift in sodium channel inactivation induced by co-treatment of VEGF-A and EG00229 (Table S2).

Similar results were obtained for the NRP-1 inhibitor EG00229, which also inhibited the VEGF-A mediated increase in total Na+ currents (Fig. 3F-H and Table S1) but had no effect on the biophysical properties (Fig. 3I, J and Tables S1, S2).

VEGF-A–mediated increase in sodium currents is normalized by Spike protein or NRP-1 inhibition in DRG neurons.Representative sodium current traces (A, F) recorded from small-sized DRGs neurons, incubated for 30 min with the indicated treatments, in response to depolarization steps from –70 to +60 mV from a holding potential of –60 mV. Summary of current-voltage curves (B, G) and normalized peak (C, H) currents (pA/pF) from DRG neurons as indicated. Boltzmann fits for normalized conductance G/Gmax voltage relationship for voltage dependent activation (D, I) and inactivation (E, J) of the sensory neurons as indicated. Error bars indicate mean ± s.e.m. Half-maximal activation and inactivation (V1/2) and slope values (k) for activation and inactivation were not different between any of the conditions (p >0.9999, Kruskal-Wallis test with Dunn’s post hoc); values presented in Table S2. P values of comparisons between treatments are as indicated; for full statistical analyses see Table S1.

As calcium channels play multiple critical roles in the transmission and processing of pain-related information within the primary afferent pain pathway [61], we evaluated if they were affected. We focused on N-type (CaV2.2) channels as these mediate neurotransmitter release at afferent fiber synapses in the dorsal horn and are critical in the pain matrix [50].

VEGF-A facilitated a 1.8–fold increase in total Ca2+ currents compared to vehicle (PBS)-treated DRGs, which was completely blocked by Spike protein (Fig. 4A-C and Table S1). Spike protein alone did not affect Ca2+ currents (Fig. 4A-C). Additionally, we did not observe any changes in activation and inactivation kinetics between the conditions tested (Fig. 4D, E and Tables S1, S2).

Similar results were obtained for the NRP-1 inhibitor EG00229, which inhibited the VEGF-A mediated increase in N-type Ca2+ currents (Fig. 4F-H and Table S1) but had no effect on the biophysical properties (Fig. 4I, J and Tables S1, S2). These data implicate Spike protein and NRP-1 in Na+ and Ca2+ (CaV2.2) channels in VEGF-A/NRP-1 signaling.

VEGF-A–mediated increase in calcium currents is normalized by Spike protein or NRP-1 inhibition in DRG neurons.Representative calcium current (via N-type channels) traces (A, F) recorded from small-sized DRGs neurons, incubated for 30 min with the indicated treatments, in response to holding voltage of –60 mV with 200-ms voltage steps applied at 5-s intervals in +10 mV increments from –70 to +60 mV. Pharmacological isolation of N-type (CaV2.2) current was achieved with a cocktail of toxins/small molecules. Summary of current-voltage curves (B, G) and normalized peak (C, H) currents (pA/pF) from DRG neurons as indicated. Boltzmann fits for normalized conductance G/Gmax voltage relations for voltage dependent activation (D, I) and inactivation (E, J) of the sensory neurons as indicated. Error bars indicate mean ± s.e.m. Half-maximal activation and inactivation (V1/2) and slope values (k) for activation and inactivation were not different between any of the conditions (p >0.9999, Kruskal-Wallis test with Dunn’s post hoc); values presented in Table S2. P values of comparisons between treatments are as indicated; for full statistical analyses see Table S1.

VEGF-A enhances synaptic activity in the lumbar dorsal horn that is normalized by inhibition of NRP-1 signaling and Spike protein
The spinal cord is an integrator of sensory transmission where incoming nociceptive signals undergo convergence and modulation [57]. Spinal presynaptic neurotransmission relies on DRG neuron action potential firing and neurotransmitter release.

From these fundamental physiological principles, as well as the results described above, we were prompted to evaluate whether synaptic activity was affected in the lumbar dorsal horn.

The amplitudes of spontaneous excitatory postsynaptic currents (sEPSCs) of neurons in the substantia gelatinosa region of the lumbar dorsal horn were not affected by VEGF-A (Fig. 5A, B and Table S1). In contrast, VEGF-A application increased sEPSC frequency by ∼3.6–fold, which was reduced by ∼57% by inhibition of NRP-1 with EG00229 and ∼50% by Spike protein (Fig. 5A, C and Table S1).

Amplitude and inter-event interval cumulative distribution curves for sEPSCs are shown in Figure 5D, E. When compared to vehicle controls, VEGF-A, with or without NRP-1 inhibitor or Spike protein, had no effect on the cumulative amplitude distribution of the spontaneous EPSCs (Fig. 5D and Table S1) but changed the cumulative frequency distribution of spontaneous EPSCs with significantly longer inter-event intervals (Fig. 5E and Table S1). Together, these data suggest a presynaptic mechanism of action of Spike protein and NRP-1.

sEPSC frequency is reduced by pharmacological antagonism of NRP-1 by EG00229.
(A) Representative traces of spontaneous excitatory postsynaptic currents (sEPSC) from neurons from the substantia gelatinosa in the superficial dorsal horn (lamina I/II) treated for at least 30 min with the indicated conditions. Summary of amplitudes (B) and frequencies (C) of sEPSCs for all groups are shown. Cumulative distribution of the sEPSCs amplitude (D) and the inter-event interval (E) recorded from cells as indicated. Perfusion of 30 μM EG00229 decreased spontaneous excitatory synaptic transmission (A-E) in lumbar dorsal horn neurons. P values of comparisons between treatments are as indicated; for full statistical analyses see Table S1.

Spike protein and inhibition of NRP-1 confer anti-nociception in the spared nerve injury model (SNI) of chronic neuropathic pain
We used the spared nerve injury (SNI) model of neuropathic pain, chosen because it produces a reliable and consistent increase in pain sensitivity [12], to evaluate the potential of disruption of the VEGF-A/NRP-1 pathway to reverse nociception. VEGF-A triggers autophosphorylation of VEGFR2 at Y1175 [54], thereby serving as a proxy for activation of VEGF-A signaling. In rats with SNI, intrathecal application of Spike, decreased the phosphorylation of VEGFR2 (Y1175) on both the contralateral (non-injured) and ipsilateral (injured) side (Fig. 6A, B). This shows that Spike can inhibit VEGF-A signaling in a rat model of chronic neuropathic pain. SNI injury efficiently reduced paw withdrawal thresholds (PWTs) (mechanical allodynia, Fig. 6C and Table S1) 10 days post injury. Spinal administration of Spike protein significantly increased PWTs (Fig. 6C and Table S1), in a dose-dependent manner, for 5 hours. Analysis of the area under the curve (AUC) confirmed the dose-dependent reversal of mechanical allodynia (Fig. 6D and Table S1) compared to vehicle-treated injured animals. Similar results were seen with female rats injected with Spike (Fig. 6E, F). Finally, inhibition of NRP-1 signaling with EG00229 also reversed paw-withdrawal thresholds (Fig. 6G, H and Table S1).

SARS-CoV-2’s Spike protein and NRP-1 antagonism reverses SNI-induced mechanical allodynia.Spared nerve injury (SNI) elicited mechanical allodynia 10 days after surgery. (A) Representative immunoblots of NRP-1, total and pY1175 VEGF-R2 levels at pre-synaptic sites in rat spinal dorsal horn after SNI. Tissues were collected 3 hours (i.e., at peak of anti-allodynia) following intrathecal injection of the receptor binding domain of the Spike protein (2.14 μg/5μl). (B) Bar graph with scatter plots showing the quantification of n= 4 samples as in A (*p<0.05, Kruskal-Wallis test). Paw withdrawal thresholds for SNI rats (male) administered saline (vehicle) or four doses of the receptor binding domain of the Spike protein (0.00214 to 2.14 μg/5μl) intrathecally (i.t.); n = 6-12) (C), 2.14 μg/5μl dose of Spike in female rats, or the NRP-1 inhibitor EG00229 in male rats (2 μg/5μl; n = 6) (E). (D, F, H) Summary of data shown in panels C, E, and G plotted as area under the curve (AUC) for 0-5 hours. P values, versus control, are indicated. Data is shown as mean ± s.e.m. and was analyzed by non-parametric two-way analysis of variance where time was the within subject factor and treatment was the between subject factor (post hoc: Sidak). AUCs were compared by Mann-Whitney test. The experiments were analyzed by an investigator blinded to the treatment. P values of comparisons between treatments are as indicated; for full statistical analyses see Table S1.

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