key: cord-0764478-3gp0txql
authors: Temerozo, Jairo R.; Sacramento, Carolina Q.; Fintelman‐Rodrigues, Natalia; Pão, Camila R. R.; de Freitas, Caroline S.; Dias, Suelen Silva Gomes; Ferreira, André C.; Mattos, Mayara; Soares, Vinicius Cardoso; Teixeira, Lívia; Azevedo‐Quintanilha, Isaclaudia G.; Hottz, Eugenio D.; Kurtz, Pedro; Bozza, Fernando A.; Bozza, Patrícia T.; Souza, Thiago Moreno L.; Bou‐Habib, Dumith Chequer
title: VIP plasma levels associate with survival in severe COVID‐19 patients, correlating with protective effects in SARS‐CoV‐2‐infected cells
date: 2022-03-24
journal: J Leukoc Biol
DOI: 10.1002/jlb.5cova1121-626r
sha: 93df6efd6fb8ec103d2d77592d70a67652d70ee1
doc_id: 764478
cord_uid: 3gp0txql

Infection by SARS‐CoV‐2 may elicit uncontrolled and damaging inflammatory responses. Thus, it is critical to identify compounds able to inhibit virus replication and thwart the inflammatory reaction. Here, we show that the plasma levels of the immunoregulatory neuropeptide VIP are elevated in patients with severe COVID‐19, correlating with reduced inflammatory mediators and with survival on those patients. In vitro, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP), highly similar neuropeptides, decreased the SARS‐CoV‐2 RNA content in human monocytes and viral production in lung epithelial cells, also reducing cell death. Both neuropeptides inhibited the production of proinflammatory mediators in lung epithelial cells and in monocytes. VIP and PACAP prevented in monocytes the SARS‐CoV‐2‐induced activation of NF‐kB and SREBP1 and SREBP2, transcriptions factors involved in proinflammatory reactions and lipid metabolism, respectively. They also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF‐kB. Specific inhibition of NF‐kB and SREBP1/2 reproduced the anti‐inflammatory, antiviral, and cell death protection effects of VIP and PACAP. Our results support further clinical investigations of these neuropeptides against COVID‐19.

also promoted CREB activation, a transcription factor with antiapoptotic activity and negative regulator of NF-kB. Specific inhibition of NF-kB and SREBP1/2 reproduced the anti-inflammatory, antiviral, and cell death protection effects of VIP and PACAP.

Our results support further clinical investigations of these neuropeptides against COVID-19. During the inflammatory response to human pathogenic coronaviruses, circulating neutrophils and monocytes migrate and infiltrate the lungs 15, 16 and other organs, contributing to potentiate and perpetuate the inflammation and eventually exacerbating the tissue damage [17] [18] [19] . Previous studies showed that MERS-CoV-and SARS-CoV-infected macrophages produce high levels of proinflammatory cytokines and chemokines 20, 21 , and, more recently, that lung monocytes from severe pneumonia caused by SARS-CoV-2 are potent producers of TNF-α and IL-6, whose levels were increased in the serum of the same patients 7 . Also, we and other authors have found that SARS-CoV-2 induces inflammasome activation and cell death by pyroptosis in monocytes, either by experimental or natural infection, which are associated with lung inflammation and are risk factors in critically ill COVID-19 patients 13, 14 .

Thus, it is critical to identify agents able to prevent the infection anti-inflammatory and prohomeostatic activities, both neuropeptides have been considered as promising therapeutic agents for autoimmune disorders and chronic inflammatory illnesses [29] [30] [31] [32] . Therefore, based on the well-known properties of both neuropeptides to regulate inflammatory reactions, and on the dysregulated immune responses that affect COVID-19 patients, we investigated whether they could present protective roles during SARS-CoV-2 infection. We report here that VIP levels are elevated in the plasma of individuals with severe manifestations of COVID-19, which correlated with survival on critically ill patients. We also verified, in in vitro assays, that VIP and PACAP inhibit the production of proinflammatory mediators in SARS-CoV-2-infected monocytes and lung epithelial cells, and reduced viral production and cell death. 

We prospectively enrolled patients with severe or mild/asymptomatic participants are presented in Table 1 . Mild and severe COVID-19 patients presented differences regarding age and presence of comorbidities, such as obesity, cardiovascular diseases, and diabetes (Table 1) , which is consistent with previously reported patient cohorts 2,34-36 .

The SARS-CoV-2-negative control group included subjects of older age and chronic noncommunicable diseases, so it is matched with mild and critical COVID-19 patients, except for hypertension ( Table 1 ). All ICUadmitted patients received usual supportive care for severe COVID-19 and respiratory support with either noninvasive oxygen supplementation (n = 5) or mechanical ventilation (n = 19) (Table S1) Table S2 ). Age and frequency of comorbidities were not different between severe patients requiring mechanical ventilation or noninvasive oxygen supplementation neither between survivors and nonsurvivors (Tables S1 and S2).

Statistics were performed using GraphPad Prism software version 

From April to May 2020, we followed up 24 critically ill COVID-19 patients, at the median age of 53-year-old (Table 1) PACAP plasma levels, we did not find significant differences between the groups analyzed, inflammatory markers, viral load or with VIP levels (data not shown). The finding that nonsurvival of severe COVID-19 patients is associated with lower levels of circulating VIP, a molecule with prohomeostasis and anti-inflammatory activities 32, 37 , moreover pointing to an application as a prognostic marker, also implies to a therapeutical potential of VIP in COVID-19. In fact, VIP has been approved for 3 clinical trials against COVID-19 in intravenous 38 and inhaled 39, 40 formulations. Our initial clinical data prompted us to evaluate the Severe COVID-19 patients admitted to the ICU were subdivided between those requiring invasive mechanical ventilation or noninvasive O 2 supplementation (J) and according to the 28-day mortality outcome as survivors or non survivors (K). Linear regression (with the 95% confidence interval) and Spearman's correlation were calculated according to the distribution of the data. Dots represent: controls, grey; asymptomatic, blue; mild, brown; severe, red. The horizontal lines in the box plots represent the median, the box edges represent the interquartile ranges, and the whiskers indicate the minimal and maximal value in each group. *p ≤ 0.05; **p ≤ 0.01; ns, not significant 41, 42 ) and the viral replication in Calu-3 cells (a lineage of lung epithelial cells highly susceptible to SARS-CoV-2) exposed to VIP or PACAP. We found that VIP reduced the SARS-CoV-2 RNA content in monocytes, achieving up to 40% and 50% inhibition at 5 and 10 nM, respectively (Figure 2(A) ). PACAP similarly decreased the levels of viral RNA synthesis with 5 and 10 nM (up to 50% for both doses) (Figure 2(B) ). We next evaluated whether VIP and PACAP could also be able to restrict virus production in pulmonary cells, one of the major targets of SARS-CoV-2. We found that VIP reduced viral replication, reaching up to 50 and 40% inhibition with 1 and 5 nM, respectively (Figures 2(C) and S1(A) ). PACAP also diminished F I G U R E 3 Receptor contribution for the VIP-and PACAP-mediated inhibition of SARS-CoV-2 replication. Monocytes (A) and Calu-3 cells (B) were exposed (overnight) or not to antagonists for VPAC1/2 (atVPAC1/2; 100 nM for monocytes; 50 nM for Calu-3 cells) and PAC1 receptors (atPAC1; 150 nM for monocytes; 300 nM for Calu-3 cells) and to VIP or PACAP (antagonists were added 10 min before exposure to neuropeptides, and were kept in the culture medium). Also, monocytes (C) and Calu-3 cells (D) were treated (overnight) or not with agonists for VIP and PACAP receptors, as indicated, at different concentrations. Upon overnight exposure (A-D), culture medium was removed and then cells were infected with SARS-CoV-2 for 1 h, as described in Material and Methods. After infection, viral input was removed, and cells were washed, and then reexposed to receptor antagonists and VIP or PACAP (A and B) or to receptor agonists, as before (C and D). Viral RNA synthesis was evaluated by qPCR in monocytes 24 h after infection. In Calu-3 cells, supernatants were collected at 48 h after infection, and viral replication was evaluated by quantifying PFUs in Vero E6 plaque assays. Data in (A and C) are shown normalized to infected cells kept only with culture medium, and in (B and D) represents means ± SD of absolute values. *p ≤ 0.05; **p ≤ 0.01; ns, not significant; (A and B) n = 5; (C and D) n = 4. Each dot represents an independent assay with 3 replicates virus production up to 40 and 50% at concentrations equivalent to 10 and 50 nM (Figures 2(D) and S1(B)). In parallel, VIP and PACAP protected monocytes and Calu-3 cells from SARS-CoV-2-mediated cytopathic effect, as measured by LDH activity in supernatants (Figures 2(E) and 2(F)). Overall, these results show that SARS-CoV-2 infection and its associated cell death are decreased in cells exposed to VIP or PACAP.

The different optimal concentrations of VIP and PACAP to reduce SARS-CoV-2 replication in Calu-3 cells might be explained by the relative abundance of the neuropeptide receptors, since it has been shown that these cells express only VPAC1 43 . However, all 3 recep-tors are reported to be expressed in lungs, with some studies showing that VPAC1 levels are higher than VPAC2 or PAC1 (to which PACAP binds with higher affinity than to VPAC1 and VPAC2 22, [44] [45] [46] . With that in mind, we evaluated the role of the individual receptors in the neuropeptide-mediated inhibition of SARS-CoV-2 in both cells. To this end, monocytes and Calu-3 cells were exposed to VIP or PACAP in the presence of antagonists for VPAC1/2 receptors (VIP antagonist) and PAC1 receptor (PACAP 6-38), and then infected with SARS-CoV-2. In both cells, only when in the presence of VPAC1/2 receptor antagonist, the inhibition of SARS-CoV-2 infection by VIP and PACAP was reverted (Figures 3(A) and 3(B) ). We also performed assays in monocytes and Calu-3 cells treated with specific agonists to VPAC1, VPAC2, and PAC1 (Ala-VIP, Bay 55-9837, and Maxadilan, respectively). Activation of VPAC1 at 1, 5, and 10 nM, and of VPAC2 at 1 nM, significantly reduced the SARS-CoV-2 RNA content in monocytes (Figure 3(C) ). In Calu-3 cells, we verified that VPAC1 is the main receptor involved the inhibition of SARS-CoV-2 in Calu-3 cells, resembling the level of inhibition achieved with VIP, while exposure to a VPAC2 agonist resulted in a more modest inhibition ( Figure 3D ). The stimulus with a PAC1 agonist had no effect on viral replication (Figures 3(C) and 3(D) ). Likewise, we verified the cytopathic effect, as measured by LDH activity in supernatants, in both uninfected and infected cells exposed to VPAC1, VPAC2, and PAC1 agonists. Uninfected cells displayed no viability alteration in the presence of VIP and PACAP receptor agonists ( Figures S2(A) and S2(B) ). In infected cells, reduced SARS-CoV-2 infection-associated cell death was observed mainly upon VPAC1 activation ( Figures S2(C) and S2(D) ). As a whole, these findings suggest that VPAC1 receptor is the main contributor for the VIP-and PACAPmediated SARS-CoV-2 inhibition in monocytes and Calu-3 cells, and that activation of this receptor can lead to a diminished viral replication similar to that induced by the own neuropeptides.

Controlling the production of proinflammatory cytokines may be critical for reducing SARS-CoV-2 replication and limiting tissue damages, and based on evidence that VIP and PACAP can regulate the inflammatory response 27,47 , we next evaluated whether both neuropeptides could attenuate the production of proinflammatory mediators by SARS-CoV-2-infected monocytes or lung epithelial cells. As shown in (Figure 4(B) ), implying that VIP and PACAP may offer a critical protection to inflamed lungs affected by SARS-CoV-2 replication. Because proinflammatory cytokines may favor SARS-CoV-2 replication, which, in turn, can amplify the cellular synthesis of these mediators, these findings may support our assumption that VIP and PACAP offer tissue protection by inhibiting virus replication and regulating the boost of cytokine production.

Given that the transcription factor NF-kB is critically involved in the cellular production of inflammatory mediators 48 CREB and NF-kB share the CREB-binding protein/p300 (CBP/p300 protein) as a cofactor, and CREB activation results in the inhibition of NF-kB 53 . We found that activation of CREB was diminished in SARS-CoV-2-infected monocytes ( Figure 5(B) ), a result coherent with NF-kB activation in the same cells. Consistent with this finding, VIP and PACAP promoted CREB activation (as measured by increase of CREB phosphorylation) in those infected monocytes, a result matching the inhibition of NF-kB and the reduction of cellular production of proinflammatory cytokines. We also evaluated in SARS-CoV-2-infected monocytes the expression of the active form of SREBP-1 and SREBP-2, transcription factors that also interact with CBP/p300 54 and are crucial for the replication of several viruses, including coronaviruses [55] [56] [57] .

In fact, we and other authors reported that SARS-CoV-2 infection promotes the activation of SREBP, and that this activation is associated with enhanced viral replication 58,59 and COVID-19 disease severity 60 . 

In this work, we identified that the plasma levels of the neuropeptide VIP are elevated in patients with severe forms of COVID-19, to express only VPAC1 43 . However, lung tissues, while reported to express high levels of VPAC1, also express VPAC2 and PAC1 44, 46 , and, more specifically, VPAC2 mRNA was detected in airway epithelial, glandular, and immune cells of the lung 45 . Therefore, while the inhibition curve of SARS-CoV-2 by VIP and PACAP in Calu-3 cells points to different optimal doses than those obtained for monocytes, it is possible that in normal lung cells and tissue, VIP and PACAP could present a broader range of action in the inhibition of SARS-CoV-2. In fact, VIP and specific agonists for VPAC1 or VPAC2 have been proposed and tested for respiratory conditions, such as asthma [77] [78] [79] , pulmonary arterial hypertension 77, 80, 81 and chronic obstructive pulmonary disease (COPD) 77, 78, 82 , demonstrating that the anti-inflammatory actions of VIP and PACAP can be achieved in lung tissues. Future studies should define which of these receptors would preferentially be activated by specific agonists to restrain SARS-CoV-2 replication in lungs or other sites. Also, as G protein-coupled receptors ligands, it is expected that VIP and PACAP curve profiles be subject to variation due receptor density in cell membrane, receptor isoforms, and subtypes of associated G proteins. Those factors can influence the threshold and outcome of activation, and have been described for a variety of G protein-coupled receptors, including VIP/PACAP receptors [83] [84] [85] . Together with the possible differences of receptor expression and self-regulatory characteristics of GPCRs, a third regulation level of VIP and PACAP action on pulmonary cells can be achieved by the activity of proteases and peptidases, as lungs are described to express high levels of several of them in both normal and pathologic conditions [86] [87] [88] . Some of these peptidases could target VIP and PACAP, thus altering the ligand/receptor ratio and modulating the signaling pathways.

As up to now the availability of antivirals specific to SARS-CoV-2 is limited, and that the hyperinflammation may persist in COVID-19 patients even after the lowering of the viral load, the searching for compounds that target the aberrant production of proinflammatory cytokines and, simultaneously, the own viral replication, should be stimulated. Our present results showing that VIP and PACAP hold these 2 critical activities point these neuropeptides or their analogue molecules as potential therapeutic agents for COVID-19.

We thank the Hemotherapy Service from Hospital Clementino Fraga Thanks are due to Oswaldo Cruz Foundation/Fiocruz under the auspicious of Inova program. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

We pay tribute to Dr. Juliana de Meis, our cherished young colleague who died prematurely, leaving a significant legacy on the knowledge of immunopathogenesis of parasitic diseases. 

The authors declare no competing financial interests.

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