key: cord-1007117-79fdqzfm
authors: Jakwerth, Constanze A.; Feuerherd, Martin; Guerth, Ferdinand M.; Oelsner, Madlen; Schellhammer, Linda; Giglberger, Johanna; Pechtold, Lisa; Jerin, Claudia; Kugler, Luisa; Mogler, Carolin; Haller, Bernhard; Erb, Anna; Wollenberg, Barbara; Spinner, Christoph D.; Buch, Thorsten; Protzer, Ulrike; Schmidt-Weber, Carsten B.; Zissler, Ulrich M.; Chaker, Adam M.
title: Early reduction of SARS-CoV-2-replication in bronchial epithelium by kinin B(2) receptor antagonism
date: 2022-03-05
journal: J Mol Med (Berl)
DOI: 10.1007/s00109-022-02182-7
sha: 13f7d109263bcc9f4c5a8f19a7b3b9601ef52b2a
doc_id: 1007117
cord_uid: 79fdqzfm

ABSTRACT: SARS-CoV-2 has evolved to enter the host via the ACE2 receptor which is part of the kinin-kallikrein pathway. This complex pathway is only poorly understood in context of immune regulation but critical to control infection. This study examines SARS-CoV-2-infection and epithelial mechanisms of the kinin-kallikrein-system at the kinin B(2) receptor level in SARS-CoV-2-infection that is of direct translational relevance. From acute SARS-CoV-2-positive study participants and -negative controls, transcriptomes of nasal curettages were analyzed. Primary airway epithelial cells (NHBEs) were infected with SARS-CoV-2 and treated with the approved B(2)R-antagonist icatibant. SARS-CoV-2 RNA RT-qPCR, cytotoxicity assays, plaque assays, and transcriptome analyses were performed. The treatment effect was further studied in a murine airway inflammation model in vivo. Here, we report a broad and strong upregulation of kallikreins and the kinin B(2) receptor (B(2)R) in the nasal mucosa of acutely symptomatic SARS-CoV-2-positive study participants. A B(2)R-antagonist impeded SARS-CoV-2 replication and spread in NHBEs, as determined in plaque assays on Vero-E6 cells. B(2)R-antagonism reduced the expression of SARS-CoV-2 entry receptor ACE2, G protein–coupled receptor signaling, and ion transport in vitro and in a murine airway inflammation in vivo model. In summary, this study provides evidence that treatment with B(2)R-antagonists protects airway epithelial cells from SARS-CoV-2 by inhibiting its replication and spread, through the reduction of ACE2 levels and the interference with several cellular signaling processes. Future clinical studies need to shed light on the airway protection potential of approved B(2)R-antagonists, like icatibant, in the treatment of early-stage COVID-19. GRAPHICAL ABSTRACT: [Image: see text] KEY MESSAGES: Induction of kinin B(2) receptor in the nose of SARS-CoV-2-positive patients. Treatment with B(2)R-antagonist protects airway epithelial cells from SARS-CoV-2. B(2)R-antagonist reduces ACE2 levels in vivo and ex vivo. Protection by B(2)R-antagonist is mediated by inhibiting viral replication and spread. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00109-022-02182-7.

SARS-CoV-2 vaccines have been approved worldwide since the end of 2020 and are starting to show their protective effects in public health [1, 2] . Even with vaccines at hand, an important medical need for therapeutic approaches for COVID-19 remains: immunocompromised individuals may not mount a sufficient immune response after vaccination and escape variants, such as the currently spreading SARS-CoV-2 variant Omicron [3] , may breach protection afforded by the vaccines [4] [5] [6] [7] .

Key factors for SARS-CoV-2 cell entry are two cell surface molecules, angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease (TMPRSS)2 [8] . TMPRSS2 cleaves the coronaviral spike protein and primes it for cell fusion, while ACE2 enables the virus particle to enter the cell by binding of its spike protein [9, 10] . The latter acts as central component in its function as terminal carboxypeptidase in the counter-regulatory axis of the reninangiotensin-system (RAS) and the contact-activation-system (CAS) [8, 11] , which initiates blood coagulation and can additionally activate the kinin-kallikrein-system (KKS) [12] . In its role in the RAS, ACE2 has anti-vasoconstrictive and anti-inflammatory effects by hydrolyzing the vasoconstrictive and tissue-damaging angiotensin II, which contributes to airway remodeling and fibrosis [13, 14] , to angiotensin (1-7) [15] . In its role in the KKS, ACE2 further hydrolyzes vasoactive peptides such as des-Arg9-bradykinin (DABK), which activates the pro-inflammatory axis of the KKS [16] via the inducible kinin B 1 receptor (BDKRB1;B 1 receptor;B 1 R) [17] . While DABK is the ligand of B 1 R, bradykinin, the end product of the KKS-cascade, activates the constitutively expressed kinin B 2 receptor (BDKRB2;B 2 receptor;B 2 R) [18] . Through this mechanism, bradykinin mediates its pro-inflammatory effects by eliciting a variety of responses, including vasodilation and edema, via the G protein-triggered phosphatidylinositol-calcium second messenger-system [19] [20] [21] [22] [23] . The fact that SARS-CoV-2 utilizes ACE2 to enter airway cells along with the fact that ACE2 is a multifunctional enzyme that counter-regulates the ACEdriven mechanisms of the RAS and balances the KKS may therefore explain the serious course of COVID-19, not only in the lungs but systemically [24, 25] .

Recent publications suggest that the KKS could play a role in COVID-19. KKS comes into play particularly in connection with the high prevalence of thromboembolic events in severely ill COVID-19 patients [7, 17, [26] [27] [28] . A recent study on a cohort of 66 COVID-19 patients admitted to the intensive care unit showed that the KKS was strongly activated, which was reflected in the consumption of factor XII (F12), pre-kallikrein (KLKB1), and high-molecular-weightkininogen (HMWK; KNG1) [26] . When activated, plasmakallikrein (KLKB1) releases kinins from HMWK (KNG1) in the peripheral blood. In tissues, however, the functional real tissue kallikrein (KLK1) generates bradykinin and kallidin [29] , but by cleavage of low-molecular-weight-kininogen [30] , which is an additional splice product of the KNG1 gene [31, 32] . It has further been hypothesized that kinindependent "local lung angioedema" involving B 1 R and B 2 R is an important characteristic of COVID-19 [33] [34] [35] [36] . This study examines the potential of an intervention in the KKS at the kinin receptor level in SARS-CoV-2-infection with translational relevance and reveals an antiviral and protective effect of B 2 R-antagonism on human bronchial epithelium.

Nasal brushings were performed as part of a larger healthcare professional observational cohort study, which was approved by the Ethics Commission of the Technical University of Munich (AZ 175/20 s) during the first COVID-19 wave in Germany in 2020. Nasal brushings were obtained from 7 healthy healthcare professionals and 4 healthcare professionals with new onset of mild to moderate respiratory symptoms and within 2 days of newly confirmed SARS-CoV-2 diagnosis. No vaccine or specific treatment was available at the time of sampling. RNA was extracted from these nasal brushings and subjected to whole-genome transcriptome analysis (see Supp.Info.). All participants gave written informed consent prior to participation (Table 1 ).

Mice received murine IL-12Fc (1 μg protein in 50μL PBS) or PBS control intranasally [37] . Intranasal application was performed under isoflurane anesthesia in two steps of 25μL per nostril. Forty-eight hours later, the mice received a single subcutaneous injection of icatibant (2 nmol per 10 g body weight; HOE-140 ((icatibant), H157, SLBX4410, Sigma) or PBS control. The experiment was terminated by CO 2 asphyxiation 6 h or 24 h after injection of icatibant. The experiment was carried out twice. Organs were snap frozen for protein extraction. Experiments were pre-registered at www. anima lstud yregi stry. org (study title "Effect of drug on ACE2 levels in mice"; https:// doi. org/ 10. 17590/ asr. 00002 25). Mice enrolled in the experiment were 6-8 weeks old, from either C57BL/6 J, BALB/c, or C3H HeN strains. Both sexes were included for each strain and means of each mouse type (strain/sex) are depicted as single values in Fig. 2A : circle:female; triangle:male. Black:C57BL/6, midgrey:C3H HeN, light gray:BALB/c strain. Experiments were performed and analyzed in a randomized and blinded fashion. Animals were obtained from Janvier Labs (Le Genest-Saint-Isle, France) and housed 5 per cage and sex in individually ventilated cages at Laboratory Animal Service Center of the University of Zurich in Schlieren (Schlieren, Zurich, Switzerland). The animal vivarium was a specificpathogen-free (SPF) holding room, that was temperatureand humidity-controlled (21 ± 3 °C, 50 ± 10%), with a 12-h light/dark cycle. All animals had ad libitum access to the same food and water throughout the entire study. All procedures described in this study had previously been approved by the Cantonal Veterinarian's Office of Zurich, Switzerland (License ZH096/20), and every effort was made to minimize the number of animals used and their suffering.

Additional methods are provided in the supporting information.

ACE2 is the central viral entry receptor for SARS-CoV-2 on human epithelial cells of the respiratory tract [8] . Recent studies showed that this receptor and its co-receptors are not only expressed in the lower airways, and thus on alveolar epithelial cells type-I and -II, but are also present in the upper airways, but predominantly in the nasal mucosa [38] .

To investigate local effects of the acute SARS-CoV-2infection on the nasal epithelium, we analyzed the transcriptome of nasal curettages from symptomatic study participants, who tested acutely positive for SARS-CoV-2 (n = 4), and from SARS-CoV-2-negative study participants (n = 7). In a transcriptome analysis, the most strongly induced genes encoding secreted factors included many members of the kallikrein family (Fig. 1A , Table S1), in particular the kallikreins KLK5, KLK9, and KLK12 (Fig. 1B, Table S2 ). Next, we focused on the central factors of the tissue-KKS as stated above. Two-thirds of the genes were upregulated including the precursor of bradykinin LMWK (KNG1), true tissue kallikrein (KLK1), responsible of hydrolyzing LMWK to kallidin/bradykinin, and further the receptor for bradykinin, B 2 R (BDKRB2), which was significantly increased (Fig. 1C , Table S3 ). Since the two plasma factors factor XII (F12) and pre-kallikrein (KLKB1) are mainly processed and act in the plasma-KKS, it was expected that these factors are not differentially expressed in the nasal mucosa. The induction of KNG1, KLK1, and BDKRB2 in primary nasal samples of SARS-CoV-2-positive study participants is evidence for an autocrine bradykinin effect via B 2 R that is triggered locally during COVID-19 disease.

This finding prompted us to investigate selective kinin B 2 receptor antagonism in connection with SARS-CoV-2-infection. We therefore hypothesized that a B 2 R-antagonist like icatibant, an approved compound for the treatment of hereditary angioedema [39] , counter-regulates the effects of bradykinin during a SARS-CoV-2-infection and thereby has a protective effect on the integrity of the airway mucosa. To circumvent limitations of cell lines like Vero-E6, A549, or Calu-3 cells that are intrinsically impaired to form an interferon response upon viral infection [40] , we infected primary human NHBEs with SARS-CoV-2.

To examine the effects of SARS-CoV-2-infection and B 2 R-antagonist treatment on the microscopic integrity of the airway epithelium, 3D-air-liquid interphase organoid cultures were differentiated from primary NHBEs (Supp.Info). After complete differentiation, epithelia were pre-treated from the basal side with the approved B 2 R-antagonist icatibant, and subsequently infected with SARS-CoV-2 from the apical side. The cultures pre-treated with B 2 R-antagonist showed less virus-induced balloon-like structures compared to untreated cultures. The epithelial layers remained qualitatively more intact, which indicates a protective effect of the B 2 R-antagonist for the bronchial epithelium (Fig. 1D ). This finding was further strengthened by cytotoxicity assays: the B 2 R-antagonist had no toxic effects on NHBEs even at high doses determined by lactate dehydrogenase (LDH) release, but rather exhibited a cellprotecting effect in uninfected cells (Fig. 1E ) and during SARS-CoV-2-infection ( Fig. 1F ). Next, the supernatants of pre-treated, infected primary NHBEs were collected and titrated onto fresh Vero-E6 cell cultures and plaque assays were performed. Strikingly, we found that in vitro treatment of NHBEs with B 2 R-antagonist prior to infection reduced the number of plaque-forming units (PFU) in a plaque assay by 87% (Fig. 1G ). The levels of total SARS-CoV-2-RNA in cells that had been pre-treated with the B 2 R-antagonist decreased by 52% compared to untreated infected NHBEs (Fig. 1H ). With regard to the virus entry process, ACE2 was reduced by pre-treatment with B 2 R-antagonist at the mRNA level ( Fig. 1I ), but just in trend at the protein level (Fig. 1J ). However, ACE2 protein levels were significantly reduced upon SARS-CoV-2-infection. The membrane-standing protease TMPRSS2 cleaves the spike protein for SARS-CoV-2 and primes it for optimized binding to its entry receptor ACE2. In contrast to ACE2, TMPRSS2 transcript levels were significantly increased in infected compared to uninfected NHBEs but were not affected by B 2 R-antagonist pre-treatment (Fig. S2A ). Further experiments on the B 2 R-antagonist effect on the SARS-CoV-2-infection of NHBE showed that pre-treatment with B 2 R-antagonist significantly reduced infection-mediated cytotoxicity measured by LDH release (Fig. 1F ).

The finding that B 2 R-antagonism leads to a downregulation of ACE2 protein levels in lung epithelial cells was confirmed in vivo in a murine airway inflammation model. To mirror COVID-19 pathogenesis, mice were treated with IL-12, which mimics virus-induced airway inflammation via activation of the IL-12/IFN-γ-axis [37, 41] . Specifically, mice received intranasal murine IL-12Fc, to generate a pro-inflammatory state in the lungs. The experiment was designed in two blocks of 24 sex-matched mice from three different strains per group, to rule out any confounding genetic effect. After 48 h, mice were injected subcutaneously (s.c.) with the B 2 R-antagonist and the experiment was terminated 6 h or 24 h later to analyze ACE2 protein levels in the lungs. IL-12Fc pre-treated mice, which were then further treated with the B 2 R-antagonist on day 2, showed reduced ACE2 protein levels in the lungs after 6 h compared to control mice, which were only treated with PBS on day 2 ( Fig. 2A) . This effect decreased after 24 h. Anticipating treatment of SARS-CoV-2-infected study participants with the B 2 R-antagonist icatibant, NHBEs were first infected with SARS-CoV-2 and then treated with the B 2 R-antagonist 6 h after infection. Confirming the results of pre-treatment, post-infection treatment with the B 2 R-antagonist also attenuated the cytopathic effect of SARS-CoV-2 ( Fig. 2B ) and reduced the number of PFU in a plaque assay on Vero-E6 cells by 84% (Fig. 2C) .

We also aimed to reflect repeated dosage [42] during Fig. 1 Induction of kallikreins and kinin receptor B 2 in the nasal mucosa of acutely positive COVID-19 study participants. A Volcano plot of significantly differentially regulated genes (DEGs = differentially expressed genes) in nasal curettages of study participants that were acute positive for SARS-CoV-2 compared to healthy individuals (negative) using human miR microarray technology. Highlighted genes have a fold change (FC) ≥ 10 with P < 0.05; genes in red are upregulated; genes in blue are downregulated. B Heat map of gene expression analysis of kallikrein genes and C of genes of the kininkallikrein-system (KKS) in nasal curettages comparing acute SARS-CoV-2-positive study participants to healthy controls. All entities are shown. Asterisks indicate significantly regulated genes (P < 0.05) in SARS-CoV-2-infected NHBEs compared to medium. Color code indicates Log2-fold change from low (blue) through 0 (white) to high (red). Duplicate gene names indicate the abundance of two or more isoforms of the same gene in the analysis. D 3D-air-liquid interphase cultures from NHBEs were pre-treated for 24 h with/without 1 nM B 2 R-antagonist from the basal side and subsequently infected with SARS-CoV-2 for 48 h from the apical side. E Lactate dehydrogenase (LDH) cytotoxicity assay using the LDH Cytotoxicity Detection Kit PLUS studying the effect of increasing doses of the B 2 R-antagonist after 48 h in primary NHBEs from 4 donors. Results are depicted as mean ± s.e.m. Statistical tests compared each dose of B 2 R-antagonist with 0 nM B 2 R-antagonist. F Cytotoxicity assay determining LDH release into the supernatants of cultures of SARS-CoV-2-infected NHBEs from 12 donors that were pre-treated for 24 h with/without 1 nM B 2 R-antagonist. G Quantification of infectious particles in the supernatants of SARS-CoV-2-infected NHBEs from 10 donors that were pre-treated with/without 1 nM B 2 R-antagonist for 24 h. Supernatants were titrated on Vero-E6 cells and plaque assay was quantified 24 h later. Results are depicted as plaque-forming units (PFU) per milliliter. H qPCR analysis of total SARS-CoV-2 RNA (viral genome and transcripts, which all contain the N1 sequence region) normalized to human ACTB of SARS-CoV-2-infected primary NHBE after 24 h of pre-treatment with/without 1 nM B 2 R-antagonist followed by 24 h of SARS-CoV-2 inoculation. For Fig. 1E , F, and H, statistical tests compared SARS-CoV-2-infected versus uninfected samples or B 2 R-antagonist-treated versus untreated samples. I Analysis of human ACE2 gene expression using qPCR (n = 10) and J of human ACE2 protein levels analyzed by ELISA from cell lysates (n = 6) after 24 h of pre-treatment of NHBEs with/without 1 nM B 2 R-antagonist, followed by SARS-CoV-2 inoculation for 24 h ◂ on average, respectively. Genomic viral RNA was detected using RT-qPCR against the sequence of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP), which is only found in virions and during the viral replication. On the other hand, total viral RNA was detected with qPCR targeting a sequence of the SARS-CoV-2 N gene that is present in the viral genome and also in every SARS-CoV-2 protein-encoding transcript. Both, total SARS-CoV-2 RNA and genomic viral RNA levels were reduced upon treatment with the B 2 R-antagonist (Fig. 2D-I) .

Severe cases of COVID-19 develop cytokine storms [43] [44] [45] [50] , though these treatments may interfere with or alter the antiviral immune response. We therefore compared the effect of B 2 R-antagonism on gene expression of SARS-CoV-2-infected bronchial epithelium with the effect of hydrocortisone. While the B 2 R-antagonist mainly suppressed epithelial gene expression during infection, the effects of hydrocortisone on gene induction and gene repression were comparable (Fig. 3A, Tables S4-5 ). This finding matches previous reports [51] .

With regard to cell-intrinsic antiviral immunity, differentially expressed genes (DEGs) in NHBEs induced by SARS-CoV-2-infection included type-I and -III interferons and IFN-inducible, antiviral APOBEC genes (Fig. S1E , Table S6 ). SARS-CoV-2-infection particularly induced antiviral cytidine deaminases APOBEC3A and B, which we previously described to be induced by type-I interferons in the treatment of hepatitis B-virus-infection [52] . APOBEC3C mRNA levels, however, were decreased in SARS-CoV-2-infected NHBE, which could indicate a novel evasion mechanism [53] . Neither B 2 R-antagonist nor hydrocortisone inhibited the expression of genes with cell-intrinsic antiviral effects, but even increased the antiviral factor APOBEC3A at the mRNA level (Fig. 3B, Table S7 ) [54] .

Our gene expression analysis shows that SARS-CoV-2-infection further induces the expression of acute-phase proteins, such as TNF-α and IL-8 (CXCL8) [55, 56] , as well as IL-17C, MIP-3α (CCL20), IL-36γ [57] , and chemokines CXCL1,-2,-3,-8,-17, CCL2,-3,-5 [57] in primary airway epithelial cells (Fig. 3C, Table S8 ). The induction of these factors most likely contributes to the recruitment and activation of relevant immune cells to the site of infection. In addition, gene expression of acute-phase proteins was not significantly affected in airway epithelial cells by B 2 R-antagonist or hydrocortisone treatment (Fig. 3C, Table S8 ). Neither drug interfered with cell-intrinsic antiviral immune mechanisms, like IFN induction, APOBEC induction, or chemokine induction, thereby showing great potential for treatment options of COVID-19 while maintaining the host's antiviral immune response.

In addition, we found that SARS-CoV-2-infection increases the expression of three known and postulated entry (co-) receptors: (1) transmembrane serine protease TMPRSS11A (Fig. S1C , Table S6 , [9] [10] [11] [12] [13] , which was described to prime the MERS coronavirus spike protein [58] , (2) transmembrane serine protease TMPRSS11D, which was shown to activate SARS-CoV-2 spike protein [59] , and (3) pathogen-associated molecular pattern-binding C-type lectin receptor DC-SIGN (CD209), which was described to serve as entry receptor for SARS-CoV [60] and has also been suggested as a receptor for SARS-CoV-2. The induction of these additional entry receptor candidates triggered by SARS-CoV-2-infection may potentiate the viral spread in the bronchial epithelium and thus represent a pathogenetic mechanism that needs further research.

Overall, treatment with the B 2 R-antagonist and hydrocortisone had no significant effects on the expression of most candidate viral entry receptors, except for hydrocortisone, which enhanced the expression of TMPRSS proteases (Fig. 3D, Table S14 ). In particular, when focusing on the known SARS-CoV-2 entry receptors, hydrocortisone treatment of uninfected cells was already sufficient to induce an increase in TMPRSS2 gene expression (Fig. 3E) . SARS-CoV-2-infection per se also increased TMPRSS2 expression, and pre-treatment of SARS-CoV-2-infected NHBEs with hydrocortisone further potentiated this effect. On the other hand, ACE2 expression showed only a slight upward trend after hydrocortisone pre-treatment (Fig. S2B) . Finally, hydrocortisone pre-treatment of SARS-CoV-2-infected NHBEs had no inhibitory effect on the release of infectious particles 24 h after infection (Fig. 3F) , which was expected, since treatment of COVID-19 study participants with corticosteroids has an immunomodulatory rationale. 

In order to identify gene networks that are attenuated by B 2 R-antagonism, DEGs were processed in a network analysis using the database "String" to identify enriched cellular processes. B 2 R-antagonism reduced the expression levels of 343 membrane-bound receptors significantly in treated versus untreated SARS-CoV-2-infected NHBEs (Table S15) . Two particular cellular processes affected by pre-treatment with the B 2 R-antagonist were identified, namely G protein-coupled receptor signaling (GO:0,007,186; Fig. 4A , S2C, Tables S4, S17-18) and ion transport (GO:0,006,811; Fig. 4B , Tables S4, S17-18). DEGs involved in both processes were significantly downregulated in treated versus untreated SARS-CoV-2-infected NHBEs (Tables S16-18) .

Notably, all 35 cell surface receptors induced by SARS-CoV-2-infection were downregulated in cells that were treated with the B 2 R-antagonist (Fig. 4C , D, Tables S19-20).

Here, we provide evidence for the effect of interference with the KKS at the kinin B 2 receptor level as a means of protecting the airway epithelium from SARS-CoV-2-infection, while maintaining cell-intrinsic antiviral host response. We initially hypothesized that through KKS interference, either feedback mechanisms or modulated signal transduction targets the virus entry receptor ACE2 and thus interferes with the spread of SARS-CoV-2. To this end, the approved B 2 R-antagonist icatibant was used in this study. We demonstrate that treatment with a B 2 R-antagonist inhibits the replication and spread of SARS-CoV-2 in primary airway epithelial cells, which was determined by a decrease in total and genomic SARS-CoV-2-RNA, resulting in less infectious particles in plaque assays, both when applied pre-and post-infection. While a low concentration of 1 nM B 2 R-antagonist was sufficient to reduce viral RNA in primary bronchial epithelial cells when cells were treated pre-infection, 100 nM B 2 R-antagonist was required to this effect, when cells were treated post-infection. In addition, the significant reduction in virus load as determined by PCR tapered off after 96 h. On the one hand, in vitro infections are performed with excess amounts of virus particles. On the other hand, the fact that, due to its constitution as a peptide analog, the B 2 R-antagonist icatibant used in this study has a short half-life in the human body [42] but also pharmacological tolerance to interference at receptor level may explain why the effect reached significance after 6 h but did not persist. Therefore, it may be required to administer higher doses of the B 2 R-antagonist to COVID-19 patients a few times per day to inhibit viral replication in the long term. Due to this necessary repetitive administration of the B 2 R-antagonist, the monoclonal anti-plasma-kallikrein (KLKB1) antibody lanadelumab [61] may also be considered a potential pharmacologic alternative. However, it is not clear whether the effects of the B 2 R blockade alone and its effects on the KKS are responsible for the SARS-CoV-2 inhibition, or whether the compound itself additionally mediates a direct antiviral effect. Therapeutic application of the B 2 R-antagonist icatibant in future dose-finding studies should therefore focus on early intervention with at least two doses daily [62] and on either optimized pharmacokinetics or increased high local tissue concentrations, e.g., through topical application.

Two potential mechanisms of action for suppressing SARS-CoV-2-replication and spread in airway epithelium are revealed by this study: Since the decrease of genomic SARS-CoV-2 RNA and total SARS-CoV-2 RNA was comparable, we conclude that the B 2 R-antagonist icatibant does probably not affect the viral transcription machinery but inhibits the infection rather on the levels of entry, protein synthesis/processing, and assembly, maturation, or budding. In comparison, the corticosteroid hydrocortisone even upregulated TMPRSS2 in infected airway epithelial cells. It is noteworthy that hydrocortisone did not change the release of infectious particles from airway epithelial cells into the supernatant. Although TMPRSS2 expression was even enhanced by hydrocortisone, our data implicate that this effect on TMPRSS2 alone is insufficient to increase susceptibility for SARS-CoV-2-infection. 2. Treatment with the B 2 R-antagonist had a broad suppressive effect on gene expression of multiple cell signaling molecules, in particular on membrane-standing factors involved in GPCR signaling and ion transport. It has recently been published that SARS-CoV-2 may use cellular GPCR signaling pathways, thereby modulate epithelial transport mechanisms involved in ion transport and thereby cause a local ion imbalance in the airways [63] . In addition, an extensive recent study described that intracellular SARS-CoV-2 protein interactions include factors involved in intracellular trafficking and transport [64] . In fact, SARS-CoV-2-infection led to a differential regulation of the gene expression of 12 potassium channel (5 upregulated/7 downregulated), 1 sodium channel (down), but in particular of 55 members of the solute carrier family (24 downregulated/31 upregulated) in primary airway epithelial cells. On the other hand, B 2 R-antagonist treatment of SARS-CoV-2-infected NHBE resulted in a downregulation of 20 potassium channels and 6 sodium channels, as well as a downregulation of 29 members of the solute carrier family. We therefore conclude that B 2 R-antagonism not only impedes the viral entry process by reducing ACE2, as we had hypothesized, but also counter-regulates cellular processes that include GPCR signaling and transmembrane ion transport, which SARS-CoV-2 may utilize for efficient cell entry, replication, and viral spread.

In conclusion, the results of this study suggest that B 2 receptor antagonism protects airway epithelial cells from SARS-CoV-2 spread by reducing ACE2 levels and by interfering with several cellular signaling processes. Further research is needed to elucidate more details about molecular mechanisms involved in the viral life cycle that kinin B 2 receptor antagonism targets and underlie its effects against SARS-CoV-2-infection. Based on these data, we speculate that the protective effects of B 2 R-antagonism could potentially prevent the early stages of COVID-19 from progressing into severe acute respiratory distress syndrome (ARDS) with structural airway damage and fibrotic changes. We therefore propose that the safe approved B 2 R-antagonist icatibant be tested in clinical trials for two important aspects: (1) Treatment of early COVID-19 disease targeting the replication and spread of the virus.

(2) Optimized dosage regimen to reflect pharmacokinetics and possible pharmacological tolerance at the receptor level. Future controlled clinical trials must provide substantial evidence for optimal dosage regimen, application, efficacy, and safety to investigate, whether KKS interference at the kinin B 2 receptor level can prevent the escalation of COVID-19 to ARDS and long-term lung damage. . 4 B 2 R-antagonism exhibits a protective and suppressive effect on gene expression profile of airway epithelial cells. GO-term enrichment analysis, which results from the string network analysis of significant DEGs from the gene expression analysis comparing infected NHBE pre-treated with B 2 R-antagonist with untreated infected NHBE (SARS-CoV-2 + B 2 R-antagonist versus SARS-CoV-2). Depicted are enrichment of A GO-term GO:0,007,186 "G proteincoupled receptor signaling pathway" and B GO-term GO:0,006,811 "Ion transport." Genes that were significantly upregulated in the comparison SARS-CoV-2 versus medium are highlighted in red. C Venn diagram showing the cut set of upregulated membrane-bound cell surface receptors in SARS-CoV-2 versus medium and of downregulated DEGs in SARS-CoV-2 + icatibant versus SARS-CoV-2 (FC ≥ 1.5; P ≤ 0.05). D Heat map of gene expression analysis of the 35 membrane-bound cell surface receptors defined in cut set from Fig. 4C , all upregulated upon SARS-CoV-2-infection and downregulated upon pre-treatment with B 2 R-antagonist are depicted. Only entities with significant changes between SARS-CoV-2-infection and medium (up) and between SARS-CoV-2 + B 2 R-antagonist and SARS-CoV-2 (down) are shown (gene expression fold change FC ≥ 1.5 with P < 0.05). Color code indicates Log2-fold change from low (blue) through 0 (white) to high (red). Duplicate gene names indicate the abundance of two or more isoforms of the same gene in the analysis ◂ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.

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Phorbol ester and neomycin dissociate bradykinin receptor-mediated arachidonic acid release and polyphosphoinositide hydrolysis in Madin-Darby canine kidney cells. Evidence that bradykinin mediates noninterdependent activation of phospholipases A2 and C

Involvement of intercellular adhesion molecule-1 up-regulation in bradykinin promotes cell motility in human prostate cancers

BDKRB2 +9/-9 bp polymorphisms influence BDKRB2 expression levels and NO production in knee osteoarthritis

Activation of Toll-like receptor 2 induces B1 and B2 kinin receptors in human gingival fibroblasts and in mouse gingiva

The discovery of angiotensinconverting enzyme 2 and its role in acute lung injury in mice

Pulmonary angiotensin-converting enzyme 2 (ACE2) and inflammatory lung disease

The outcome of critically ill COVID-19 patients is linked to thromboinflammation dominated by the kallikrein/kinin system

Safety and outcomes associated with the pharmacological inhibition of the kinin-kallikrein system in severe COVID-19

Understanding the pathophysiology of COVID-19: could the contact system be the key?

Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms

Mechanisms of disease: the tissue kallikrein-kinin system in hypertension and vascular remodeling

Structural organization of the human kininogen gene and a model for its evolution

Pathways for bradykinin formation and inflammatory disease

Impaired breakdown of bradykinin and its metabolites as a possible cause for pulmonary edema in COVID-19 infection

A mechanistic model and therapeutic interventions for COVID-19 involving a RAS-mediated bradykinin storm

Pathways for bradykinin formation and interrelationship with complement as a cause of edematous lung in COVID-19 patients

A hypothesized role for dysregulated bradykinin signaling in COVID-19 respiratory complications

IL-12 initiates tumor rejection via lymphoid tissueinducer cells bearing the natural cytotoxicity receptor NKp46

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

Novel pharmacotherapy of acute hereditary angioedema with bradykinin B2-receptor antagonist icatibant

Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection

The signal pathways and treatment of cytokine storm in COVID-19

Pharmacokinetics of single and repeat doses of icatibant

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Intensive care risk estimation in COVID-19 pneumonia based on clinical and imaging parameters: experiences from the Munich cohort

Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients

A dynamic COVID-19 immune signature includes associations with poor prognosis

Lessons learned: new insights on the role of cytokines in COVID-19

Immune response to SARS-CoV-2 and mechanisms of immunopathological changes in COVID-19

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, openlabel, platform trial

Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA

APOBEC3-mediated restriction of RNA virus replication

Role of the single deaminase domain APOBEC3A in virus restriction, retrotransposition, DNA damage and cancer

Bradykinin increases IL-8 generation in airway epithelial cells via COX-2-derived prostanoids

Monocyte-derived IL-1 and IL-6 are differentially required for cytokine-release syndrome and neurotoxicity due to CAR T cells

The trinity of COVID-19: immunity, inflammation and intervention

TMPRSS11A activates the influenza A virus hemagglutinin and the MERS coronavirus spike protein and is insensitive against blockade by HAI-1

TMPRSS11D and TMPRSS13 activate the SARS-CoV-2 spike protein Viruses 13

Specific asparagine-linked glycosylation sites are critical for DC-SIGN-and L-SIGN-mediated severe acute respiratory syndrome coronavirus entry

Lanadelumab for the prophylactic treatment of hereditary angioedema with C1 inhibitor deficiency: a review of preclinical and phase I studies

Outcomes associated with use of a kinin B2 receptor antagonist among patients with COVID-19

SARS-CoV-2 may hijack GPCR signaling pathways to dysregulate lung ion and fluid transport

Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV