Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate

ABSTRACT A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.


Introduction
By mid-May of 2021, SARS-CoV-2 has infected over 160 million people and caused >3.3 million deaths. Until now, more than 1200 million vaccines have been administered. However, the efficacy of available vaccines against emerging variants remains unclear [1,2]. Therefore, the need of effective treatments to prevent the infection or to manage the disease in severe cases of COVID-19 remains an urgent medical need.
The spike protein of SARS-CoV-2 interacts with the angiotensin-converting enzyme 2 (ACE2), a keyreceptor widely expressed on the respiratory tract, through the receptor-binding domain (RBD) [3][4][5]. Other factors such as neuropilin-1 (NRP1) [6,7] and the transmembrane protease serine 2 (TMPRSS2) [8] contribute to the entry of SARS-CoV-2 into host cells. Another layer of interaction between the virus and the host that facilitates viral entry is the attachment of the spike protein to cellular heparan sulfate (HS) [5]. HS is part of the cellular glycocalyx and decorates the HS-containing proteoglycans (HSPGs). The family of syndecans are HSPGs expressed on several cell types and organs [9], being syndecan-1 the most relevant in terms of expression in the lung [10]. The interaction between the spike and HS can be efficiently blocked by lactoferrin [11].
A common feature of COVID-19 patients is moderate to severe hypoxia [12][13][14][15]. Disease severity correlates with the level of naturally occurring anti-SARS-CoV-2 antibodies in serum [16], and patients with severe disease are characterized by low oxygen saturation that often results in the need for oxygen supplementation [17]. Currently, the impact of hypoxia on SARS-CoV-2 infectivity and COVID-19 pathogenesis is unclear, but there are emerging indications that low oxygen concentrations might prevent infection or disease severity [18,19]. This notion is supported, in part, by the fact that epidemiological data indicate that COVID-19 severity decreases in high altitude areas [20].
There is evidence that hypoxia regulates the expression of ACE2, but the underlying mechanisms and impact on physiology and disease remain controversial. Previous studies have demonstrated that regulation of ACE2 expression by hypoxia is contextspecific and cell type-and time-dependent [21][22][23][24][25]. Elucidating these mechanisms in lung epithelial cells would help to understand the cellular susceptibility to infection by SARS-CoV-2 under limited oxygen availability. Additionally, the members of the syndecan family, the main cellular HSPGs, are also differentially regulated by hypoxia. For example, the expression of syndecan-1 in the lungs is reduced under hypoxia [26,27].
Cell responses to hypoxia are mediated by the family of hypoxia inducible factors (HIF). COVID-19 disease leads to the accumulation of HIF in the lung, providing a molecular link between SARS-CoV-2 infection and hypoxia in patients with severe disease [28]. Our hypothesis is that hypoxia modulates the expression of entry receptors and attachment factors on host cells. In this context, elucidating the underlying mechanisms of this regulation could lead to the development of novel therapeutic strategies for the management of COVID-19, including those targeting the hypoxia response pathway.

Lactoferrin assay
Binding of RBD to the surface of cells was measured by flow cytometry after incubation with increasing doses of human lactoferrin (0, 1, 5 and 10 mM) (Sigma Aldrich Cat#: L1294) in a final volume of 100 μL of culture medium. The incubation was done for 45 min in a cell incubator with standard conditions (37°C, 21% O 2 and 5% CO 2 ).

Titration of the pseudotyped lentiviral particles and infection of Vero E6 cells
Viral titration was performed in Vero E6 cells as described in Crawford et al. [29]. For single-infection experiments, 150,000 cells were seeded in 6-well plates and cultured overnight in normoxia (21% O 2 ) and incubated for additional 24 h under normoxia or hypoxia (1% O 2 ). After that, cells were incubated with the pseudotyped lentiviral particles (MOI: 0.1) for 48 h under normoxic conditions. The infected cells expressed ZsGreen, allowing their detection by flow cytometry.

Deletion of HIF-1α and syndecan-1 in NCI-H460 cells via CRISPR/Cas9
Deletion of HIF-1α in NCI-H460 cells was performed using a pool of two sgRNAs from Synthego (sgRNA sequences: gaugguaagccucaucacag and guuuuccaaacuccgacauu) and the TrueCut Cas9 protein v2 (Invitrogen Cat#: A36497). Deletion of syndecan-1 was performed using the following TrueGuide Synthetic sgRNAs from Invitrogen: CRISPR913712_SGM and CRISPR913714_SGM. RNP-complexes were generated with 7.5 pmol of Cas9, 7.5 pmol of the sgRNA-1 and 7.5 pmol of the sgRNA-2. RNP-complexes were introduced into cells using the Lipofectamine CRISPRMAX Cas9 Transfection reagent (Invitrogen Cat#: CMAX00001) and Opti-MEM reduced serum medium (Gibco Cat#: 31985062) following the Pub. No. MAN0017058 by Invitrogen (pages 5 and 6). A nontargeting sgRNA that does not recognize any sequence in the human genome was used as a negative control (Invitrogen Cat#: A35526). The deletion efficiency of HIF-1α and syndecan-1 proteins was measured by western blot and flow cytometry, respectively, and the pool of edited cells was used for downstream experiments.

Quantitative PCR
Total RNA was isolated using the NucleoSpin RNA kit (Macherey-Nagel Cat#: 740955.250). The cDNA was synthesized by RT-PCR from 1 μg of purified RNA with the M-MLV reverse transcriptase (Thermo Fisher Cat#: 28025-013) and random primers (Thermo Fisher Cat#: 58875). Quantitative PCR (Q-PCR) reactions were conducted in triplicate on a ViiA 7 Real-Time PCR system (Thermo Fisher) from 1 μL of cDNA using the PerfeCTa SYBR Green SuperMix reagent (Quantabio Cat#: 95056-500) and genespecific primers. The amplification programme consisted of initial denaturation at 95°C for 3 min followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 60 s and extension at 72°C for 60 s. Data were analysed using the QuantStudio software version 1.3 (Thermo Fisher). The relative quantification in gene expression was determined using the 2 -ΔΔCt method by using the RPLP0 gene as a housekeeping gene (forward: 5 ′ -CGACCTG-GAAGTCCAACTAC-3 ′ and reverse: 5 ′ -ATCTGCTG-CATCTGCTTG-3 ′ ). PGK1, a HIF-1α target gene, was used as a control for hypoxia (forward: 5 ′ -CCGCTTTCATGTGGAGGAAGAAG-3 ′ and reverse: 5 ′ -CTCTGTGAGCAGTGCCAAAAGC-3 ′ ). Primer sequences for measuring ACE2 and SDC1 expression on human NCI-H460 cells were previously described by Ma et al. [30] and Reynolds et al. [31], respectively. As these oligos presented several mismatches with the corresponding genes on the reference genome of the African green monkey, we designed the following primers to amplify ACE2 from Vero E6 cells (forward: 5 ′ -AGCACTCACGATTGTTGGGA-3 ′ and reverse: 5 ′ -CCACCCCAACTATCTCTCGC-3 ′ ).

Statistics
Statistical analyses were performed using GraphPad Prism version 8.0. The test applied in each panel is specified on the figure legends.

Results
Hypoxia reduces the binding of the SARS-CoV-2 spike to epithelial cells In order to understand if hypoxia could affect the cellular binding capacity of the SARS-CoV-2 spike protein, we subjected Vero E6 cells or human lung epithelial cells (NCI-H460) to different oxygen concentrations (21% O 2 or 1% O 2 ) and measured the binding ability of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein to the surface of these cells in vitro. Figure 1 shows a significant decrease in the binding of RBD and S1 after culturing Vero E6 (Figure 1(a)) or NCI-H460 (Figure 1(b)) cells under hypoxia (1% O 2 ) compared to normoxia (21% O 2 ).

Hypoxia decreases ACE2 and NRP1 protein levels on Vero E6 cells
To further explore the mechanism underlying the observed reduced binding of the spike under hypoxia, we assessed ACE2 gene and protein expression on Vero E6 and NCI-H460 cells by Q-PCR and western blot, respectively. Vero E6 cells, but not NCI-H460 cells, present detectable levels of ACE2 transcripts (Figure 2(a)). Accordingly, ACE2 protein is only detectable on Vero E6 cells (Figure 2(b)), suggesting that the observed binding of the spike to the surface of NCI-H460 cells is ACE2-independent. We then explored if hypoxia modulates ACE2 expression on Vero E6 cells. Hypoxia culture results in a transient accumulation of HIF-1α and upregulation of the expression of ACE2 (Figure 2(b,c)). After 48 h of exposure to a limited oxygen supply, the level of ACE2 protein on Vero E6 cells is significantly reduced (Figure 2(b)). On a similar manner, we measured the expression of other important factors that mediate the initial steps of SARS-CoV-2 infection: NRP1 and TMPRSS2. NRP1 (but not TMPRSS2) is significantly reduced on Vero E6 cells after culture for 48 h under hypoxia (Figure 2(d)), as previously described by Casazza et al. [32] on the hypoxic tumour microenvironment. NCI-H460 cells do not express detectable levels of NRP1.
Hypoxia prevents the infection of Vero E6 cells with pseudotyped viral particles expressing the spike protein of SARS-CoV-2 We then assessed the influence of oxygen availability on the infection capacity of pseudotyped lentiviral particles that express the full-length spike of SARS-CoV-2. Vero E6 cells exposed to hypoxia show a significant decrease in the rate of infection (Figure 3 (a)) compared to normoxia. NCI-H460 cells, which lack expression of ACE2 and NRP1 (Figure 2(b,d)), are not amenable to infection (Figure 3(b)).

Lactoferrin blocks the binding of RBD to the surface of epithelial cells
Recently, it has been demonstrated that the spike of SARS-CoV-2 can bind to HS expressed on the host cell surfaces [5]. We reasoned that this phenomenon might explain the observed binding of RBD to NCI-H460 epithelial cells in the absence of ACE2 and NRP1. To confirm this interaction, we blocked cellular HS with human lactoferrin, a natural glycoprotein that binds to HS and presents antiviral activity against coronaviruses [11,33]. Figure 4 shows that lactoferrin was able to reduce the attachment of the RBD of SARS-CoV-2 to Vero E6 (Figure 4(a)) and NCI-H460 (Figure 4(b)) cells in a dose-dependent manner. Interestingly, the degree of blockade by lower doses of lactoferrin was higher on NCI-H460 compared to Vero E6 cells.

Hypoxia decreases the total level of heparan sulfate on the surface of epithelial cells
Considering that cellular HS mediates the binding of RBD in NCI-H460 cells lacking the expression of ACE2 and NRP1, we explored if this interaction was altered by hypoxia. Hypoxia significantly decreases the total level of cellular HS present on Vero E6 cells ( Figure 5(a)) and, to a higher extent, on NCI-H460 cells ( Figure 5(b)). The syndecan family is a major contributor to the pool of HS on the surface of cells. In this context, we assessed the expression levels of syndecan-1, syndecan-2, syndecan-3 and syndecan-4 on NCI-H460 cells. We found that syndecan-1 and syndecan-3 are the main syndecans expressed on this cell model and that hypoxia significantly reduces their expression at protein level ( Figure 5(c)).

Hypoxia reduces the binding of RBD to NCI-H460 epithelial cells by downregulating syndecan-1 expression on a HIF-1α-dependent mechanism
Based on the finding that syndecan-1 is the most abundantly expressed syndecan on NCI-H460 cells ( Figure 5(c)), we sought to ascertain its role in the attachment of the spike protein to host cells. Figure  6(a) shows that NCI-H460 cells expressing high levels of syndecan-1 capture higher amounts of RBD compared to cells expressing low levels of syndecan-1. To further demonstrate the binding of RBD to syndecan-1, we generated a cell line with reduced expression of syndecan-1 via CRISPR/Cas9 (SDC1-KO). A pool of SDC1-edited cells binds less RBD compared to control NCI-H460 cells (Figure 6(b)). Given that HIF-1α is the main transcriptional mediator of the response to hypoxia, we checked if it was responsible for the observed downregulation of syndecan-1 under hypoxia (Figure 5(c) and Figure 6(c)). To this end, we generated HIF-1α deficient NCI-H460 cells via CRISPR/Cas9 (Figure 6(d)). Deletion of HIF-1α on NCI-H460 cells partially rescued the observed inhibition of syndecan-1 expression under hypoxia ( Figure 6(e)), resulting in a marked increase in the total levels of cellular HS (Figure 6(f)). Consequently, the level of RBD binding to HIF1-KO cells under hypoxia was higher than in control HIF-1α competent cells (Figure 6(g)).
Discussion COVID-19 disease is associated with hypoxia [12,28,34], which results in decreased oxygen availability for tissues. This phenomenon affects the susceptibility of host cells to viral infections [35] and shapes immune responses [36]. Here, we describe the binding ability of the spike of SARS-CoV-2 to epithelial cells cultured under different oxygen concentrations. We show that hypoxia inhibits the binding of the RBD and the S1 subunit of the spike of SARS-CoV-2 to host cells by modulating the expression of several entry and attachment factors. In this study, we consider two different and complementary cellular models. First, Vero E6 cells, a widely used cell type in SARS-CoV-2 biology research because of its expression of ACE2, NRP1 and TMPRSS2 factors, which are components of the cellular machinery that allow the entry of SARS-CoV-2 into host target cells. Second, NCI-H460 human lung epithelial cells, which lack the expression of ACE2 and NRP1 and as such, do not get infected by pseudotyped viral particles expressing the spike of SARS-CoV-2, but permits the binding of the spike and RBD to cellular HS [11,37]. This dual approach allowed us to investigate the mechanism underlying the observed reduction of RBD binding and infectivity.
Using the Vero E6 model we showed that prolonged hypoxia reduces the expression levels of cell surface ACE2 and NRP1, resulting in a reduced  susceptibility to infection by pseudotyped viral particles expressing the spike of SARS-CoV-2. We have not observed changes in the expression of TMPRSS2 under hypoxia. Previous studies suggest that the expression of ACE2 under hypoxia is context dependent [21][22][23][24][25]. Hypoxia exposure time is one of the variables that influence ACE2 levels. We show that acute hypoxia (24 h) can induce a transient increase in ACE2 expression, probably mediated by the binding of HIF-1α to the hypoxia response elements found in the ACE2 promoter [22,38]. However, chronic hypoxia (48 h) leads to a significant reduction of ACE2 and HIF-1α levels on Vero E6 cells. In line with these findings, a recent study shows that hypoxia reduces the protein levels of ACE2 on several cell lines [38]. Cellular HS is known to cooperate with the entry receptor ACE2 as an attachment factor, facilitating the initial capture of viral particles by the cellular glycocalyx [5]. We used NCI-H460 human lung epithelial cells to disentangle the contribution of cellular HS to the observed reduction of infection under hypoxia on an ACE2-independent manner.
First, we confirmed that the RBD binds to HS expressed on target cells by blocking this interaction with lactoferrin. Vero E6 cells are less sensitive than NCI-H460 cells to the blockade exerted by low doses of lactoferrin. This difference could be attributed to the interaction of RBD with other cellular components that are absent in NCI-H460 cells, such as ACE2 and NRP1. These findings prompted us to use NCI-H460 cells as a model to assess the effect of hypoxia on the levels of cellular HS and its interaction with the spike of SARS-CoV-2.
We showed that hypoxia leads to a significant reduction of the cellular pool of HS on NCI-H460 cells. Interestingly, syndecan-1, which is expressed in the lung [10], is responsible for this decrease under hypoxia. We confirmed this finding by measuring the binding of RBD to cells expressing different amounts of syndecan-1. We then wanted to ascertain if HIF-1α was responsible for the reduction of the expression of syndecan-1 observed under hypoxia at both transcriptional and protein expression levels. Ablation of HIF-1α in NCI-H460 cells demonstrated that HIF-1α is necessary for the inhibition of syndecan-1 expression under hypoxia. Moreover, deletion of HIF-1α results in an increase of cellular HS levels and higher binding capacity of RBD to hypoxic NCI-H460 cells.
Together, these results indicate that the hypoxia response governed by HIF-1α in the host target cells leads to a protective effect against SARS-CoV-2 infection. In this context, the administration of HIF prolylhydroxylase (PHD) inhibitors that result in the accumulation of HIF-1α may serve as a potential treatment to limit the initial attachment and entry of the virus into host cells [38,39]. It should be noted that as the infection progresses into COVID-19 disease, exacerbated inflammatory responses contribute to severity. Inflammation and the response to hypoxia are closely related processes that coexist in certain microenvironments [40,41]. Macrophages are important mediators of lung inflammation during COVID-19 disease [42]. As a result of hypoxia in the lung, elevated HIF-1α activity on immune innate cells contributes to severity [43,44]. In this scenario, promoting systemic HIF-1α activity through pharmacological intervention in severe COVID-19 patients might be detrimental. In summary, in this study we show that hypoxia decreases the binding of the spike of SARS-CoV-2 to epithelial cells by at least two different mechanisms: (1) decreasing the level of ACE2 and NRP1 expression; and (2) reducing the total amount of cellular HS (attachment factor) and syndecan-1 on the cell surface of epithelial cells.

Limitations of the study
All experiments presented here have been performed in vitro with immortalized cell lines. The significance of these findings at physiopathological oxygen levels in vivo remains unexplored. In this context, the use of humanized mouse models and real SARS-CoV-2 virus would reinforce the findings of this study.

Acknowledgements
The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). Vero E6 monkey kidney cells were kindly provided by Dr. Nicola G.A. Abrescia (CIC bioGUNE).

Disclosure statement
No potential conflict of interest was reported by the author (s).