Introduction

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread globally, with at least 755 million people diagnosed and the death toll is over 6.8 million. SARS-CoV-2 variants of concern, including Alpha, Beta, Delta, and Omicron, continue to evolve and increase transmissibility and the ability to escape host immune responses. These variants have brought huge challenges to the design and development of vaccines and therapeutic reagents (Zhou et al., 2020). In order to discover novel strategies to control the virus, it is important to understand host responses to SARS-CoV-2 infection.

SARS-CoV-2 infection induces cell-cell fusion (also known as syncytia formation) in multiple cell types including lung epithelial cells, neurons and glia (Martínez-Mármol et al., 2023). Syncytia of SARS-CoV-2 infected cells with neighboring cells could potentially contribute to increased viral transmission and pathogenicity in the infected host (Rajah, Bernier, Buchrieser, & Schwartz, 2022), which also makes the virus insensitive to extracellular neutralizing antibodies (Li et al., 2022; Yu et al., 2023). Moreover, syncytia formation among pneumocytes with long-term persistence of viral RNA has been observed in the lung autopsy of deceased COVID-19 donors, which may contribute to prolonged clearance of the virus and long COVID symptoms (Bussani et al., 2020). Therefore, inhibiting syncytia formation is critical to ensure viral clearance and to control viral transmission.

It has been reported that SARS-CoV-2 mediated syncytia formation is effectively inhibited by multiple interferon (IFN)-stimulating genes (ISGs) (Pfaender et al., 2020; Wang et al., 2020). However, low IFN levels with impaired ISG responses were observed during early SARS-CoV-2 infection, which may have compromised the antiviral responses of IFN in severe COVID-19 patients (Blanco-Melo et al., 2020; Hadjadj et al., 2020). Thus, identifying endogenous host factors that regulate syncytia formation is of great significance for harnessing the transmission of SARS-CoV-2.

Of note, a variety of cells are involved in the host responses to SARS-CoV-2 infection (Ren et al., 2021), lung epithelial cells are the primary target of SARS-CoV-2 infection and transmission, which subsequently recruit and activate innate immune cells (Barnett et al., 2023). Tissue-resident macrophages and circulating monocytes contribute to local and systemic inflammation largely through releasing inflammatory cytokines (Sefik et al., 2022). Among these cytokines induced by SARS-CoV-2 infection, the combination of TNF-α and IFN-γ induces inflammatory cell death, resulting in clear tissue damage, while other cytokines’ function remains obscure (Karki et al., 2021). In addition, when corticosteroids were used to suppress the inflammatory response in patients infected by SARS-CoV (N. Lee et al., 2004) or MERS-CoV (Arabi et al., 2018), the clearance of viral RNA was obviously delayed, suggesting the importance of innate immune factors including cytokines in viral clearance.

Innate immune cells express toll-like receptors (TLRs), and TLR-mediated signaling induces robust production of inflammatory cytokines (Medzhitov, 2001). In the current work, we screened the role of soluble pro-inflammatory cytokines on the SARS-CoV-2 spike-induced cell-cell fusion. Notably, we identified that IL-1β, which is the key factor of inflammatory response, inhibited SARS-CoV-2 spike-induced syncytia formation in various cells by activating RhoA/ROCK pathway to initiate actin bundle formation at cell-cell interface between SARS-CoV-2 infected cells and neighboring cells. Importantly, IL-1β significantly reduced SARS-CoV-2 transmission among lung epithelia in experimental mice in vivo. Therefore, our data highlight an important role for pro-inflammatory cytokines against viral infection.

Results

Host factors secreted by activated innate immune cells inhibit SARS-CoV-2 induced cell-cell fusion

We have previously established quantitative and qualitative models for SARS-CoV-2 spike-induced cell-cell fusion by bioluminescence assay, immunoblotting and fluorescence imaging (Yu et al., 2022). In order to explore the potential effect of cytokines on SARS-CoV-2-induced cell-cell fusion, human monocyte cell line THP-1 and human peripheral blood mononuclear cells (PBMCs) were used in this study. We applied several TLR ligands to stimulate such innate immune cells and collected the cell culture supernatants for subsequent experiments (Fig. 1A). Of note, cell culture supernatants of THP-1 cells stimulated by TLR ligands significantly reduced the bioluminescence signal, while neither untreated THP-1 cell culture supernatant nor the medium control had any effect on the bioluminescence signal reflecting cell-cell fusion (Fig. 1B). SARS-CoV-2 spike engagement of ACE2 primed the cleavage of S2’ fragment in target cells, a key proteolytic event coupled with spike-mediated membrane fusion (Yu et al., 2022). In parallel with bioluminescence assay, a large amount of enriched S2’ cleavage was detected in HEK293T-Spike and HEK293T-ACE2 co-cultured group and co-culture incubated with untreated THP-1 cell culture supernatant, while S2’ cleavage was significantly reduced upon treatment with TLR ligands-stimulated THP-1 cell culture supernatants (Fig. 1C). Syncytia formation was also visualized using cells co-expressing spike and a ZsGreen fluorescent reporter. The number and area of syncytium were significantly reduced by the treatment with TLR ligands-stimulated THP-1 cell culture supernatants (Fig. 1D). Considering the presence of TLR ligands in cell culture supernatant, we tested their potential direct effects. As expected, TLR ligands alone did not reduce the bioluminescence signal and S2’ cleavage compared to the control groups (Fig. S1 A and B), as well as no effect on syncytia formation (Fig. S1C).

Fig. 1.

Concurrently, we also tested the effect of PBMCs culture supernatants on SARS-CoV-2 spike-induced cell-cell fusion. Consistent with the results from THP-1 cells, TLR ligands stimulated PBMCs culture supernatants treatment also significantly reduced the bioluminescence signal, S2’ cleavage, and the number and area of syncytium compared with the medium group (Fig. S1 D-F). These results suggested that activated innate immune cells released host factors to inhibit SARS-CoV-2 spike-induced cell-cell fusion.

To validate the effect of innate immune cell culture supernatants on cell-cell fusion in authentic SARS-CoV-2 infection, we pre-treated ACE2-expressing cells with THP-1 cell culture supernatants before inoculation with SARS-CoV-2 B.1.617.2 (Delta) or Wuhan-Hu-1 (wild type, WT) strains. Cell lysates were used for the detection of SARS-CoV-2 spike and N protein 24 hours post infection (hpi) (Fig. 1E). This experiment revealed that TLR ligands stimulated THP-1 cell culture supernatants reduced S2’ cleavage and N protein during Delta or WT SARS-CoV-2 infection in HEK293T-ACE2 cells, while untreated THP-1 cell culture supernatant had no effect (Fig. 1F and Fig. S2A). In addition, TLR ligands stimulated THP-1 cell culture supernatants significantly reduced the number and area of syncytium induced by Delta or WT SARS-CoV-2 infection (Fig. S2 B and C). Furthermore, we infected the human colon epithelial carcinoma cell line Caco-2 with WT SARS-CoV-2, and found that S2’ cleavage and N protein were reduced after TLR ligands stimulated THP-1 cell culture supernatants pre-treatment (Fig. 1G). Accordingly, immunofluorescent staining also showed that TLR ligands stimulated THP-1 cell culture supernatants significantly reduced the number and area of syncytium during SARS-CoV-2 infection in Caco-2 cells (Fig. 1H). These data suggested that host factors secreted by activated innate immune cells inhibit authentic SARS-CoV-2 induced cell-cell fusion.

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion

To explore which host factors inhibited SARS-CoV-2 induced cell-cell fusion, we first detected mRNA levels of different cytokines in THP-1 cells stimulated by TLR ligands. It was found that the expression levels of IL1A, IL1B, IL6 and IL8 were significantly increased upon TLR ligands stimulation, while IL4, IL12A, IFNA1, IFNB1 and IFNG mRNA levels were relatively unchanged or undetected (Fig. S3A). In addition, we also detected the mRNA levels of cytokine receptors in HEK293T modelling cells, confirming that IL1R1, IL4R, IL6ST, IL8RA, IFNAR1, IFNGR1 were expressed in such cells, while IL2RA and IL12RB1 were undetectable (Fig. S3B).

We next selected recombinant IL-1α, IL-1β, IL-6, and IL-8 to test whether individual cytokine may play a role in affecting SARS-CoV-2 spike induced cell-cell fusion (Fig. 2A). Interestingly, IL-1α and IL-1β significantly reduced the bioluminescence signal compared to the control group, while IL-6 and IL-8 had little or no effect (Fig. 2B). In addition, fluorescence images of cells expressing Zsgreen reporter also confirmed that IL-1α and IL-1β significantly inhibited SARS-CoV-2 spike induced syncytia formation (Fig. S3C). Furthermore, IL-1β and IL-1α both reduced the bioluminescence signal and S2’ cleavage (Fig. 2 C and D, and Fig. S4A) in cell lysates in a dose-dependent manner. Moreover, the syncytia formation was inhibited with increasing concentrations of IL-1β or IL-1α (Fig. S4 B and C). Intriguingly, when we added both IL-1α and IL-1β, there was no synergistic inhibition on cell-cell fusion compared to either cytokine alone (Fig. S4 D and E), suggesting a potential saturation of IL-1 receptor binding to these homologues. Since both IL-1α and IL-1β activate the downstream pathway through the same receptor IL-1R1, it is suggested that IL-1α or IL-1β may inhibit cell-cell fusion through the same pathway. Considering the higher mRNA level of IL1B than IL1A, as well as the classical release pathway of IL-1β from innate immune cells (Weber, Wasiliew, & Kracht, 2010), we mainly used IL-1β for further experiments.

Fig. 2.

In order to validate the effect of IL-1β on cell-cell fusion during authentic SARS-CoV-2 infection, we pre-treated ACE2-expressing cells with IL-1β before inoculating with Delta or WT authentic SARS-CoV-2. Cell lysates were used for the detection of SARS-CoV-2 spike and N protein 24 hpi (Fig. 2E). To this end, it was found that IL-1β reduced S2’ cleavage and N protein compared to the control group during such infection both in HEK293T-ACE2 (Fig. 2F and Fig. S5A) and Caco-2 cells (Fig. 2G). Meanwhile, IL-1β inhibited authentic SARS-CoV-2 induced syncytia formation (Fig. 2H and Fig. S5 B and C). These results verified that IL-1β inhibits authentic SARS-CoV-2 induced cell-cell fusion in various target cells.

As expected, innate immune cells activated by TLR ligands secreted IL-1β into the cell culture supernatants (Fig. S6, A and B), and TLR2 was essential for THP-1 cells to release IL-1β in response to TLR2 ligands (Fig. S6C). More importantly, the cell culture supernatants of TLR2-knockout THP-1 cells stimulated by TLR2 Ligands had no effect on the bioluminescence signal, while the cell culture supernatants from WT THP-1 cells stimulated by the same TLR2 Ligands significantly reduced the bioluminescence signal (Fig. S6D). In addition, pre-treatment with TAK1 inhibitor (5Z-7) or IKKβ inhibitor (TPCA1) in WT THP-1 cells prevented IL-1β secretion after PGN stimulation (Fig. S6E), as well as eliminated the inhibitory effect of PGN-stimulated WT THP-1 cell culture supernatant on SARS-CoV-2 spike-induced cell-cell fusion (Fig. S6F). In parallel, pre-treatment with these inhibitors in PBMCs showed the same results (Fig. S6 G and H). These data suggested that TLR-knockout or inhibitors targeting the respective TLR signaling prevented innate immune cells from releasing IL-1β into supernatants, which led to failed inhibition of SARS-CoV-2 spike induced cell-cell fusion. These findings thus further verify that IL-1β is an important host factor inhibiting SARS-CoV-2 induced cell-cell fusion.

To investigate the effector function of IL-1 on cells expressing SARS-CoV-2 spike (donor cells) and neighboring cells expressing ACE2 (acceptor cells), we pre-treated HEK293T-S or HEK293T-ACE2 cells or both with IL-1β, then co-cultured after washing with PBS. Cells were then analyzed by the quantitative and qualitative models (Fig. S7A). Notably, pre-treatment of either HEK293T-S or HEK293T-ACE2 cells with IL-1β alone reduced bioluminescence signal and S2’ cleavage; when IL-1β pre-treatment on both HEK293T-S and HEK293T-ACE2 cells was applied, bioluminescence signal and S2’ cleavage were further reduced (Fig. S7B). Furthermore, we used Vero E6-overexpressing ACE2 cell line (Vero E6-ACE2) and human Calu-3 cells as acceptor cells. In this case, pre-treatment of either HEK293T-S or Vero E6-ACE2 cells with IL-1β alone reduced part of S2’ cleavage, while IL-1β pre-treatment of both HEK293T-S and Vero E6-ACE2 cells led to further reduction of S2’ cleavage (Fig. S7C). And the same results were observed in the case of Calu-3 as acceptor cells (Fig. S7D). Accordingly, fluorescence imaging showed that IL-1β significantly reduced the number and area of syncytia (Fig. S8A). Notably, IL-1β reduced the bioluminescence signal and S2’ cleavage in different SARS-CoV-2 variants (Fig. S8 B-D). These results suggest that IL-1β acts on both donor and acceptor cells to inhibit SARS-CoV-2 spike induced cell-cell fusion in various cell lines.

SARS-CoV (Belouzard, Chu, & Whittaker, 2009) and MERS-CoV (Straus et al., 2020) spike protein also induce cell-cell fusion in target cells. Therefore, we further explored whether IL-1β could also inhibit SARS-CoV and MERS-CoV spike-induced cell-cell fusion in ACE2-or Dipeptidyl peptidase-4 (DPP4)-expressing cells by bioluminescence assay, immunoblotting and a modified stop-mCherry fluorescent model, wherein mCherry reporter is only expressed when Cre excises the Stop cassette inside the fused syncytia (Fig. S9A). Similar to SARS-CoV-2 spike-induced cell-cell fusion, IL-1β also reduced bioluminescence signal (Fig. S9 B and C), S2’ cleavage (Fig. S9 D and E) and the number and area of syncytium (Fig. S9F). These data suggested that IL-1β possesses a broad spectrum to inhibit cell-cell fusion induced by different coronaviruses.

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion through IL-1R1/MyD88/IRAK/TRAF6 pathway

To investigate the mechanism of IL-1β inhibition on SARS-CoV-2 induced cell-cell fusion, we performed gene knockout using CRISPR-Cas9 technology, in conjunction with inhibitors targeting the IL-1 receptor pathway (Fig. 3A). In the presence of IL-1 receptor antagonist (IL-1RA), IL-1β was unable to reduce bioluminescence signal and S2’ cleavage (Fig. 3B). MyD88 is the downstream adaptor of IL-1R1. We then generated MyD88 knockout HEK293T cell line, wherein IL-1β was unable to reduce bioluminescence signal (Fig. 3C) and S2’ cleavage (Fig. S10A). In addition, we found that IL-1β was unable to reduce bioluminescence signal and S2’ cleavage in the presence of IRAK1/4 inhibitor (Fig. 3D). Furthermore, IL-1β was also unable to reduce bioluminescence signal (Fig. 3E) and S2’ cleavage (Fig. S10B) in TRAF6 knockout HEK293T cell line. These results suggested that IL-1β inhibits SARS-CoV-2 spike induced cell-cell fusion through IL-1R1-MyD88-IRAK-TRAF6 pathway.

Fig. 3.

Intriguingly, when we tested TAK1, a downstream molecule of TRAF6 for the potential involvement in the signaling, it was found that IL-1β still reduced bioluminescence signal and S2’ cleavage in TAK1 knockout (sgMAP3K7) HEK293T cell line (Fig. S11A). Moreover, we found that in the presence of TPCA1, an IKKβ inhibitor, IL-1β still inhibited bioluminescence signal and S2’ cleavage as well (Fig. S11B). Although IL-1β upregulates the mRNA transcription levels of NF-κB pathway-related genes, such as RELB, NFKBIA, and NFKB1 (Fig. S11C), IL-1β still reduced the bioluminescence signal after these NF-κB pathway-related genes knockout (Fig. S11D). Taken together, these results demonstrated that IL-1β inhibits SARS-CoV-2 spike induced cell-cell fusion independent from the TAK1-IKKβ-NF-κB signaling cascade.

Furthermore, we validated these findings in authentic SARS-CoV-2 infected Caco-2 and Calu-3 cells. Consist with the results from HEK293T cells, IL-1β failed to reduce S2’ cleavage and N protein in the presence of IRAK1/4 inhibitor, whereas it still reduced S2’ cleavage and N protein in the presence of the IKKβ inhibitor TPCA1 in Caco-2 (Fig. 3F) and Calu-3 cells (Fig. 3G).

IL-1β inhibits SARS-CoV-2 induced cell-cell fusion through RhoA/ROCK mediated actin bundle formation at the cell-cell junction

It has been reported that IL-1β activates RhoA signaling via MyD88 and IRAK, which is a pathway independent from IKKβ (Chen, Zuraw, Liu, Huang, & Pan, 2002). As a major downstream effector of RhoA, ROCK phosphorylates substrates that are involved in the regulation of the actin cytoskeleton, cell attachment, and cell motility (Riento & Ridley, 2003). Therefore, we set out to detect the active level of RhoA through pull-down assay. To this end, we verified that IL-1β activated RhoA signaling in sgControl HEK293T cells but not in sgMyD88-or sgTRAF6-HEK293T cells (Fig. 4A). To directly visualize the distribution of endogenous GTP-RhoA (active RhoA), we used a location biosensor derived from the carboxy terminus of anillin (GFP-AHPH) (Priya et al., 2015; Sun et al., 2015). Interestingly, IL-1β significantly increased the fluorescence intensity of GFP-AHPH in sgControl HEK293T cells, but had no effect in sgMyD88– and sgTRAF6-HEK293T cells (Fig. 4 B and C).

Fig. 4.

To investigate whether IL-1β inhibits SARS-CoV-2 spike-induced cell-cell fusion through RhoA/ROCK pathway, we co-transfected GFP-AHPH in ACE2-expressing cells, then co-cultured with S-expressing cells at different time points. In the process of syncytia formation, cell-cell contact established between S-expressing cells and ACE2-expressing cells, and GFP-AHPH localized distally from cell-cell junction in the early stage of syncytia formation. With the enlargement of syncytium, GFP-AHPH is visualized at the periphery of syncytium (Fig. 4D, top panel). However, in IL-1β treated group, GFP-AHPH foci is enriched to the cell-cell junction in the early stage. Over time, GFP-AHPH was recruited more to the cell-cell junction between S-expressing cells and ACE2-expressing cells, preventing further cell-cell fusion (Fig. 4D, bottom panel). Cartoon schematics inserted in the imaging data illustrate such findings in a modeled manner.

ROCK-mediated actin rearrangement is through the downstream substrates, which induces cross-linking of actin bundles with myosin and generate contractile forces along these bundles (Watanabe, Kato, Fujita, Ishizaki, & Narumiya, 1999). It has been reported that RhoA initiates actin arc formation (Dupraz et al., 2019; Stern et al., 2021), so we further explored the changes of actin cytoskeleton during SARS-CoV-2 spike-induced cell-cell fusion. Phalloidin staining showed that actin filaments (F-actin) at cell-cell junction between S-expressing cells and ACE2-expressing cells was gradually disappeared along with cell-cell fusion in the early stages of syncytia formation. With the formation and enlargement of syncytium, F-actin of syncytium is preferably distributed peripherally (Fig. 4E, top panel). However, IL-1β activated RhoA to initiate actin bundles formation at cell-cell junction, the formation of these actin bundles potentially generates barriers and prevents membrane fusion between S-expressing cells and ACE2-expressing cells. Even with the prolonged co-culture time, IL-1β-induced actin bundles formed at cell junctions consistently inhibited further syncytia formation (Fig. 4E, bottom panel).

Consistent with our previous results, immunofluorescence staining showed GFP-AHPH moving to the opposite of cell-cell junction and locating peripherally with syncytia formation (Fig. 4F, top panel), while upon IL-1β treatment, GFP-AHPH located at the cell-cell junction of authentic SARS-CoV-2 infected cells and neighboring cells (Fig. 4F, bottom panel). In parallel, staining results showed that F-actin at the cell-cell junction were disassembled during authentic SARS-CoV-2 infection; with the formation of syncytium, F-actin of syncytium was mainly distributed peripherally (Fig. 4G, top panel). However, actin bundles formed at cell-cell junction induced by IL-1β inhibited membrane fusion and further syncytia formation (Fig. 4G, bottom panel). Furthermore, we also observed the same results in authentic SARS-CoV-2 infected Caco-2 cells. F-actin of syncytium in control group was also distributed peripherally, the formation of syncytium promoted the transmission of SARS-CoV-2, as N protein was widely distributed in the cytoplasm of the syncytium (Fig. 4H, top panel). While in IL-1β treated group, actin bundles formed at cell-cell junction between SARS-CoV-2 infected cells and neighboring cells prevented syncytia formation and further viral transmission (Fig. 4H, bottom panel). Together, these data revealed that IL-1β induced the formation of actin bundles at the cell-cell junction of SARS-CoV-2 infected cells and neighboring cells through RhoA/ROCK pathway, which inhibited SARS-CoV-2 induced cell-cell fusion.

To further investigate the role of RhoA/ROCK pathway in inhibiting SARS-CoV-2 induced cell-cell fusion, we co-transfected constitutively activated RhoA L63 (Nobes & Hall, 1999) (RhoA-CA) plasmid with spike or ACE2 in HEK293T cells, and found that RhoA-CA significantly reduced the bioluminescence signal and S2’ cleavage in a dose-dependent manner (Fig. 5A). HEK293T-ACE2 (Fig. S12A), Caco-2 (Fig. 5B) and Calu-3 cells (Fig. 5C) expressing RhoA-CA also significantly reduced S2’ cleavage and N protein compared to the control group during authentic SARS-CoV-2 infection. Meanwhile, we observed that constitutive activation of RhoA enriches actin bundles at cell-cell junction, thus preventing SARS-CoV-2 induced cell-cell fusion in HEK293T-ACE2 (Fig. S12B), Caco-2 (Fig. 5D) and Calu-3 cells (Fig. 5E).

Fig. 5.

ROCK inhibitor Y-27632 treatment prevents the formation of actin bundles (van der Heijden et al., 2008), so we hypothesized that Y-27632 could affect the inhibitory effect of IL-1β on SARS-CoV-2 spike-induced cell-cell fusion. Indeed, Y-27632 treatment increased bioluminescence signal and S2’ cleavage in a dose-dependent manner, promoting syncytia formation. When treated with lower concentrations of Y-27632, IL-1β can eliminate Y-27632-enhanced cell-cell fusion. However, IL-1β was unable to inhibit cell-cell fusion in the presence of higher concentrations of Y-27632 (Fig. S12C). Immunofluorescence results showed that although IL-1β induced actin bundles inhibited cell-cell fusion (Fig. S12D, top panel), higher concentrations of ROCK inhibitor Y-27632 treatment prevented the formation of IL-1β-induced actin bundles at cell-cell junctions, promoting membrane fusion and cytoplasmic exchange between S-expressing cells and ACE2-expressing cells, which restored syncytia formation (Fig. S12D, bottom panel). Furthermore, we verified that IL-1β was unable to reduce S2’ cleavage and N protein in the presence of Y-27632 in authentic SARS-CoV-2 infected Caco-2 (Fig. 5F), Calu-3 cells (Fig. 5G) and human lung cells (Fig. 5H). Immunofluorescence results also confirmed that the elimination of IL-1β induced actin bundles by Y-27632 in Caco-2 (Fig. 5I) and Calu-3 cells (Fig. 5J). These results indicated that preventing the formation of RhoA/ROCK mediated actin bundles at cell-cell junction promotes SARS-CoV-2 induced cell-cell fusion.

IL-1β restricts SARS-CoV-2 transmission via induction of actin bundles in vivo

To demonstrate the role of IL-1β in controlling SARS-CoV-2 transmission in vivo, BALB/c mice were infected with authentic SARS-CoV-2 B.1.351 after IL-1β or IL-1RA+IL-1β pre-treatment (Fig. S13A). Interestingly, the results of this experiment showed that in mice with IL-1β treatment, the body weight loss was less than in the control group, while IL-1β was unable to improve body weight in the presence of IL-1RA (Fig. S13B). According to hematoxylin and eosin (H&E) staining, tissue histopathology analysis demonstrated that the mice with IL-1β treatment carry less pulmonary injury compared to the control and IL-1RA+IL-1β group (Fig. 6 A and B). In addition, the expression level of SARS-CoV-2 N gene in the lung from IL-1β treated mice was significantly lower than in the control and IL-1RA+IL-1β treated mice (Fig. 6C). In addition, immunohistochemistry staining showed that the infected area in the epithelial linings of lung tissue was significantly reduced by IL-1β treatment compared to the control and IL-1RA+IL-1β groups (Fig. 6 D and E), indicating that IL-1β restricted the transmission of SARS-CoV-2 in the lung. Moreover, fluorescence staining showed that SARS-CoV-2 infected lung epithelial cells fused with neighboring cells, promoting viral transmission in the airway epithelial cells, while IL-1β induced the formation of actin bundles to restrict the syncytia formation and further viral transmission (Fig. 6F, Fig. S13 C and D).

Fig. 6.

To further verify the function and mechanism of IL-1β in controlling SARS-CoV-2 transmission, we treated BALB/c mice with PBS, IL-1β or ROCK inhibitor Y-26732 + IL-1β (Fig. S14A), then isolated the lung tissue cells for authentic SARS-CoV-2 infection. We found that S2’ cleavage and N protein were significantly reduced in IL-1β treated mice compared to control mice at day 2, while Y-26732 treatment abolished the inhibitory effect of IL-1β on S2’ cleavage and N protein (Fig. 6G). Of note, the lung tissue cells in IL-1β treated mice remained resistant to SARS-CoV-2 infection at day 7, while the protective effect of IL-1β was abolished by Y-26732 treatment (Fig. S14B).

In addition, we found that IL-1β treated mice have no significant changes in body weight, liver and spleen weight compared to control mice (Fig. S15 A-D), indicating that this dose of IL-1β did not cause toxicity. Of note, we isolated the tissue cells and then infected with authentic SARS-CoV-2, the results showed that S2’ cleavage and N protein were significantly reduced in IL-1β treated mice compared to control mice both in isolated lung and intestine tissue cells (Fig.S15 E and F), suggesting that IL-1β may have protective effects on various tissue cells during SARS-CoV-2 infection. Taken together, IL-1β prevents the transmission of SARS-CoV-2 in vivo by inducing the formation of actin bundles.

Discussion

In the present study, we explored the functional roles of innate immune factors against SARS-CoV-2 infection. Notably, IL-1β inhibited various SARS-CoV-2 variants and other beta-coronaviruses spike-induced cell-cell fusion. Mechanistically, IL-1β activates and enriches RhoA to the cell-cell junction between SARS-CoV-2-infected cells and neighboring cells via the IL-1R-mediated signal to initiate actin bundle formation, preventing cell-cell fusion and viral spreading (Fig. S16). These findings revealed a critical antiviral function for pro-inflammatory cytokines to control viral infection.

Elevated IL-1β levels in severe COVID-19 patients is central to innate immune response as it induces the expression of other pro-inflammatory cytokines (Tahtinen et al., 2022). In addition, IL-1α is also secreted during SARS-CoV-2 infection (Xiao et al., 2021). Of note, several therapeutic strategies have employed the inhibition of IL-1 signal in an attempt to treat SARS-CoV-2 infection (Huet et al., 2020; Ucciferri et al., 2020). Intriguingly, although anakinra, a recombinant human IL-1 receptor antagonist, improved clinical outcomes and reduced mortality in severe COVID-19 patients (Cavalli et al., 2020), it did not reduce mortality in mild-to-moderate COVID-19 patients, and even increased the probability of serious adverse events (Tharaux et al., 2021). With another note, IL-1 blockade significantly decreased the neutralizing activity of serous anti-SARS-CoV-2 antibodies in severe COVID-19 patients (Della-Torre et al., 2021). According to our finding that both IL-1β and IL-1α are able to inhibit SARS-CoV-2-induced cell-cell fusion, inhibition of IL-1 signaling may have abolished the antiviral function of IL-1, thus failing to restrict virus-induced syncytia formation and transmission.

Notably, IL-1β plays a key role in triggering vaccine-induced innate immunity, suggesting that innate immune responses play important roles in the antiviral defense by enhancing the protective efficacy of vaccines (Eisenbarth, Colegio, O’Connor, Sutterwala, & Flavell, 2008; Tahtinen et al., 2022). In addition, vaccination with Bacillus Calmette-Guérin (BCG) has been reported to confer nonspecific protection against heterologous pathogens, including protection against SARS-CoV-2 infection in human and mice (Hilligan et al., 2022; A. Lee et al., 2023; Rivas et al., 2021). In addition, Lipid nanoparticle (LNP) in mRNA vaccine (Han et al., 2023) and penton base in adenovirus vaccine (Di Paolo et al., 2009) can both activate innate immune cells to amplify the protective effect of vaccines, which may also be attributed to IL-1β-mediated inhibition of SARS-CoV-2-induced cell-cell fusion on top of adaptive immune responses induced by the vaccines.

With another note, patients with inherited MyD88 or IRAK4 deficiency have been reported to be selectively vulnerable to COVID-19 pneumonia. It was found that these patients’ susceptibility to SARS-CoV-2 can be attributed to impaired type I IFN production, which do not sense the virus correctly in the absence of MyD88 or IRAK4 (García-García et al., 2023). In our study, MyD88 or IRAK4 deficiency abolished the inhibitory effect of IL-1β on SARS-CoV-2 induced cell-cell fusion, suggesting that these innate immune molecules are critical to contain SARS-CoV-2 infection, and this may be another mechanism accounting for the disease of those patients. Moreover, MyD88 signaling was essential for BCG induced innate and type 1 helper T cell (TH1 cell) responses, and protection against SARS-CoV-2, which is consistent with our fundings.

Of note, cell-cell fusion is not limited to the process of viral infection, both normal and cancerous cells can utilize this physiological process in tissue regeneration or tumor evolution (Delespaul et al., 2020; Powell et al., 2011). For example, myoblast fusion is the key process of skeletal muscle terminal differentiation, inactivation of RhoA/ROCK signaling is crucial for myoblast fusion (Nishiyama, Kii, & Kudo, 2004). Our current work revealed that inhibition of RhoA/ROCK signaling promoted virus-induced cell-cell fusion, possibly due to the virus hijacking of such biological process. In turn, activated RhoA/ROCK signaling inhibits virus-induced cell-cell fusion, so it can be targeted for future therapeutic development to control viral transmission. Cell-cell fusion is mediated by actin cytoskeletal rearrangements, the dissolution of F-actin focus is essential for cell-cell fusion; in contrast, syncytia formation cannot proceed if disassembly of actin filaments or bundles is prevented (Doherty et al., 2011; Rodríguez-Pérez et al., 2021). We uncovered that preventing actin bundles dissolution inhibited virus-induced cell-cell fusion, and IL-1β induced RhoA/ROCK signal promotes actin bundle formation at cell-cell junctions. As RhoA is ubiquitously expressed by all cell types, it is currently unclear whether IL-1-mediated RhoA activation is specific towards viral infection-associated cytoskeleton modification, or may regulate other RhoA-related processes, which is a limitation of the current work and remains to be investigated in future.

In summary, this study demonstrated the function and mechanism of IL-1β in inhibiting SARS-CoV-2 induced syncytia formation, and highlighted the function of innate immune factors including cytokines against coronaviruses transmission, thus provide potential therapeutic targets for viral control.

Materials and Methods

Reagents and plasmids

The antibodies used for immunoblotting include: rabbit anti-SARS-CoV-2 S2 (Sino Biological, 40590-T62, 1:2000), mouse anti-SARS-CoV-2 N (Sino Biological, 40143-MM05, 1:1000), rabbit anti-ACE2 (Proteintech, 21115-1-AP, 1:2000), rabbit anti-MERS-CoV S2 (Sino Biological, 40070-T62, 1:1000), rabbit anti-MyD88 (Cell Signaling Technology, 4283, 1:1000), rabbit anti-TRAF6 (Abcam, ab33915, 1:5000), rabbit anti-TAK1 (Cell Signaling Technology, 4505, 1:1000), mouse anti-Myc-Tag (Abclonal, AE010,1:2000), HRP-conjugated β-tubulin (Abclonal, AC030, 1:5000), mouse anti-β-actin (Proteintech, 66009-1-Ig, 1:5000), anti-rabbit/anti-mouse (Jackson Immuno Research, 111-035-003, 1:5000). The antibodies and regents used for immunofluorescence include: rabbit anti-SARS-CoV-2 S2 (Sino Biological, 40590-T62, 1:200), mouse anti-SARS-CoV-2 N (Sino Biological, 40143-MM05, 1:200), rabbit anti-ACE2 (Proteintech, 21115-1-AP, 1:200), mouse anti-HA-Tag (Abclonal, AE008, 1:200). Actin-Tracker Green-488 (Beyotime, C2201S,1:100), goat α-mouse IgG-555 (Invitrogen, A-21424, 1: 400) and goat α-rabbit IgG-647 (Invitrogen, A-21236, 1: 400), DAPI (Abcam, ab228549, 1:2000) and antifade mounting medium (vectorlabs, H-1400-10). Purified LTA from S. aureus (Invitrogen, tlrl-pslta), Pam3CSK4 (Invitrogen, tlrl-pms), Peptidoglycan from S. aureus (Sigma-Aldrich, 77140), LPS (Invitrogen, tlrl-eklps), TPCA1 (Selleck, S2824), 5Z-7-Oxozeaenol (Sigma-Aldrich, O9890), IRAK1/4 inhibitor (Selleck, S6598), Y-27632 (Selleck, S6390). Inhibitors were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, D2650), and DMSO was added as solvent control. Recombinant human IL-1α (200-01A), human IL-1β (200-01B), mouse IL-1β (211-11B), human IL-1RA (200-01RA), human IL-6 (200-06) and human IL-8 (200-08M) were purchased from Peprotech. mouse IL-1RA (769706) was purchased from BioLegend. IL-1β concentrations in supernatants from THP-1 and PBMCs were determined using ELISA kits, according to the manufacturer’s instructions (R&D Systems, DY201). RhoA pull-down activation assay Biochem Kit (BK036-S) was purchased from Cytoskeleton, Inc. Collagenase, type I (17100017) was purchased from Gibco and B-ALI Growth Media (00193516) was purchased from Lonza Bioscience.

SARS-CoV-2 spike (Wuhan-Hu-1, GenBank: QHD43419.1) was homo sapiens codon-optimized and generated de novo into pVAX1 vector by recursive polymerase chain reaction (PCR). WT, Alpha, Beta and Delta variants containing point and deletion mutations were generated using stepwise mutagenesis using spike construct containing the truncated 19 amino acids at the C-terminal (CTΔ19). The latest human codon optimized Omicron was purchased from Genescripts, and subcloned into the pVAX1 backbone with CTΔ19 for comparison. Human ACE2 assembled in a pcDNA4.0 vector was used for transient expression of ACE2. GFP-AHPH (Addgene plasmid # 71368; http://n2t.net/addgene:71368; RRID: Addgene_71368) and pRK5myc RhoA L63 (Addgene plasmid # 15900; http://n2t.net/addgene:15900; RRID: Addgene_15900) were from Addgene.

Cell culture and stimulation

HEK293T cells were purchased from the National Science & Technology Infrastructure (NSTI) cell bank (www.cellbank.org.cn). Caco-2 cells (catalog no. SCSP-5027) were obtained from Cell Bank/Stem Cell Bank, Chinese Academy of Sciences. Vero E6-ACE2 and Calu-3 cells were gifted from Prof. Dimitri Lavillette (Applied Molecular Virology Laboratory, Discovery Biology Department, Institut Pasteur Korea). HEK293T and Vero E6-ACE2 were cultured in Gibco Dulbecco’s Modified Eagle Medium (DMEM) (GE Healthcare) supplemented with 10% fetal bovine serum (FBS) (Sigma) and 1% Penicillin/streptomycin (P/S) (Life Technologies) at 37°C with 5% CO₂ in a humidified incubator. Human colon epithelial carcinoma cell line Caco-2 and human lung cancer cell line Calu-3 cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% FBS, 1% non-essential amino acids and 1% P/S at 37°C with 5% CO₂ in a humidified incubator. Human monocytic cell line THP-1 was cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% FBS, 1% P/S and 50 μM 2-ME at 37°C with 5% CO₂ in a humidified incubator. All cells were routinely tested for mycoplasma contamination; passages between 4 ^th to 25 ^th were used. Human PBMCs were isolated from the peripheral blood of healthy doners (Shanghai Blood Center). This study was performed in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects and the principles expressed in the Declaration of Helsinki. Briefly, fresh human PBMCs were separated using Ficoll-Paque PLUS reagent (cytiva, 17144003) at 1200 g for 10 min at room temperature with SepMateTM-50 (SepMate, 86450). PBMCs were washed three times with filtered PBS containing 0.5% BSA and 2 mM EDTA. PBMCs was counted and resuspended in RPMI 1640 medium supplemented with 1% FBS and 1% P/S.

For stimulation, THP-1 cells were seeded at 2 × 10⁶ cells per ml in FBS free RPMI 1640 and PBMCs were seeded at 1 × 10⁷ cells per ml in 1% FBS RPMI 1640, then stimulated with LTA (10 μg/ml), Pam3CSK4 (1 μg/ml), PGN (2 μg/ml), LPS (1 μg/ml) for 24 hours, cell culture supernatants were collected after centrifugation at 2000 g for 5 min for subsequent experiments.

Transient transfection and cell-cell fusion assays

For transient transfections, HEK293T cells were seeded in flat bottom 24-well plates at 0.5 x 10⁶ cells /mL overnight. 250 ng plasmids encoding SARS-CoV-2 spike mutants or ACE2 variants were packaged in Lipofectamine 2000 (Life technologies) and transfected for 24 hours. For luciferase assays, S-mediated membrane fusion, a Cre-loxp Firefly luciferase (Stop-Luc) co-expression system was introduced to enable the detection of DNA recombination events during cell-cell-cell fusion. 200 ng Cre plasmids were co-transfected into S-HEK293T cells and 200 ng Stop-Luc plasmid were co-transfected into ±ACE2-HEK293T cells, respectively. For visualization of syncytia formation, 100 ng ZsGreen plasmid was co-transfected with spike variants. HEK293T cells in the 24-well plates were then detached using ice-cold calcium-free PBS in the absence of trypsin and centrifuged at 600 g for 4 min.

For cell-cell fusion assays, cell pellets were resuspended into complete DMEM and mixed with control HEK293T cells, or HEK293T-ACE2, Vero E6-ACE2 or Calu-3 cells at 1:1 ratio before adhesion to the 48-well or 96-well plates, cell mixes were incubated for 16 hours at 37°C. Quantification of cell-cell-cell fusion was performed by measuring luciferase expression as relative luminescence units (RLU) 1 min by mixing cell lysates with the Steady-Glo luciferase substrate (E2520, Promega) on a Synergy H1 plate reader (Biotek). Fluorescent images showing syncytia formation were captured at endpoint using a 10x objective and 12-bit monochrome CMOS camera installed on the IX73 inverted microscope (Olympus). Attached cells and syncytia were lysed in a NP-40 lysis buffer containing 0.5% (v/v) NP-40, 25 mM Tris pH 7.3, 150 mM NaCl, 5% glycerol and 1x EDTA-free protease inhibitor cocktail (PIC) (Roche).

Immunoblotting

Tissue culture plates containing adherent syncytia and cell mixes were directly lysed on ice in 2x reducing Laemmli loading buffer before boiled at 95°C for 5 min. Protein samples were separated by standard Tris-glycine SDS-PAGE on 7.5% or 9.5% Tris-glycine polyacrylamide gels. Proteins were then transferred onto 0.45 μm PVDF membranes (Millipore) for wet transfer using Towbin transfer buffer. All membranes were blocked in PBS supplemented with 0.1% Tween20 (PBST) and 2.5% bovine serum albumin (BSA) or 5% non-fat dry milk, before overnight incubation in primary antibodies at 4°C. Blots were labelled with HRP-tagged secondary antibodies (Jackson ImmnuoResearch) and visualized with PicoLight substrate enhanced chemiluminescence (ECL) solution (Epizyme Scientific). Immunoblot images were captured digitally using a 5200 chemiluminescent imaging system (Tanon) with molecular weight markers indicated.

Real time PCR

HEK293T cells of 0.5 x 10⁶ cells /mL were inoculated in 24 wells overnight. After the cells were about 80% covered, the specified stimulant was added, washed with PBS for three times, and 1 mL TRIzol Reagent (15596018; Thermo Fisher Scientific) was added for full lysis at room temperature for 5 min, add 250 μL chloroform fully mixed at room temperature for 5 min, centrifuge at 10000 r/min, centrifuge at 4°C for 10 min. Carefully remove the aqueous phase using a pipette. Leave behind some of the aqueous phase (about 1 mm above DNA layer to prevent DNA contamination). Place in another 1.5 mL Eppendorf tube. Add 550 µl isopropanol to the aqueous phase and mix gently. Leave at –20°C for 30 min. 14000 r/min, centrifuge at 4°C for 20 min, wash with 75% ethanol twice. Dissolve in 30 µL DEPC water. Then reverse transcribed to cDNA using a GoSript Reverse Transcription kit (Promega). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix (TOYOBO) on ABI QuantStudio 6 flex Real-time PCR System (Thermo Fisher Scientific). The RT-qPCR Primer sequences for targeting respective genes are displayed in Table S1. Target genes Relative quantification were normalized to GAPDH as relative unit (RU) via formula.

CRISPR/Cas9-Mediated Gene Targeting

Gene-deficient THP-1 or HEK293T cells were generated using CRISPR/Cas9-mediated gene targeting technology. Briefly, LentiCRISPR v2 (52961; addgene) containing sgRNA specifically targeting indicated genes were constructed. The sgRNA sequences for targeting respective genes are displayed in Table S2. The Lentiviral particles were produced in HEK293T cells by transfection with LentiCRISPR v2-sg gene, psPAX2, VSV-G at 2:1.5:1 ratio using Lipofectamine 2000. The lentiviral particles were then used to infect THP-1 or HEK293T cells. One day post infection, the cells were subjected to puromycin selection at a concentration of 2 μg/ml for 72 hours. Surviving cells were subjected to limiting dilution and seed in 96-well plates to obtain single clones stably knocking out respective genes.

RhoA pull-down assay

After 1 ng/mL IL-1β treatment for 30 min, place culture vessel on ice, aspirate media, wash with ice cold PBS. Aspirate off PBS. Tilt plates on ice for an additional 1 min to remove all remnants of PBS. Residual PBS will adversely affect the assay. Lysis cells in an ice-cold cell lysis buffer with protease inhibitor cocktail. Harvest cell lysates with a cell scraper. Transfer lysates into the pre-labeled sample tubes on ice. Immediately clarify by centrifugation at 10000 g, 4°C for 1 min. Save 20 µL of lysate for total RhoA. For pulldown assay, add 600 µg total cell protein to 10 μL rhotekin-RBD beads, then incubate at 4°C on a rotator for 1 hour. Pellet the rhotekin-RBD beads by centrifugation at 5000 g at 4°C for 1 min. Very carefully remove 90% of the supernatant, then wash the beads once with 500 μL each of Wash Buffer. Pellet the rhotekin-RBD beads by centrifugation at 5000 g at 4°C for 3 min. Very carefully remove the supernatant. Add 20 μL of 2x Laemmli sample buffer to each tube and thoroughly resuspend the beads. Boil the bead samples for 2 min. The samples were analyzed by Immunoblots.

Immunostaining and confocal microscopy

HEK293T cells were seeded overnight onto sterilized poly-D-lysine (100 ug/mL) (Beyotime, ST508) treated 12 mm coverslips (fisher scientific, 1254580) in 24-well plates. After transfection with spike mutants, cells were washed with PBS once before fixed with 4% (w/v) paraformaldehyde (PFA) for 20 min. Then, cells were washed twice with PBS and permeabilized by 0.1% Triton at room temperature for 10 min. Next, cells were washed twice with PBS and blocked with Immunol Staining Blocking Buffer (Beyotime, P0102) at room temperature for 1 hour. Primary antibodies were incubated at room temperature for 1 hour. Coverslips were then washed twice with PBS before incubation with Actin-Tracker Green-488 or second antibodies for 1 hour at room temperature. Coverslips were washed twice with PBS before DAPI staining for 10 min. Coverslips were washed twice with PBS before mounted in antifade mounting medium. Fluorescent images covering various areas on the coverslips were captured at 12-bit depth in monochrome using a 100x oil immersion objective on the Olympus SpinSR10 confocal microscope and subsequently processed using imageJ software (NIH) with scale bars labeled.

Authentic SARS-CoV-2 Infection of cells

All experiments involving authentic SARS-CoV-2 virus in vitro were conducted in the biosafety level 3 (BSL3) laboratory of the Shanghai municipal center for disease control and prevention (CDC). The experiments and protocols in this study were approved by the Ethical Review Committee of the Shanghai CDC. Briefly, HEK293T-ACE2 or Caco-2 cells were seeded into 24-well or 96-well plates at a density of 4 x 10⁵ cells per mL overnight, then pre-treated cells with different treatments for 1 hour before infected with 0.5 multiplicity of infection (MOI) Delta or WT authentic SARS-CoV-2 (B.1.617.2 and Wuhan-Hu-1) for 24 hours. Calu-3 cells were seeded into 24-well or 96-well plates at a density of 4 x 10⁵ cells per mL overnight, infected with 0.5 MOI WT authentic SARS-CoV-2 for 1 hour, then washed with PBS before treated cells with different treatments for 24 hours. Brightfield images were captured to indicate the syncytia formation, cell lysates were collected for spike S2’ cleavage and N protein immunoblots.

For mice tissue cells, specific pathogen-free 6-week-old female BALB/c mice were lightly anesthetized with isoflurane and intranasal with PBS, mIL-1β (1 μg/kg) or Y-26732 (1 mg/kg) + mIL-1β (1 μg/kg) at day 0, then mice were Intraperitoneal injection with PBS, mIL-1β (1 μg/kg) or Y-26732 (1 mg/kg) + mIL-1β (1 μg/kg) at day 1 and 2. At day 7, mice were anesthetized by intraperitoneal injection of Avertin (2,2,2-tribromoethanol, Sigma-Aldrich), then the thoracic cavity and abdominal cavity of the mice were opened, an outlet was cut in the left ventricle of the mice, and then the right ventricle of the mice was perfused with phosphate buffer saline (PBS) through the pulmonary artery to remove blood cells in the lung. Next, the lung digestive solution with HBSS 1ml, 1 mg/ml collagenase IA, DnaseI (200 mg/ml; Roche), DispaseII (4 U/ml; Gibco) and 5% FBS was injected into the lung cavity; and the intestine digestive solution containing DMEM 10mL, 1 μM DTT, 0.25 μM EDTA and 5% FBS, then peeled off the lung or intestine organs, and digested for 30 min with shaking at 37°C. After digestion, the cells were resuspended in DMEM supplemented with 10% FBS and 1% P/S, subsequently infected cells with 1 MOI authentic SARS-CoV-2 B.1.351 or BF.7 for 24 hours. All procedures were conducted in compliance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences.

For human lung cells, the human lung tissues were cut into small pieces of about 2mm and washed three times with HBSS solution containing 1% PS, digested with collagenase type I (100 mg+50 mL PBS) in an incubator at 37°C for 4 hours. Then filtered through a 70 μm filter and centrifuged at 500 g for 5 min at room temperature. Lysed with 3 mL Red Blood Cell Lysis Buffer for 5min at room temperature, and then centrifuged at 500 g for 5 min at room temperature, washed twice with HBSS solution containing 1% PS. Human lung cells were resuspended in B-ALI Growth Media and seeded into 96-well plates at a density of 4 x 10⁵ cells per mL overnight, infected with 0.5 MOI WT authentic SARS-CoV-2 for 1 hour, then washed with PBS before treated cells with different treatments for 24 hours. The experiments and protocols were approved by the Ethical Review Committee of the Shanghai CDC.

Authentic SARS-CoV-2 infection of BALB/c mice

Specific pathogen-free 6-week-old female BALB/c mice were lightly anesthetized with isoflurane and intranasal with PBS, mIL-1β (1 μg/kg) or mIL-1RA (150 μg/kg) + mIL-1β (1 μg/kg) for 1 hour, then intranasal challenge with 5 × 10⁴ FFU of SARS-CoV-2 B.1.351. For booster injection, mice were Intraperitoneal injection with PBS, mIL-1β (1 μg/kg) or mIL-1RA (150 μg/kg) + mIL-1β (1 μg/kg) at 1– and 2-days post infection (dpi). Mice were monitored daily for weight loss. Lungs were removed into trizol or 4% PFA at 4 dpi. All protocols were approved by the Institutional Animal Care and Use Committee of the Guangzhou Medical University.

Pulmonary histopathology

Lungs were collected from mice infected by SARS-CoV-2 at 4 dpi and fixed in 4% PFA (Bioss) for 12 hours followed by dehydrating, embedded in paraffin for sectioning, then stained with hematoxylin and eosin (H&E), immunohistochemistry (IHC) or immunofluorescence (IF). H&E and IHC analyzed by PerkinElmer Vectra 3, IF analyzed by Olympus SpinSR10 confocal microscope. The pathological scores were judged according to this paper (Curtis, Warnock, Arraj, & Kaltreider, 1990).

Statistics analysis

Bar graphs were presented as mean values ± standard error of mean (SEM) with individual data points. All statistical analyses were carried out with the Prism software v8.0.2 (GraphPad). Data with multiple groups were analyzed using matched one-way ANOVA followed by Sidak’s post hoc comparisons. Statistical significance P values were indicated between compared groups and shown on Figures.

Author Contributions

Conceptualization, X.Z., S.Y. and G.M.; Methodology, X.Z., S.Y., Y.J. and G.M.; Investigation, X.Z., S.Y., Y.Z., K.Y., Y.G., Me.C., D.D., Y.L., J.M., X.C., Y.Y., X.W., Y.J. and J.Z.; Writing – Original Draft, X.Z., S.Y.; Writing – Review & Editing, X.Z., S.Y., Y.J. and G.M.; Funding Acquisition, Y.Z., Y.J., M.C. J.Z and G.M.; Resources, Y.J., M.C. J.Z and G.M.; Supervision, G.M.

Competing Interest Statement

Authors declare that they have no competing interests.

Acknowledgements

We thank Qiuhong Guo, Prof. Dimitri Lavillette and Prof. Gary Wong for their experimental supports and key reagents used in this work. This study is supported by grants from Natural Science Foundation of China (92269202, 82825001, 92054104), National Key R&D Program of China (2022YFC2304700, 2022YFC2303200, 2022YFC2303502), Three-Year Initiative Plan for Strengthening Public Health System Construction in Shanghai (2023-2025) Key Discipline Project (GWVI-11.1-09), as well as the Shanghai Municipal Science and Technology Major Project (2019SHZDZX02). Guangdong Basic and Applied Basic Research Foundation (2023A1515010152) and Young Scientists Fund of the Guangzhou National Laboratory (QNPG23-03).