SARS-CoV-2 S protein triggers lung and intestinal epithelial cell damage via TGF-β/Smad2/3-mediated inflammatory cytokine production

doi:10.21203/rs.3.rs-1999623/v1

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SARS-CoV-2 S protein triggers lung and intestinal epithelial cell damage via TGF-β/Smad2/3-mediated inflammatory cytokine production

https://doi.org/10.21203/rs.3.rs-1999623/v1

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Background and objective Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to ravage the world. Despite many prevention and control measures, morbidity and mortality have not decreased due to SARS-CoV-2-induced organ damage, which occur via unknown mechanisms.

Methods Primary Human small intestinal mucosa epithelial cells (HSIMECs), human colonic epithelial cells (HCoEpiCs), and human type II alveolar epithelial cells (hTIIAECs) were treated with recombinant SARS-CoV-2 S protein for 48 h. Cell morphology, permeability, and viability were detected. The expression of cysteinyl aspartate specific proteinase 3 (caspase 3) and B-cell lymphoma-2 (Bcl-2) was examined using Western blotting. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of inflammatory cytokines in the supernatant. Apoptosis was observed using a Hoechst 33258 Staining Kit. SB431542 and BAY11-7082 were used to inhibit transforming growth factor-β (TGF-β)/suppressor of mothers against decapentaplegics (Smads) and the inhibitor of kappa B kinase (IKK)/nuclear factor-κB (NF-κB) pathways, respectively.

Results S protein produced no obvious changes in morphology, but decreased cell viability and increased permeability were observed in a concentration- and time-dependent manner compared to the control (P<0.05). Apoptosis occurred with increased caspase 3 and decreased Bcl-2 (P<0.05). S protein stimulated a disordered secretion of cytokines, including interleukin (IL)-6, IL-10, tumour necrosis factor α (TNF-α), and IL-13 (P<0.05). Suppression of TGF-β/Smad3, but not the IKK/NF-κB, pathway relieved the damage to colon cells caused by the S protein in HSIMECs and HCoEpiCs and inhibited apoptosis mediated by TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) in hTIIAECs.

Conclusion The SARS-CoV-2 S protein damaged intestine and lung cells, which was associated with cytokine production and the induction of apoptosis mediated by the TGF-β/Smad3, but not the NF-κB, pathway.

COVID-19

SARS-CoV-2

S protein

Inflammation

Transforming growth factor-β

Tissue damage

Coronavirus disease 2019 (COVID-19) pneumonia, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is rapidly spreading to hundreds of countries worldwide (1). COVID-19 has a high fatality rate due to organ and tissue damage (2), which have dramatically influenced the living and working conditions of billions of people worldwide. Therefore, the World Health Organization (WHO) has declared COVID-19 a public health emergency of international concern. However, the mechanisms of cell damage triggered by SARS-CoV-2 are not clear.

Increasing evidence suggests that the lungs are the primary target organs of SARS-CoV-2, and infection results in pneumonia and respiratory failure due to congestion and alveolar exudation (3). Although COVID-19 presents with a range of clinical manifestations, the initial appearance is characterized by systemic and respiratory symptoms. Other symptoms, including digestive system-related symptoms, also exist due to increased circulating proinflammatory cytokines and the aggregation of inflammatory cells in target organs, such as the intestine (4-6). The pathological damage caused by SARS-CoV-2 infection occurs via its intensively glycosylated spike (S) proteins and its receptor binding domain (RBD), which bind to angiotensin-converting enzyme 2 (ACE2) receptors that are primarily expressed in human type II alveolar epithelial cells (7-9). This binding leads to reinforcement of surface tension and further acute respiratory distress syndrome (ARDS). The injured pneumocytes activate several immune cells to release a cascade of cytokines that promote inflammation. These inflammatory responses further exacerbate other tissue damage (10-12). A large number of studies showed that the inflammatory response induced by the S protein was related to the activation of several signalling pathways, but the specific mechanisms are not known.

Cells and primary reagents

Primary human small intestinal mucosa epithelial cells (HSIMECs, HUM-iCell-d007) and human type II alveolar epithelial cells (hTIIAECs, HUM-iCell-a002) were obtained from Guangzhou Taylor Biological Technology Co., Ltd (Guangzhou, Guangdong, China). Human colonic epithelial cells (HCoEpiCs) were obtained from Guangzhou Genio Biological Technology Co., Ltd. (Guangzhou, Guangdong, China, JNO-1787). Human normal intestinal epithelial cell NCM460 was from BFB (Shanghai, China, BFN608006385). Recombinant SARS-CoV-2 (2019-nCOV) spike RNA-binding domain (RBD)-His protein (S protein) was purchased from Sino Biological Inc. (0.25 mg/ml, 40592-V08H70-100). A human interleukin 6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit (MM-0049H1), human interleukin 10 (IL-10) ELISA kit (MM-0066H1), human interleukin 13 (IL-13) ELISA kit (MM-0062H1), human tumour necrosis factor α (TNF-α) ELISA kit (MM-0122H1), and granulocyte-macrophage colony-stimulating factor (GM-CSF) ELISA kit (ml025475) were purchased from Wuhan Meimian Biotechnology Co., Ltd. Horse radish peroxidase (HRP) was purchased from Solarbio (P8020, China). Foetal bovine serum was purchased from Gibco (42G3279K). Modified Eagle’s medium (MEM) was purchased from Procell (PM150410). Trypsin enzyme was purchased from HyClone (SH30042.01). Rabbit anti-cysteinyl aspartate specific proteinase 3 (caspase 3, ab32351), B-cell lymphoma-2 (Bcl-2) antibody (ab32124), and anti-GAPDH rabbit pAb (ab181602) were purchased from Abcam. Rabbit anti-ph-nuclear factor-κB (NF-κB) p65 (ab76302), anti-ph-suppressor of mothers against decapentaplegic 2 (Smad2, ab280888), and anti-ph-Smad3 (ab52903) antibodies were purchased from Abcam. Rabbit anti-ph-IκBα (13776-1) was purchased from SAB (USA). Horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG (P010104) and a Hoechst staining kit (C0003) were purchased from Beyotime (China).

Treatment of intestinal epithelial cells with S protein

HSIMECs, HCoEpiCs and hTIIAECs were plated in six-well plates overnight at 37 ℃ with 5% CO₂. S protein at different concentrations was added to the wells, and the final concentrations were 50 ng/ml, 100 ng/ml, and 500 ng/ml. The cells were cultured for 24 h, 36 h, and 48 h, and cell morphology was observed using a microscope. All experiments were duplicated at least three times (the same below).

Detection of cell activity using cell counting kit-8 (CCK-8)

Cell activity was detected using enhanced CCK-8 (Beyotime, China, C0042) according to the manufacturer’s instructions. Briefly, 100 μl/well CCK-8 mixture (diluted 10 times with serum-free medium) was added to cells treated with S protein and incubated at 37 °C in 5% CO₂ for 3 h. The absorbance value was detected at 450 nm.

Cell permeability test

HRP was used as a tracer to detect cell permeability. The cells were inoculated in a double permeable Transwell (0.4 μm) at a density of 3×10⁵/well, treated with different conditions and cultured for 24 h, 36 h, and 48 h. The medium in the upper and lower chambers was replaced, and 0.5 μm HRP serum-free medium was added to the upper chamber of the Transwell and incubated at 37 °C for 1 hour. The lower chamber liquid was collected and added to a 96-well plate (10 μl/well) for measurement of HRP permeability. The tetramethylbenzidine (TMB) substrate (100 μl) was added and incubated for 3 min away from light. TMB stop buffer (100 μl) was added, and the absorbance was measured at 450 nm.

Measurement of inflammatory cytokines in the cell supernatant using ELISA

Cell cultures and treatments were the same as described above. The cell supernatants were collected and added to a 96-well plate. A standard solution was prepared according to the instructions. Fifty microlitres of sample diluent was added to each well and incubated at 37 °C for 30 min. HRP-conjugated reagent (50 μl) was added to each well. Chromogenic reagent A (50 μl) and 50 μl chromogenic reagent B were added to each well for 10 min, followed by 50 μl stop buffer. The absorption value was detected at 450 nm.

Intervention effect of TGF-ß/Smads and NF-kB pathway inhibitors on S protein induction using a neuronal conditioned medium 460 (NCM460) cell model

Human normal intestinal epithelial cell NCM460 was used to detect the effects of TGF-β/Smads and NF-kB pathway inhibitors on S protein-induced biological effects. NCM460 cells were cultured in six-well plates overnight at 37 ℃ with 5% CO₂. S protein (500 ng/ml), SB431542 (8 μM) or BAY11-7082 (50 μM) were added to the medium and treated for 24 h, 48 h, 72 h, and 96 h. The cells and supernatant were collected. The expression of related proteins and inflammatory factors, including IL-6, IL-13, GM-CSF, TNF-α, and IL-10, was detected using Western blotting (WB) and ELISA.

Observation of apoptosis using a Hoechst 33258 staining kit

Sterile cover slides were placed in a six-well plate, and hTIIAECs were plated on these slides overnight. The cells were treated with S protein or inhibitors, the culture medium was removed, and 0.5 ml fixative was added for 10 min. The fixative was removed, and the cells were washed twice with PBS. Hoechst 33258 staining solution (0.5 ml) was added for 5 min. The staining solution was removed, and the slides were washed twice with PBS. The cell nuclei were observed using fluorescence microscopy.

Western blotting

The cells were treated with S protein in the same manner as described above and inoculated in 6-well plates. The plates were washed 2 times, a ristocetin-induced platelet agglutination (RIPA) plus 1% phenylmethanesulfonylfluoride lysis solution was added to the cells. Centrifugation was performed at 12000 rpm at 4 °C for 20 min to obtain the supernatant. A Bradford protein assay kit was used to determine the protein concentration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed, and the proteins were transferred to membranes. The membranes were blocked with 5% skim milk blocking buffer at room temperature for 2 h. The membranes were incubated with anti-Bcl-2, anti-caspase-3, anti-ph-NF-κB p65, anti-ph-IκBα, anti-ph-Smad2, anti-ph-Smad3 and anti-GAPDH antibodies (1:1000) and an HRP-labelled second antibody (1:10000) for 1.5 h. The ECL exposure solution was a 1:1 mixture of liquid A:liquid B, and it was evenly applied to cover the entire membrane for 2 min, followed by exposure to detect the protein bands.

Statistical analysis

All data were analysed using the Solutions Statistical Package for the Social Sciences (SPSS) 23.0, and the results are presented as the mean (M) ± standard deviation (SD). Comparisons between multiple groups were performed using one-way analysis of variance (ANOVA), and post hoc multiple comparisons were performed using LSD-t. P values < 0.05 were considered statistically significant.

Introduction of SARS-CoV-2 S protein significantly promoted cell permeability and inhibited cell survival

To observe whether the S protein affected the morphologies of intestine and lung cells, cells were incubated with the S protein for 24 h, 36 h, and 48 h. As shown in Fig. 1, the treatment of HSIMECs and HCoEpiCs with the S protein produced no obvious changes in morphology compared with the cells without the S protein. S protein induced morphological changes of hTIIAECs to some extent (Fig. 1), and this effect was dose-dependent rather than time-dependent.

Although the morphological changes induced by the S protein were not significant, the cell permeability of intestinal epithelial cells was obviously increased (Fig. 2a-b). S protein significantly increased the optical density (OD) value of HSIMECs and HCoEpiCs, and these effects were concentration-dependent (P<0.05) and time-dependent (P<0.05). S protein promoted cell permeability and had a significant effect on cell viability. As shown in Fig. 2c-d, treatment with the S protein obviously reduced the viability of HSIMECs and HCoEpiCs compared to the control (without S protein) (P<0.05), and this inhibition of proliferation was concentration-dependent (P<0.05). Consistent with this observation, S protein significantly increased the expression of the pro-apoptosis protein caspase 3 in intestinal cells in a concentration-dependent (P<0.05) and time-dependent (P<0.05) manner (Fig. 2e-h). S protein obviously decreased the expression of Bcl-2 (P<0.05), and this decrease was also concentration- and time-dependent.

Cell damage decreases cell viability and increases cell permeability. The S protein also reduced cell viability (Fig. 3a) and increased cell permeability (Fig. 3b) of hTIIAECs in a dose-dependent manner, especially at 500 ng/ml. The reduction in cell viability may be related to apoptosis. As shown in Fig. 3c-e, the S protein significantly induced the expression of the proapoptotic protein caspase 3 in hTIIAECs and decreased the expression of the antiapoptotic protein Bcl-2, especially at 500 ng/ml, compared to the hTIIAECs without S protein treatment (P<0.05). The influence of the S protein on the expression of caspase 3 and Bcl-2 was also time-dependent. We observed morphological changes using Hoechst 33258 staining. The S protein induced apoptosis (Fig. 3f), which was consistent with the WB results.

SARS-CoV-2 S protein stimulated dysregulation of several inflammatory profiles in small intestine and colon cells

Inflammation is a pathological process after SARS-CoV-2 infection (13) and results in tissue and organ damage (14-16). NF-κB and TGF-β signalling mediate the production of several cytokines involved in the inflammatory response. S protein induced an abnormal expression of several inflammatory cytokines in HSIMECs, including IL-6, TNF-α, and IL-10 (P<0.05), but the S protein-induced levels of NF-κB, IL-13, and TGF-β1 were not significant compared with the control (Fig. 4a). Similar to the HSIMECs, S protein also stimulated an increase in IL-6 and TNF-α and a decrease in IL-13 (P<0.05), but no significant influence on NF-κB, IL-10, and TGF-β1 was observed compared with the control (Fig. 4b). These results suggest that the S protein does not affect the exocrine expression of NF-κB and TGF-β1. We speculated that NF-κB and TGF-β1 did not participate in the S protein-induced production of cytokines. BAY11-7082 and SB431542 are inhibitors of IKK/NF-κB and TGF-β signalling, respectively, and were used to observe the effects of the S protein in another normal intestinal cell model, NCM460 (Fig. 4c). Our results revealed that SB431542 reversed the reduced cell viability caused by the S protein, while BAY11-7082 enhanced the damage to colon epithelial cells induced by the S protein (Fig. 4c). Western blotting further demonstrated that S protein did not cause changes in p-IκBα, p-NF-κB p65, p-Smad2, or p-Smad3 expression (Fig. 4d). Treatment with BAY11-7082 and SB431542 influenced inflammatory factors, including IL-6, IL-13, IL-10, GM-CSF, and TNF-α (Fig. 4e). SB431542 increased the secretory levels of GM-CSF and TNF-α in the supernatant, which was opposite to the effect of BAY11-7082.

SARS-CoV-2 S protein caused dysregulation of inflammatory cytokine secretion in hTIIAECs, which was associated with activation of the TGF-β/Smad3, but not the NF-kB, pathway

We also observed the influence of the S protein on the levels of inflammatory factors in lung cells. As shown in Fig. 5a, the S protein upregulated the expression of IL-6 and TNF-α but downregulated the levels of IL-13 and TGF-β1 (P<0.05). The effects of the S protein on the TGF-β and NF-κB pathways were further examined in lung epithelial cells. S protein did not cause changes in the expression level of NF-kB pathway molecules in hTIIAECs, including p-NF-kB p65 and p-IkBα, as measured WB (Fig. 5b-c), but S protein upregulated the expression of p-Smad3, but not p-Smad2, compared to the control (P<0.05). These results suggested that the S protein induced activation of the TGF-β/Smad3 pathway, but not the NF-kB pathway, which was consistent with subsequent observations. Two inhibitors, SB431542 (8 μM) and BAY11-7082 (50 μM), were used to suppress the expression of TGF-β1 and IKK/NF-kB pathway components, respectively (Fig. 5b-c). Notably, SB431542 but not BAY11-7082 inhibited S protein-induced apoptosis in hTIIAECs (Fig. 5d). These data suggested that the S protein activated the TGF-β/Smad3 pathway in hTIIAECs. Inhibition of the TGF-β/Smad3 pathway by SB431542 decreased the levels of TNF-α, GM-CSF, IL-6, and IL-13 and increased the level of IL-10 (Fig. 5e-f). However, these phenomena were not observed in the cells treated with BAY11-7082, whose effect was consistent with the S model.

Clinical and experimental studies showed that the lungs were the primary target of SARS-CoV-2. SARS-CoV-2 infection causes extensive cellular damage and inflammatory cascades, termed cytokine storms, which are the pathological basis for severe cases (17-18). Our study showed that the S protein induced damage to lung and intestinal epithelial cells via the inhibition of cell viability, alterations in cell permeability, and promotion of apoptosis in time- and concentration-dependent manners. These processes were most significant at 500 ng/ml, which suggests that the injury of target cells resulting from SARS-CoV-2 is positively related to virulence (19-20). Recent studies found that infection and induction of SARS-CoV-2 caused cytotoxic effects in several human cells, such as cardiomyocytes (21), immune cells (22), and intestinal cells (23). Increasing clinical and experimental data demonstrated that SARS-CoV-2 induced injury in a variety of cells an\j tissues, including myocardial cells (24), endothelial cells (25), pulmonary microvascular transendothelial cells (25), and alveolar type 2 cells (26).

SARS-CoV-2 triggers inflammatory responses and cell death in lung epithelial cells via the activation of caspase-8 (27). We also observed that the S protein promoted caspase 3 expression, which suggests that caspase 3 is also involved in the induction of cell damage. The S protein also triggered apoptosis of intestinal cells by increasing caspase-3 and decreasing Bcl-2 in our study, which may underlie S protein promotion of cell damage. A number of recent studies also showed that cell death triggered by SARS-CoV-2 was associated with the induction of apoptosis via several mechanisms, such as caspase-8 and inflammasome activation (28-29) and autophagy promotion (30). These results suggest that the SARS-CoV-2-induced inflammatory response is an important factor in cell damage (30-31), and TNF-α, interferon-γ (IFN-γ), reactive oxygen species (ROS), and phosphatidylinositol 3-hydroxykinase (PI3K)/threonine kinase (AKT)/mechanistic target of rapamycin (mTOR) signalling are involved in these mechanisms (28-30). Extensive studies demonstrated that the entry of the virus into target cells caused a cytokine “storm”, which led to severe organ or tissue injury, including lung injury (31-33). Therefore, we used three cell lines derived from different tissues, HSIMECs, HCoEpiCs and hTIIAECs, and demonstrated that S protein induced the dysregulation of several inflammatory cytokines in these small intestine, colon and lung cells, including IL-6 and TNF-α. However, the S protein decreased the level of IL-10 instead of IL-13 in HSIMECs, but reduced the level of IL-13 instead of IL-10 in HCoEpiCs, which was consistent with hTIIAECs. These results suggest that the changes in IL-10 and IL-13 induced by the S protein are associated with the cell type. S protein reduced TGF-β1 but not NF-κB levels in the supernatant of hTIIAECs, but this phenomenon was not observed in HSIMECs or HCoEpiCs. These results suggest that TGF-β1 is related to S protein-induced biological effects in lung cells. Extensive studies reported that cell damage induced by the S protein was related to TGF-β1 and NF-κB signalling in inflammation.

We further observed that treatment with an inhibitor of the TGF-β1, but not NF-κB, signalling pathway relieved the S protein-induced reduction in intestinal cell viability, which was promoted by the IKK/NF-κB pathway inhibitor BAY11-7082. These data suggest that the TGF-β pathway contributes to intestinal cell damage via other pathways or mechanisms, and the NF-κB pathway does not play a significant role in this process. Inhibition of the TGF-β pathway, but not the NF-kB pathway, also relieved the S protein-induced apoptotic injury in hTIIAECs and improved inflammatory cytokines, which suggest that the S protein-activated TGF-β pathway, but not NF-kB p65, is involved in the process of lung cell damage. These results are consistent with other studies (34-35). Notably, a reduced concentration of TGF-β1 was found in the supernatant of hTIIAECs, which was inconsistent with the results of p-Smad2 and p-Smad3 in WB. We propose that the S protein, or other factors, deplete extracellular TGF-β1, and the intracellular Smad pathway is activated by other mechanisms. Ferreira-Gomes et al. observed that SARS-CoV-2 in severe COVID-19 induced a TGF-β-dominated chronic immune response that did not target itself (36), which is consistent with the results of multilevel proteomics analysis showing that the TGF-β pathway was specifically dysregulated by SARS-CoV-2 (37). An in vitro experiment further demonstrated that the S protein triggered a transcriptional response related to activation of TGF-β signalling, which is required for S protein-mediated barrier dysfunction (38), via the induction of host factor proteins, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin (39).

In conclusion, the S protein of SARS-CoV-2 triggered intestinal and lung epithelial cell injury and disorder of inflammatory factor secretion in vitro. Although the NF-κB and TGF-β pathways may not be involved in S protein induction of cell damage, blockade of the TGF-β, but not the NF-κB, pathway alleviated S protein-induced cell damage via other unknown mechanisms., Further research is needed to investigate these mechanisms.

ACE2: angiotensin-converting enzyme 2

ARDS: acute respiratory distress syndrome

AKT: threonine kinase

Bcl-2: B-cell lymphoma-2

Caspase 3: cysteinyl aspartate specific proteinase 3

COVID-19: coronavirus disease 2019

ELISA: enzyme-linked immunosorbent assay

GM-CSF: granulocyte-macrophage colony-stimulating factor

HSIMEC: Human Small Intestinal Mucosa Epithelial Cell

HCoEpiC: human colonic epithelial cells

HRP: horse radish peroxidase

hTIIAEC: human type II alveolar epithelial cell

IL: interleukin

IFN: Interferon

IKK: inhibitor of kappa B kinase

MEM:modified eagle medium

mTOR: Mechanistic Target Of Rapamycin

NF-κB: nuclear factor-κB

OD:optical density

PI3K: phosphatidylinositol 3-hydroxykinase

RBD: receptor binding domain

ROS: reactive oxygen species

SARS-CoV-2: severe acute respiratory syndrome coronavirus 2

SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis

Smad: small mothers against decapentaplegic

TGF-β: Transforming growth factor-β

TNF-a: tumor necrosis factor α

TMB: tetramethylbenzidine

WB: western blotting

Funding

This work was supported by “Bao'an Traditional Chinese Medicine Development Foundation-Special fund for research on COVID-19 treatment and epidemic prevention and control technologies and application of Traditional Chinese Medicine (2020KJCX-KTYJ-302)”.

Conflict of interest statement

All authors declare no conflict of interest.

Compliance with Ethical Standards

Ethical approval: This article does not contain any studies with human participants or animals performed by any of the authors.

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SARS-CoV-2 S protein triggers lung and intestinal epithelial cell damage via TGF-β/Smad2/3-mediated inflammatory cytokine production

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