Data from a new study by researchers from Shenzhen Baoan Second People’s Hospital in China has alarmingly validated that not only does the spike proteins of the SARS-CoV-2 virus damage lung cells but they also damage and destroy the intestinal cells as well.
The study findings clearly showed that the SARS-CoV-2 spike protein damaged human small intestinal mucosa epithelial cells (HSIMECs), human colonic epithelial cells (HCoEpiCs), and human type II alveolar epithelial cells (hTIIAECs) which was associated with cytokine production and the induction of apoptosis mediated by the TGF-β/Smad3, but not the NF-κB, pathway.
The study findings were published on a preprint server and is currently being peer reviewed for publication into the journal: Molecular Biology Reports By Springer.
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.