SARS-CoV-2 spike protein interacts with and activates TLR41

Abstract

Dear Editor, Accumulating clinical data suggest the main causes of death by COVID-19 include respiratory failure and the onset of sepsis. 1 Importantly, sepsis has been observed in nearly all deceased patients. 2-5 It remains elusive how SARS-CoV-2 infection results in viral sepsis in humans. Toll-like receptor 4 (TLR4) mediates antigram-negative bacterial immune responses by recognizing lipopolysaccharide (LPS) from bacteria. 6 We recently found that SARS-CoV-2 infection provoked an anti-bacterial like response at the very early stage of infection via TLR4. However, the identity of the original trigger initiating these abnormal immune responses during SARS-CoV-2 infection is unknown. Previous in silico studies predicted cell surface TLRs, especially TLR4, are most likely to be involved in recognizing molecular patterns, probably spike protein, from SARS-CoV-2 to induce inflammatory responses. 7,8 Consistently, we found that the induction of IL1B by SARS-CoV-2 was completely blocked by TLR4-specific inhibitor Resatorvid (Fig. 1a). Combined with our recent data that TLR4 signaling was activated by SARS-CoV-2, we hypothesized that spike protein could activate TLR4 pathway. A recent study has reported that trimeric SARS-CoV-2 spike proteins are high quality antigens. 9 To this end, we purified the trimeric spike protein (1-1208 aa) (Fig. 1b; Supplementary information, Fig. S1a), as this form of spike protein presents on the surface of viral particle, which most likely interacts with the proteins on the cell surface. Results of the surface plasmon resonance (SPR) assay showed that SARS-CoV-2 spike trimer directly bound to TLR4 with an affinity of~300 nM (Fig. 1b), comparable to many virus-receptor interactions. We then treated THP-1 cells, a cell line of human monocytes, with purified spike protein. IL1B was robustly induced by spike protein in a dose-dependent manner (Fig. 1c), which was comparable to LPS (Supplementary information, Fig. S1b). IL6 was also induced by spike protein (Supplementary information, Fig. S1c). As IL1B induction was much more robust than that of IL6, we chose IL1B production as a marker of immune activation. Moreover, the pseudovirus expressing spike protein can also induce IL1B production (Fig. 1d). Neutrophils also express TLR4 on their cell surface and play an important role in the development of sepsis. We utilized all-trans retinoic acid (ATRA) to treat HL-60 cell (a promyelocytic leukemia cell line), which directed those cells to differentiate into neutrophils. Spike proteins significantly induced IL1B production in HL-60 cells after ATRA treatment (Fig. 1e; Supplementary information, Fig. S1d). We treated THP-1 cells with the N-terminal domain (NTD) or the receptor-binding domain (RBD) of spike protein. Only the trimeric protein could induce IL1B and IL6 (Fig. 1f; Supplementary information, Fig. S1e). To examine if this activation was mediated by TLR4, we treated cells with Resatorvid. Resatorvid greatly blocked induction of IL1B by spike protein and LPS (Fig. 1g). Moreover, spike pro...

Selectively expressing SARS-CoV-2 Spike protein S1 subunit in cardiomyocytes induces cardiac hypertrophy in mice

“…Although both NL63 and SARS-CoV-2 (CoV-2) use ACE2 as their receptor to infect host cells, NL63 is much less virulent than CoV-2, indicating that tissue tropism may not account for their dramatic virulent differences. Because CoV-2 but not NL63 causes severe cytokine storm (Mehta et al, 2020), and two independent studies have shown that CoV-2 Spike protein (CoV-2-S) activates TLR4/NF-kB signaling in macrophages (Zhao et al, 2021), we then suspected that CoV-2-S and NL63-S might have different effects on TLR4/NF-kB signaling.…”

“…In toxic shock syndrome, this motif interacts with both T cell receptor (TCR) and CD28 to elicit T lymphocyte activation (Cheng et al, 2020). Two independent in vitro studies have shown CoV-2-S to activate TLR4 innate immune signaling in cultured macrophages (Shirato and Kizaki, 2021; Zhao et al, 2021). These studies highlight CoV-2-S as a potent culprit that could possibly induce systemic inflammation; however, the underlying molecular mechanism is not fully understood.…”

“…To better understand how CoV-2 damages the heart, it is critical to elucidate the biology of CoV-2 encoded proteins, each of which may play multiple pathological roles. For example, CoV-2 Spike glycoprotein (CoV-2-S) not only engages angiotensin-converting enzyme 2 (ACE2) to mediate virus infection (Zhou et al, 2020), but also directly impairs endothelial function (Lei et al, 2021) and triggers TLR4/NF-kB signaling in cultured macrophages (Shirato and Kizaki, 2021; Zhao et al, 2021). ACE2 is a crucial enzyme of the renin angiotensin system (RAS) that maintains blood pressure homeostasis (Teerlink, 1996), and is critical in modulating cardiac hypertrophy through Angiotensin 2 regulation.…”

Proinflammatory responses in SARS-CoV-2 infected and soluble spike glycoprotein S1 subunit activated human macrophages

“…Reports using non-soluble SARS-CoV-2 trimeric spike (S) glycoprotein 10 and purified S1 subunit produced in E.coli 11 suggest that the S protein activates inflammation in macrophages. Prefusion trimeric S is cleaved by cellular proteases 12 that dissociate the S1 subunit during virion assembly 13 or after engagement of the ACE2 receptor 14 .…”

Targeting TLR4 Signaling to Blunt Viral-Mediated Acute Lung Injury

“…By understanding the mechanisms by which the virus-induced "cytokine storm" is mitigated, we expect to discover therapeutic targets in the influenza model that will be applicable also to patients with severe SARS-CoV-2 and other virus-induced lung diseases. A recent study reported that the SARS-CoV-2 trimeric spike (S) protein directly interacted with and activated TLR4 in vitro in human and murine macrophagelike cell lines, and in bone marrow-derived macrophages from wildtype, but not TLR4 -/-, mice (143). Moreover, treatment of cells with Resatorvid (TAK-242), that blocks MyD88 interaction with TLR4 (31), also blocked S protein-induced Il1b mRNA (143).…”