SARS-CoV-2 spike stimulates hyperinflammation via Toll-like receptor 2

The coronavirus disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been ravaging the world for a year and a half, causing well over 2.6 million deaths.

A new study, released on the bioRxiv* preprint server, describes the mechanism of inflammation in severe COVID-19, besides revealing a potential new therapeutic target to modulate the severity of COVID-19.

The significant percentage of severe and critical disease following infection by the virus has been a cause of great concern. It is associated with dysregulated inflammation, but the pathogenesis remains unclear.

The spike protein

The current study demonstrates the inflammatory potential of the spike protein of the virus as being key to the cytokine storm. The spike protein not only forms the characteristic corona of the virus, but mediates receptor binding to the host cell angiotensin-converting enzyme 2 (ACE2) receptor on alveolar epithelial cells.

This is followed by viral entry and productive infection, with the replication of the genome and the transcription of genomic and subgenomic ribonucleic acid (RNA). The eventual outcome is the death of the infected cells.

Inflammatory injury in COVID-19

The lung tissue is further injured by the inflammatory infiltrate of macrophages, monocytes and neutrophils, all of which are responding to the expression or presence of viral proteins in the host cell. They are also activated by the products of dying cells.

This is called innate immunity and is essential to provide protection against the virus. In severe COVID-19, the excessive release of cytokines and chemokines produces deleterious effects on the host tissue, particularly in the lung and the endothelium of the vasculature.

Higher levels of inflammatory interleukins (IL) such as IL-2, IL-6, IL-8, the tumor necrosis factor (TNF), and chemokines like the monocyte chemotactic proteins (MCP1), with the potent growth factor granulocyte monocyte colony-stimulating factor (GMCSF) have been detected in the blood of COVID-19 patients.

Both IL-6 and TNF-alpha have been correlated with morbidity and mortality in COVID-19.

Pathway of inflammatory damage

The pattern of innate inflammation begins with the recognition of pathogen-associated molecular patterns (PAMPs). This is in response to pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), NOD-like receptors (NLRs), and RIG-I like receptors (RLRs).

The presence of viral RNA is detected by RNA sensing receptors, namely, TLR7, RIG-I, and MDA5. Following activation, PRRs set off multiple signaling molecules that mediate the activation of transcription factors for genes involved in immunity and inflammation. These include NF-kB, AP1, and IRF3.

IRF3 and NF-kB induce interferon (IFN) responses, both alpha and beta interferons, which are fundamental to antiviral responses, both innate and adaptive.

Earlier studies have shown that both dendritic cells and macrophages in the innate immune pathway produce inflammatory cytokines and chemokines in response to SARS-CoV infection. However, type I IFNs were not seen to be produced. This pattern of high inflammatory cytokines but low type I IFN is reproduced in COVID-19 patients with severe disease.

SARS-CoV-2 spike causes inflammation of innate immune cells

The current study demonstrates that macrophages stimulated with the spike, as well as with either of its subunits, the S1 or S2, produce IL-6, TNFa, and IL-1b. S2 is more potent in this respect.

Macrophages are key to the hyperinflammatory response in COVID-19. They produce signaling molecules that recruit T cells and other immune cells to the site of inflammation, further worsening the damage.

Chemokines like CXCL1, CXCL2, and CCL2 were released by the macrophages in response to S1 and S2, in a dose-dependent fashion. Other viral proteins failed to do so, and if the spike S2 was denatured by heat, no inflammatory response occurred either.

These results were confirmed by stimulating human peripheral blood mononuclear cells (hPBMC) with S2, when cytokines and chemokines were potently induced.

In lieu of mouse macrophages, that lack the human ACE2 receptor, the researchers challenged mouse bone marrow-derived macrophages (mBMDMs) with S1 and S2. Again, the same response was found, but no type I or type II IFN response.

Epithelial inflammatory response to spike

The virus also infects epithelium of the lungs, kidneys, gut and the vascular endothelium. Both S1 and S2 proteins resulted in inflammation in a delayed fashion, with the highest level being at 24 hours after stimulation. This was followed by gamma-interferon induction, but not type I IFN.

The presence of the spike within the cytosol did not, however, induce inflammation in epithelial cells.

Spike-expressing epithelial cells trigger macrophage inflammation

The results also showed that cytosolic spike within epithelial cells, but not kidney cells, induced inflammatory responses in co-cultured innate immune cells.

Spike activates the NF-kB pathway

The researchers observed that spike stimulation led to the activation of the NF-kB pathway, which is, along with MAPK, STAT3, and AKT, one of the signaling pathways concerned with activating transcription factors for inflammatory genes. STAT3 activation was also seen, but not MAPK or AKT.

TLR2-dependent NF-kB activation was observed. Typically, TLRs are triggered as a result of recognizing PAMPs on the cell surface or within the endosome, and this activation occurs via the adapter protein MyD88. The researchers found that spike protein activated the NF-kB pathway via this recognized pathway.

Specifically, TLR2, which is known to recognize lipoproteins, was identified to be the receptor responsible. In TLR2 knockout mice, intraperitoneal challenge with S1 or S2 resulted in increased levels of inflammatory cytokines IL-6, IL-1b, and TNFa.

Mechanism of activation of innate immune cells

The researchers suggest that innate immune cells like monocytes and macrophages recognize the spike protein at the cell surface via their TLR2 receptors, as described above. Secondly, spike-expressing epithelial cells can activate macrophages in their close vicinity.

Thirdly, epithelial cells can be activated by extracellular spike protein, inducing inflammatory signaling molecules, though not so potent as innate immune cells. However, this phenomenon can occur in the SARS-CoV2-infected lungs, which are the primary site of disease in COVID-19 patients.

What are the implications?

These data suggest that TLR2 is the immune sensor for SARS-CoV-2 S protein, which potentially triggers inflammatory responses through the activation of the NF-kB pathway.”

The spike protein is thus a powerful PAMP that induces hyperinflammation via this pathway.

Many COVID-19 vaccines have been developed to advanced clinical trial stages within a short period. However, most of them use the spike antigen, which may be important in view of the above findings.

Secondly, the emergence of new variants with extensive mutations in the spike protein may lead to more severe disease, possibly resistant to existing antibodies and to those elicited by current vaccines. This makes it imperative to develop effective therapies against the virus.

The study suggests TLR2 or its downstream adapters as ideal therapeutic targets to reduce the level of inflammation, and its consequences on the host, in COVID-19.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.