SARS-CoV-2 Spike S1 glycoprotein is a TLR4 agonist, upregulates ACE2 expression and induces pro-inflammatory M1macrophage polarisation
Authors: Aboudounya Mohamed M. , Holt Mark R. , Heads Richard J. 
Published: August 2021

Background and aims

TLR4 is an important innate immune receptor that recognizes bacterial LPS, viral proteins and other pathogen associated molecular patterns (PAMPs). It is expressed on tissue-resident and immune cells. We previously proposed a model whereby SARS-CoV-2 activation of TLR4 via its spike glycoprotein S1 domain increases ACE2 expression, viral loads and hyperinflammation with COVID-19 disease [1]. Here we test this hypothesisin vitroand demonstrate that the SARS-CoV-2 spike S1 domain is a TLR4 agonist in rat and human cells and induces a pro-inflammatory M1 macrophage phenotype in human THP-1 monocyte-derived macrophages.

Methods

Adult rat cardiac tissue resident macrophage-derived fibrocytes (rcTMFs) were treated with either bacterial LPS or recombinant SARS-CoV-2 spike S1 glycoprotein. The expression of ACE2 and other inflammatory and fibrosis markers were assessed by immunoblotting. S1/TLR4 co-localisation/binding was assessed by immunocytochemistry and proximity ligation assays on rcTMFs and human HEK-293 HA-TLR4-expressing cells. THP-1 monocytes were differentiated into M1 or M2 macrophages with LPS/IFNγ, S1/IFNγ or IL-4 and RNA was extracted for RT-qPCR of M1/M2 markers and ACE2.

Results

TLR4 activation by spike S1 or LPS resulted in the upregulation of ACE2 in rcTMFs as shown by immunoblotting. Likewise, spike S1 caused TLR4-mediated induction of the inflammatory/wound healing marker COX-2 and concomitant downregulation of the fibrosis markers CTGF and Col3a1, similar to LPS. The specific TLR4 TIR domain signalling inhibitor CLI-095 (Resatorvid®), blocked the effects of spike S1 and LPS, confirming that spike S1 is a TLR4 agonist and viral PAMP (VAMP). ACE2 expression was also inhibited by the dynamin inhibitor Dynasore®, suggesting ACE2 expression is mediated by the alternative endosomal/β-interferon pathway. Confocal immunofluorescence microscopy confirmed 1:1 stoichiometric spike S1 co-localisation with TLR4 in rat and human cells. Furthermore, proximity ligation assays confirmed spike S1 and TLR4 binding in human and rat cells. Spike S1/IFN-γ treatment of THP-1-derived macrophages induced pro-inflammatory M1polarisation as shown by an increase in IL-1β and IL-6 mRNA.

Conclusions

These results confirm that TLR4 is activated by the SARS-CoV-2 spike protein S1 domain and therefore TLR4 may be a receptor/accessory factor for the virus. By binding to and activating TLR4, spike S1 caused upregulation of ACE2, which may facilitate viral entry into cells. In addition, pro-inflammatory M1macrophage polarisation via TLR4 activation, links TLR4 activation by spike S1 to inflammation. The clinical trial testing of CLI-095 (Resatorvid®) and other TLR4 antagonists in severe COVID-19, to reduce both viral entry into cells and hyperinflammation, is warranted. Our findings likely represent an important development in COVID-19 pathophysiology and treatment, particularly regarding cardiac complications and the role of macrophages.