Background <p>Recent findings indicate that hyperinflammatory responses to SARS-CoV-2 are major contributors to the severity and fatality of COVID-19. Pattern recognition receptors, particularly Toll-like receptors 2 (TLR2) and 4 (TLR4), have been implicated in detecting SARS-CoV-2 proteins, especially the spike protein. However, the role of viral structural components in triggering innate immune responses remains poorly understood.</p> Methods and results <p>HEK293 reporter cells, engineered to stably express TLR2 or TLR4, and THP-1 differentiated macrophages were exposed to various spike protein components and SARS-CoV-2 variants. Protein levels of cytokines were detected by ELISA and mRNA expression was evaluated by quantitative real-time RT-PCR. We demonstrate that the S1 subunit and full-length spike protein elicit TLR4-dependent pro-inflammatory responses. TLR4 activation was triggered by the monomeric, but not the homotrimeric, form of the SARS-CoV-2 spike protein.</p> Conclusions <p>These findings suggest that distinct elements of the SARS-CoV-2 spike protein differentially activate TLR4 signaling pathways, driving innate immune and inflammatory responses.</p>