TLR2 And TLR4 Are Novel Activating Receptors For SARS-CoV-2 Spike Protein In NK Cells

COVID-19 News: The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has profoundly impacted global health, affecting millions of people worldwide. While initially identified as a respiratory virus, COVID-19 is now recognized as a complex, multi-organ disease with significant immune system involvement. A critical aspect of the body's defense against viral infections involves natural killer (NK) cells. These cells play a vital role in the innate immune response, particularly in the early stages of infection. In COVID-19 patients, particularly those with severe disease, a notable reduction in circulating NK cells has been observed, even though these cells appear highly activated or exhausted. This new study by researchers from Bambino Gesù Children’s Hospital, IRCCS-Italy, that is covered in this COVID-19 News report, aims to explore whether the SARS-CoV-2 spike protein (SP) directly activates NK cells and, if so, through which receptors.


rSP binds and activates NK cells. (A) Mean values ± SEM and representative dot plots of binding of biotinylated rSP (Wuhan) to freshly isolated NK cell subsets. (B) Expression of NKG2A, NKG2C, and CD57 on rSP-streptavidin positive CD56bright and CD56dim NK cell subsets. One representative dot plot (left panels) and the mean values ± SEM of three independent experiments (right panels) are reported. (C) Expression of CD69, NKG2A, NKG2C, and CD57 on untreated (CTRL) and rSP (G614)-stimulated CD56bright and CD56dim NK cells. Data of one representative experiment (left panels) and the mean values ± SEM of seven experiments (right panels) are reported. Statistical analysis was performed using a paired t-test. (D) CD25 and CD69 expression on the CD56bright and CD56dim NK cell subsets stimulated with different VOCs. Data are plotted as percent of expression on at least 20 HD. Statistical analysis was performed using paired ANOVA Kruskal–Wallis multiple comparison test *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. ns: not significant.
 
NK Cells in COVID-19
NK cells are crucial in the immune response against viral infections, including SARS-CoV-2. They are categorized into two main subsets: CD56brightCD16− and CD56dimCD16+. These subsets have different distributions and functions within the immune system. In COVID-19 patients, especially those with severe disease, a marked reduction in NK cell numbers has been reported. Interestingly, an inverse correlation exists between NK cell count and viral load, suggesting that NK cell numbers could serve as a prognostic marker for disease outcomes.
 
Activation and Exhaustion
Despite the reduction in NK cell numbers, those present in COVID-19 patients exhibit strong activation, as evidenced by increased expression of activation markers like CD69 and Ki-67. Patients who progress to severe disease typically show elevated levels of pro-inflammatory cytokines, such as IFN-γ, wh ich are primarily released by activated NK cells. However, these cells also display increased expression of inhibitory receptors, indicating a state of dysfunction or exhaustion. This phenomenon suggests that rather than combating the virus, NK cells might contribute to disease progression.
 
Direct Activation of NK Cells by SARS-CoV-2 Spike Protein
To investigate the direct effects of SARS-CoV-2 SP on NK cells, various experiments were conducted. Human NK cells were stimulated with SP from the Wuhan strain and other variants of concern (VOCs). The researchers evaluated NK cell activation markers, cytokine release, cytotoxic activity, and gene expression profiles. Additionally, small interfering RNAs (siRNAs) were used to silence specific genes to understand the involvement of various receptors in SP-induced NK cell activation.
 
Key Findings
The study revealed that SP from the Wuhan strain and other VOCs could directly bind and stimulate purified NK cells, enhancing their activation, cytokine release, and cytolytic activity. This effect was particularly notable in the CD56bright NK cell subset. Among the VOCs, the G614 and Delta-Plus strains exhibited the strongest activity in most donors. Interestingly, SP-driven NK cell functions were not mediated through the ACE2 receptor, which is not expressed on NK cells, or other known activating receptors. Instead, SPs were found to bind directly to Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), suggesting these receptors play a pivotal role in NK cell activation by SARS-CoV-2 SP.
 
Implications of TLR2 and TLR4 in NK Cell Activation
The direct binding of SP to TLR2 and TLR4 was confirmed through various experiments. SP-induced NK cell functions were significantly inhibited when these receptors were masked or when their respective genes were silenced. Additionally, phosphorylation of NF-kB and cytokine release, crucial markers of NK cell activation, were downregulated in TLR2 and TLR4 gene-silenced NK cells stimulated with SP. These findings underscore the novel role of TLR2 and TLR4 as activating receptors for SARS-CoV-2 SP on NK cells.
 
Differential Response in Recovered Patients
The study also examined the response of NK cells from COVID-19 recovered individuals to SP stimulation. It was found that VOC-SPs significantly upregulated activation markers and cytokine release in NK cells from recovered, but not uninfected, individuals. This suggests that NK cells from recovered patients retain a heightened ability to respond to SARS-CoV-2 SP, potentially contributing to immune memory and future protection against reinfection.
 
Novel Insights into NK Cell Function
This research provides crucial insights into the direct interaction between SARS-CoV-2 SP and NK cells, mediated by TLR2 and TLR4. These findings highlight a previously unrecognized role of these receptors in NK cell activation and suggest that NK cells play a more complex role in the pathophysiology of COVID-19 than previously thought. The study also raises the possibility that SP-driven NK cell activation might contribute to both the early immune response and the excessive inflammation observed in severe COVID-19 cases.
 
Therapeutic Implications
Understanding the mechanisms behind NK cell activation by SARS-CoV-2 SP opens new avenues for therapeutic intervention. Targeting TLR2 and TLR4, or their downstream signaling pathways, could potentially modulate NK cell responses, offering new strategies to manage severe COVID-19 and its complications. Moreover, these findings emphasize the importance of considering NK cell function in the development of COVID-19 treatments and vaccines.
 
Conclusion
This study uncovers a novel mechanism of NK cell activation by SARS-CoV-2 SP, mediated through TLR2 and TLR4. These receptors emerge as critical players in the immune response against COVID-19, providing new insights into the disease's pathophysiology and potential therapeutic targets. As the understanding of COVID-19 continues to evolve, these findings underscore the complexity of the immune response and the need for targeted strategies to enhance immune function while mitigating excessive inflammation.
 
The study findings were published in the peer reviewed journal: Frontiers in Immunology.
https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2024.1368946/full
 
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