Mechanisms of IL-6 expression in peripheral monocyte-derived macrophages infected with SARS-CoV-2

*Important notice: Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Monocytes are often the first cells of the immune system to contact any viral pathogen, including SARS-CoV-2. However, the detailed mechanisms of the expression of IL-6 in peripheral monocyte-derived macrophages (MDM) infected with SARS-CoV-2 remain unclear.

About the study

In the present study, researchers added the freeze/thawed lysate of SARS-CoV-2-infected cells to cultured macrophages (MDM) or into pluripotent myeloid cells to mimic SARS-CoV-2-infection in alveoli, following which these cells secreted IL-6. They studied the underlying mechanisms controlling IL-6 expression in peripheral monocyte-derived macrophages (MDM) infected with SARS-CoV-2. They also demonstrated that anti-nucleocapsid (N) antibodies enhanced nucleocapsid (N) protein-mediated induction of IL-6. 

Study findings

While examining the effects of SARS-CoV-2-infected cells on MDM (as a model of SARS-CoV-2 infection in alveoli), lysates of transmembrane serine protease 2 (TMPRSS2)/VeroE6 cells infected with SARS-CoV-2 were added to MDM together with SARS-CoV-2. 

Despite the fact that the levels of SARS-CoV-2 RNA decreased over time, the authors noted elevated levels of IL-6 in the supernatants of MDM incubated with SARS-CoV-2-infected cell lysate, but not in those with uninfected cell lysate. Interestingly, three different strains of SARS-CoV-2 isolates were able to induce IL-6 production by MDMs.

Testing myelomonocytic leukemia cells (K-ML2) cells as a substitute for MD, the authors noted that SARS-CoV-2-infected cell lysates induced IL-6 production from K-ML2 cells, suggesting a specific component of SARS-CoV-2-infected cells stimulated MDM and K-ML2 cells to produce IL-6.

Screening of SARS-CoV-2 protein-expressing plasmids transfected into derivative of human embryonic kidney 293 cells containing the SV40 T-antigen (293T cells) revealed that SARS-CoV-2 N protein, including recombinant N protein, induced IL-6 production more efficiently than SARS_CoV-2 spike (S) protein. Further, using plasmids encoding truncated versions of the N protein, authors were able to identify that the C-terminal domain (CTD) of the N protein was responsible for IL-6-inducing activity.

In the culture supernatants of both granulocyte-macrophage colony-stimulating factor (GM-CSF)-stimulated and macrophage colony-stimulating factor (M-CSF)-stimulated MDM, two days after N protein stimulation, assay results showed elevated levels of multiple cytokines. All these cytokines, including IL-6, IL-8, tumor necrosis factor (TNF-α), macrophage inflammatory protein-1 beta (MIP-1β), pentraxin-3, and thymic stromal lymphopoietin (TSLP), elevate during severe COVID-19. 

In GM-CSF-stimulated MDM, an N protein-induced increase in IP-10, a predictive marker for severe disease in COVID-19, was observed, indicating that MDM can be a source of cytokines in COVID-19 patients even without productive infection of SARS-CoV-2.

Upon transfecting N protein-containing K-ML2 cells with serially diluted patient sera from actual SARS-CoV-2 patients, although 0.01–10% of the COVID-19 patient sera enhanced the IL-6 production, sera from five healthy donors did not affect the IL-6 production. 

Further, when researchers evaluated 203 serum samples from mild, moderate, and severe COVID-19 cases, they observed that sera of severe patients induced significantly more IL-6 than others; likewise, anti-N antibodies levels were elevated.

Conclusions

The study demonstrated that SARS-CoV-2-infected epithelial cells induced IL-6 production via bystander macrophages or myeloid cells (due to the presence of SARS-CoV-2 N protein). Thus, COVID-19 symptoms rapidly worsen in some cases from five to eight days of the symptom onset, and anti-N antibodies enhance this phenomenon.

The results showed that the proposed mechanism for the antibody-dependent enhancement (ADE) of IL-6 production from macrophages could occur without actual productive SARS-CoV-2 infection. The study, thus, provided evidence for relatively lesser efficacy of the inactivated whole viruses-based COVID-19 vaccines than messenger ribonucleic acid (mRNA)-based vaccines, such as the widely used Moderna vaccine.

Regarding the COVID-19 treatment, steroids, anti-IL-6 receptor antibodies are recommended to suppress excessive immune responses. Five anti-N antibodies, including N1, N4, N5 N9, and N12 may be useful as novel treatment strategies for COVID-19, although this is possible only when future research establishes COVID-19 treatments based on IL-6 suppression.

*Important notice: Research Square 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.