RNA virus-induced excessive inflammation and impaired antiviral interferon (IFN-I) responses are associated with severe disease. This innate immune response, also referred to as “dysregulated immunity” is caused by viral single-stranded RNA (ssRNA)- and double-stranded-RNA (dsRNA)-mediated exuberant inflammation and viral protein-induced IFN antagonism. However, key host factors and the underlying mechanism driving viral RNA-mediated dysregulated immunity are poorly defined. Here, using viral ssRNA and dsRNA mimics, which activate toll-like receptor 7 (TLR7) and TLR3, respectively, we evaluated the role of viral RNAs in causing dysregulated immunity. We observed that murine bone marrow-derived macrophages (BMDMs), when stimulated with TLR3 and TLR7 agonists, induced differential inflammatory and antiviral cytokine response. TLR7 activation triggered a robust inflammatory cytokine/chemokine induction compared to TLR3 activation, whereas TLR3 stimulation induced significantly increased IFN/IFN stimulated gene (ISG) response relative to TLR7 activation. To define the mechanistic basis for dysregulated immunity, we examined cell-surface and endosomal TLR levels and downstream mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-kB) activation. We identified significantly higher cell-surface and endosomal TLR7 levels compared to TLR3, which were associated with early and robust MAPK (p-ERK1/2, p-P38, and p-JNK) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2 and NF-kB activity reduced TLR3/7-induced inflammatory cytokine/chemokine levels, whereas only ERK1/2 inhibition enhanced viral RNA mimic-induced IFN/ISG responses. Collectively, our results illustrate that high cell-surface and endosomal TLR7 expression and robust ERK1/2 activation drive viral ssRNA mimic-induced excessive inflammatory and reduced IFN/ISG response and blocking ERK1/2 activity would likely mitigate viral-RNA/TLR-induced dysregulated immunity.

An excessive inflammatory response, also known as “cytokine storm,” is a major cause of severe COVID-19. Viral RNA and its structural proteins are key mediators of SARS-CoV-2-induced inflammation. While the immune sensors and host inflammatory responses induced by SARS-CoV-2 RNA and the structural proteins spike (S), envelope (E), and nucleocapsid (N) are well defined, the role of membrane (M) protein in inflammatory/antiviral response and the signaling pathway facilitating M protein-mediated immune response is not well characterized. Here, we show that SARS-CoV-2 M protein induces robust inflammatory cytokine responses in murine and human macrophages and lungs. We also show that the M protein-induced inflammatory response relies on TLR4 signaling and that MyD88 serves as the key adaptor driving inflammation, while TRIF is primarily required for the antiviral IFN response. Detailed evaluation of downstream signaling pathways highlighted the critical roles of mitogen-activated protein kinase (MAPK) molecules JNK and ERK1/2, followed by nuclear factor kappa B (NF-κB) and p38 signaling in the M protein-TLR4-mediated inflammatory response. Finally, we show that abrogation of M protein-induced ERK1/2 and p38 signaling reduced inflammatory cytokine production and rescued type I interferon production in wild-type macrophages, while inhibition of JNK and NF-κB suppressed the M protein-induced inflammatory and antiviral cytokine responses. Collectively, our results demonstrate that SARS-CoV-2 M protein induces pro-inflammatory cytokines and type I interferons via TLR4-MyD88/TRIF activation, with MAPK activation playing a dominant role, and offer specific targeting of MAPK signaling to mitigate SARS-CoV-2-induced excessive inflammation and pathology. IMPORTANCE: Severe COVID-19 is often caused by an excessive inflammatory response, but the role of the SARS-CoV-2 membrane (M) protein in this process has not been well understood. We show that the M protein strongly activates inflammatory and antiviral immune responses through TLR4 signaling and identify the key signaling pathways involved. We also demonstrate that selectively targeting MAPK signaling can reduce harmful inflammation while restoring antiviral interferon responses. These findings improve our understanding of how SARS-CoV-2 causes disease and identify potential therapeutic targets to limit excessive inflammation without compromising antiviral immunity.