RESUMEN
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 show that murine bone marrow-derived macrophages (BMDMs) stimulated with TLR3 and TLR7 agonists induce 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 a significantly higher cell-surface and endosomal TLR7 expression compared to TLR3, which further correlated with early and robust MAPK (pERK1/2 and p-P38) and NF-kB activation in TLR7-stimulated macrophages. Furthermore, blocking EKR1/2, p38, 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 responses, and blocking ERK1/2 activity would mitigate viral-RNA/TLR-induced dysregulated immunity.
RESUMEN
SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses cause fatal pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and lethal inflammatory responses in-vivo are not well defined. CoVs possess single-stranded RNA (ssRNA) genome that is abundantly produced during infection and stimulates both antiviral interferon (IFN) and inflammatory cytokine/ chemokine responses. Therefore, in this study, using wild-type control and TLR7 deficient BALB/c mice infected with a mouse-adapted SARS-COV-2 (MA-CoV-2), we evaluated the role of TLR7 signaling in MA-CoV-2-induced antiviral and inflammatory responses and disease outcome. We show that TLR7-deficient mice are more susceptible to MA-CoV-2 infection as compared to infected control mice. Further evaluation of MA-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral type I (IFNα/ß) and type III (IFNλ) IFNs, IFN stimulated genes (ISGs, ISG15 and CXCL10), and several pro-inflammatory cytokines/chemokines in TLR7 deficient compared to control mice. Reduced lung IFN/ISG levels and increased morbidity/mortality in TLR7 deficient mice correlated with high lung viral titer. Detailed examination of total cells from MA-CoV-2 infected lungs showed high neutrophil count in TLR7 deficient mice compared to control mice. Additionally, blocking TLR7 activity post-MA-CoV-2 infection using a specific inhibitor also enhanced disease severity. In summary, our results conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite robust inflammatory response, TLR7-mediated IFN/ISG responses likely protect the host from lethal disease. Given similar outcomes in control and TLR7 deficient humans and mice, these results show that MA-CoV-2 infected mice serve as excellent model to study COVID-19.