Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection
Authors: Totura Allison L. , Whitmore Alan , Agnihothram Sudhakar , Schäfer Alexandra , Katze Michael G. , Heise Mark T. , Baric Ralph S. 
Published: May 2015

Toll-like receptors (TLRs) are sensors that recognize molecular patterns from viruses, bacteria, and fungi to initiate innate immune responses to invading pathogens. The emergence of highly pathogenic coronaviruses severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) is a concern for global public health, as there is a lack of efficacious vaccine platforms and antiviral therapeutic strategies. Previously, it was shown that MyD88, an adaptor protein necessary for signaling by multiple TLRs, is a required component of the innate immune response to mouse-adapted SARS-CoV infection
in vivo
. Here, we demonstrate that TLR3
−/−
, TLR4
−/−
, and TRAM
−/−
mice are more susceptible to SARS-CoV than wild-type mice but experience only transient weight loss with no mortality in response to infection. In contrast, mice deficient in the TLR3/TLR4 adaptor TRIF are highly susceptible to SARS-CoV infection, showing increased weight loss, mortality, reduced lung function, increased lung pathology, and higher viral titers. Distinct alterations in inflammation were present in TRIF
−/−
mice infected with SARS-CoV, including excess infiltration of neutrophils and inflammatory cell types that correlate with increased pathology of other known causes of acute respiratory distress syndrome (ARDS), including influenza virus infections. Aberrant proinflammatory cytokine, chemokine, and interferon-stimulated gene (ISG) signaling programs were also noted following infection of TRIF
−/−
mice that were similar to those seen in human patients with poor disease outcome following SARS-CoV or MERS-CoV infection. These findings highlight the importance of TLR adaptor signaling in generating a balanced protective innate immune response to highly pathogenic coronavirus infections.

IMPORTANCE
Toll-like receptors are a family of sensor proteins that enable the immune system to differentiate between “self” and “non-self.” Agonists and antagonists of TLRs have been proposed to have utility as vaccine adjuvants or antiviral compounds. In the last 15 years, the emergence of highly pathogenic coronaviruses SARS-CoV and MERS-CoV has caused significant disease accompanied by high mortality rates in human populations, but no approved therapeutic treatments or vaccines currently exist. Here, we demonstrate that TLR signaling through the TRIF adaptor protein protects mice from lethal SARS-CoV disease. Our findings indicate that a balanced immune response operating through both TRIF-driven and MyD88-driven pathways likely provides the most effective host cell intrinsic antiviral defense responses to severe SARS-CoV disease, while removal of either branch of TLR signaling causes lethal SARS-CoV disease in our mouse model. These data should inform the design and use of TLR agonists and antagonists in coronavirus-specific vaccine and antiviral strategies.