Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection

Abstract

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.

FIG 1

Two discrete TLR pathways regulate SARS-CoV pathogenesis. (A and B) Profiles from microarray analysis of MyD88 (A) and TLR3 (B) RNA expression results in 20-week-old C57BL/6J mice infected with 10³, 10⁴, or 10⁵ PFU of SARS-CoV indicate that differential levels of gene expression occurred at day 2 postinfection (a single asterisk [*] indicates differential expression determined by a >1.5 log_2-fold increase in expression compared to the results seen with mock infections; P < 0.05). (C and D) Infection of TLR3^−/− and C57BL/6NJ mice with SARS-CoV showed significantly greater weight loss in TLR3^−/− mice than in wild-type mice (**, P < 0.01; ***, P < 0.001, by nonparametric Mann-Whitney test, where values indicate the mean percent starting weight and error bars indicate standard deviation) (C), and viral titers were significantly higher in the TLR3^−/− mice than in wild-type mice (***, P < 0.001 [by Student’s unpaired t test]) (D). TLR3 signaling through the TRIF adaptor protein activated innate immune antiviral signaling programs in a MyD88-independent manner, indicating that at least two discrete TLR signaling pathways are involved in SARS-CoV pathogenesis. DPI, day postinfection.

FIG 2

TLR3^−/− mice show few alterations in cytokine and IFN signaling responses to SARS-CoV infection compared to wild-type mice. (A) TLR3 is a pattern recognition receptor that recognizes viral pathogen-associated molecular patterns and initiates antiviral signaling programs of IFNs, cytokines, and chemokines via the TRIF adaptor molecule. (B to I) RNA expression profiles of cytokines and ISGs downstream of TLR3 signaling measured by microarray analysis of IL-6 (B), TNF (C), CCL5 (D), IFN-γ (E), IFN-β (F), RSAD2 (G), CXCL10 (H), and IFIT1 (I) from TLR3^−/− or C57BL/6NJ mice infected with 10⁵ PFU of SARS-CoV normalized to the corresponding mock-infected, PBS-inoculated TLR3^−/− or C57BL/6NJ mice (n = 4 to 5 mice per group). Differentially expressed genes indicated by a single asterisk (*) showed a >1.5-fold change in expression levels between wild-type and knockout mice, with P < 0.05.

FIG 3

TRIF^−/− mice are highly susceptible to SARS-CoV infection. (A to C) TRIF^−/− mice infected with SARS-CoV have significantly greater weight loss (**, P < 0.01; ***, P < 0.001 [by a nonparametric Mann-Whitney test, where values indicate mean percent starting weight and error bars indicate standard deviation]) (A), viral titers (***, P < 0.001 [by unpaired Student’s t test]) (B), and lung hemorrhage scores (scored from 0 to 4; ***, P < 0.001 [by unpaired Student’s t test]) (C) than C57BL/6J mice infected with SARS-CoV over a 6-day course of infection. (D to F) Whole-body plethysmography analysis showed that SARS-CoV-infected TRIF^−/− mice (solid red line) have alterations in lung functions compared to SARS-CoV-infected C57BL/6J mice (solid black line), including enhanced pause (P_ENH) (D), T_PEF/T_e ratio (R_PEF) (E), and midtidal expiratory flow (EF₅₀) (F) levels over the course of 6 days (dashed red line, TRIF^−/− mock-infected mice; dashed black line, C57BL/6J mock-infected mice) (*, P < 0.05; **, P < 0.01; ***, P < 0.001 [by unpaired Student’s t test]).

FIG 4

Increased presence of viral antigen in the lungs of TRIF^−/− mice. (A to C) Immunohistochemistry was used to stain for SARS-CoV nucleocapsid antigen in the lungs of TRIF^−/− SARS-CoV-infected mice (first column in panels A to C) and C57BL/6J SARS-CoV-infected mice (second column in panels A to C) on day 2 postinfection with SARS-CoV. TRIF^−/− mock-inoculated mice and C57BL/6J mock-inoculated mice were also evaluated as negative controls and showed no viral antigen staining (data not shown). (D to F) Immunohistochemistry lung sections from day 2 postinfection were scored for the presence of SARS nucleocapsid antigen in the large airways (D) and lung parenchyma (E) and for overall staining (F). Sections were scored in a blinded manner, and scores were evaluated for significance by an unpaired Student’s t test (*, P < 0.05; **, P < 0.01; NS, not significant).

FIG 5

TLR4^−/− and TRAM^−/− mice are more susceptible to SARS-CoV infection than wild-type mice. (A) TLR4^−/−, TRAM^−/−, and wild-type mice were infected intranasally with 10⁵ PFU of SARS-CoV. Weight loss was measured each day postinfection, and TLR4^−/− mice lost significantly more weight than wild-type mice on days 3 to 7 postinfection (*, P < 0.05; ***, P < 0.001 [by nonparametric Mann-Whitney test; values indicate mean percent starting weight and error bars indicate standard deviation]). (B) TRAM^−/− mice lost significantly more weight than wild-type mice on days 2 to 10 postinfection (***, P < 0.001 [by nonparametric Mann-Whitney test, values indicate the mean percent starting weight and error bars indicate standard deviation]). (C) Virus titer in the lung was measured by plaque assay, and TLR4^−/− mice had significantly higher virus titers in the lungs at days 2 and 4 postinfection but had cleared the virus by day 7 postinfection, similarly to wild-type mice. (D) TRAM^−/− mice had significantly higher virus titers in the lungs at day 2 postinfection as measured by plaque assay, but the differences were not significant at day 4 postinfection, and both TRAM^−/− mice and wild-type mice had no detectable virus in the lungs by day 7 postinfection (*, P < 0.05; ***, P < 0.001 [by unpaired Student’s t test]; ND, not detected by plaque assay).

FIG 6

Aberrant proinflammatory cytokine, interferon, and interferon-stimulated gene signaling responses in TRIF^−/− mice infected with SARS-CoV. (A to D) RNA expression profiles of cytokines and chemokines downstream of TRIF and TLR signaling programs measured by qPCR analysis of IL-6 (A), TNF (B), CCL5 (C), and IFN-γ (D) from TRIF^−/− mice (red bars) or wild-type C57BL/6J mice (black bars) infected with 10⁵ PFU of SARS-CoV normalized to mock-infected TRIF^−/− or wild-type mice (n = 4 mice per group) at day 2 and day 4 postinfection. (E) TRIF^−/− mice infected with SARS-CoV have significantly higher protein levels of IFN-β measured by ELISA on day 2 and day 4 postinfection in lung homogenates. (F to H) RNA expression profiles of ISGs measured by qPCR analysis of RSAD2 (F), CXCL10 (G), and IFIT1 (H) in TRIF^−/− mice (red bars) or wild-type C57BL/6J mice (black bars) infected with 10⁵ PFU of SARS-CoV normalized to mock-infected TRIF^−/− mice or wild-type mice at day 2 and day 4 postinfection. Significant differences between groups were evaluated by an unpaired Student’s t test, bar graphs show the mean normalized fold change on the day postinfection, and the error bars indicate 1 standard deviation from the mean (*, P < 0.05, **, P < 0.01; ***, P < 0.001; NS, not significant).