Necroptosis does not drive disease pathogenesis in a mouse infective model of SARS-CoV-2 in vivo

Abstract

Necroptosis, a type of lytic cell death executed by the pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) has been implicated in the detrimental inflammation caused by SARS-CoV-2 infection. We minimally and extensively passaged a single clinical SARS-CoV-2 isolate to create models of mild and severe disease in mice allowing us to dissect the role of necroptosis in SARS-CoV-2 disease pathogenesis. We infected wild-type and MLKL-deficient mice and found no significant differences in viral loads or lung pathology. In our model of severe COVID-19, MLKL-deficiency did not alter the host response, ameliorate weight loss, diminish systemic pro-inflammatory cytokines levels, or prevent lethality in aged animals. Our in vivo models indicate that necroptosis is dispensable in the pathogenesis of mild and severe COVID-19.

Fig. 1. Necroptosis does not contribute to viral clearance or disease upon infection with a clinical isolate of SARS-CoV-2 (P2).

A, B Mice were challenged intranasally with 10⁴ TCID50 of a clinical SARS-CoV-2 isolate (P2) A Animals were euthanised at defined days post-infection (dpi) and lungs were collected for viral quantification by TCID50 assay. (n = 5–12 mice per group, pooled from 2 independent experiments) B Daily percent weight change of infected animals compared to initial weight. (n = 4–6 mice per group) C Lungs were collected and fixed for histological analysis at 2 and 4 dpi. Representative images of hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stained lungs with SARS-CoV-2 nucleocapsid. Histological images are representative of at least 3 animals. Scale bar = 500 µm. D Western blot analysis of homogenized lungs from wild-type (WT) and MLK knockout (Mlkl^−/−) animals 3 days after P2 infection. Samples were probed for phosphorylated MLKL (p-MLKL), MLKL, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a house-keeping gene, phosphorylated RIPK1 (p-RIPK1), phosphorylated RIPK3 (p-RIPK3), RIPK1 and RIPK3. Whole cell lysates of bone marrow derived macrophages treated with Tumor Necrosis Factor (TNF), Smac mimetic IAP antagonist and pan-caspase inhibitor were used as a positive controls for necroptosis (n = 4 mice per group and are representative of 2 independent experiments).

Fig. 2. Severe disease caused by a mouse-adapted strain of SARS-CoV-2 (P21) is not affected by Mlkl knockout.

A, B, C WT and Mlkl^−/− mice were challenged intranasally with 10⁴ TCID50 of SARS-CoV-2 P21 and monitored 3 days post infection for A lung viral burden using TCID50 assay B percent weight change compared to initial weight (n = 9 mice per group), and C the sum of twenty-six cytokines/chemokines expressed in supernatants of lung homogenates (n = 4 mice per group; mean ± SD of each cytokine are shown). D Western blot analysis of homogenized lungs from mock (intranasal DMEM inoculation), wild-type (WT) and MLK knockout (Mlkl^−/−) animals 3 days after intranasal P21 infection (10,000 TCID50). Samples were probed for phosphorylated MLKL (p-MLKL), MLKL, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a house-keeping gene, phosphorylated RIPK1 (p-RIPK1), phosphorylated RIPK3 (p-RIPK3), RIPK1, RIPK3 and SARS-CoV-2 nucleocapsid. Whole cell lysates of bone marrow derived macrophages treated with Tumor Necrosis Factor (TNF), Smac mimetic IAP antagonist (LCL-161) and pan-caspase inhibitor (IDN-6556) were used as positive controls for necroptosis (n = 2 mice per group and are representative of 2 independent experiments). E Representative images of hematoxylin and eosin (H&E) and immunohistochemistry (IHC) stained lungs with SARS-CoV-2 nucleocapsid. Mice were challenged intranasally with 10⁴ TCID50 of SARS-CoV-2 P21 and lungs were collected and fixed for histological analysis at 3 dpi. Histological images are representative of at least 3 animals. Scale bars = 500 µm. Unpaired two-tailed student’s t-test (B) after performing log₁₀ transformation (A) and one-way ANOVA with multiple comparisons (C).

Fig. 3. Lack of necroptosis pathways does not affect age-related disease severity or response to re-challenge.

A, B Aged C57BL/6 and Mlkl^−/− mice (6–8 months) were intranasally inoculated with 10⁴ TCID50 SARS-CoV-2 P21 and monitored for A the proportion of mice that became moribund, reaching humane endpoint and B daily percentual weight change over time, relative to the initial weight. (n = 6–8 mice per group; Data is representative of 2 independent experiments) C, B Mock or P21-infected C57BL/6 and Mlkl^−/− mice were rechallenged 28 days later with P21 and analyzed 3 days post re-challenge for C lung viral load (TCID50) and D percent weight change, compared to weight before second infection (n = 4 mice per group; Data are representative of 2 independent experiments). One-way ANOVA with multiple comparisons (D), after log₁₀ transformation (C) was performed. Mean ± SD are shown. ***p < 0.001, ****p < 0.0001.