IFITM3 protects the heart during influenza virus infection

Abstract

Influenza virus can disseminate from the lungs to the heart in severe infections and can induce cardiac pathology, but this has been difficult to study due to a lack of small animal models. In humans, polymorphisms in the gene encoding the antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) are associated with susceptibility to severe influenza, but whether IFITM3 deficiencies contribute to cardiac dysfunction during infection is unclear. We show that IFITM3 deficiency in a new knockout (KO) mouse model increases weight loss and mortality following influenza virus infections. We investigated this enhanced pathogenesis with the A/PR/8/34 (H1N1) (PR8) influenza virus strain, which is lethal in KO mice even at low doses, and observed increased replication of virus in the lungs, spleens, and hearts of KO mice compared with wild-type (WT) mice. Infected IFITM3 KO mice developed aberrant cardiac electrical activity, including decreased heart rate and irregular, arrhythmic RR (interbeat) intervals, whereas WT mice exhibited a mild decrease in heart rate without irregular RR intervals. Cardiac electrical dysfunction in PR8-infected KO mice was accompanied by increased activation of fibrotic pathways and fibrotic lesions in the heart. Infection with a sublethal dose of a less virulent influenza virus strain (A/WSN/33 [H1N1]) resulted in a milder cardiac electrical dysfunction in KO mice that subsided as the mice recovered. Our findings reveal an essential role for IFITM3 in limiting influenza virus replication and pathogenesis in heart tissue and establish IFITM3 KO mice as a powerful model for studying mild and severe influenza virus-induced cardiac dysfunction.

Keywords: IFITM3; heart; influenza; interferon.

Fig. 1.

Generation and validation of our IFITM3 KO C57BL/6 mouse model. (A) C57BL/6 zygotes were injected with a mix of Cas9 mRNA and 2 guide RNAs to target exon 1 of the Ifitm3 gene. As a result of a 53-bp deletion and consequent frame shift, the mutant Δ53 IFITM3 KO gene bears a nonsense mutation at codon 18 and a subsequent stop codon at position 37. The protein depicted for the mutant is hypothetical. (B) Example genotyping PCR on genomic DNA from WT, heterozygous (het), and KO mice. Sequencing of the PCR products revealed a 53-bp deletion in the KO allele. (C) RT-PCR for IFITM1, IFITM2, and IFITM3 performed on mRNA extracted from IFNβ-treated MEFs obtained from mice of the indicated genotypes. Sequencing of the PCR products confirmed the 53-bp deletion in the IFITM3 mRNA in IFITM3 KO cells and no mutation of IFITM1 and IFITM2 sequences. (D) Western blot analysis of tissue lysates from WT and IFITM3 KO mice. (E) MEFs derived from embryos of the indicated genotypes were treated with IFNβ or mock-treated, and lysates were subjected to Western blot analysis. (F) MEFs treated as in E were infected with IAV or VSV for 24 h, and percent infection was determined by flow cytometry. Graphs depict normalized measurements from at least 3 separate experiments. Relevant comparisons were analyzed by unpaired t tests as indicated by lines. *P < 0.001. NS, not significant.

Fig. 2.

IFITM3 KO mice experience increased morbidity and mortality on influenza virus infection. (A and B) WT (n = 29) and IFITM3 KO (n = 21) mice were intranasally infected with IAV strain PR8 (10 TCID₅₀) and followed daily for weight loss (A) and survival (B). Points in A depict mean values, and error bars represent SD of the mean. *P < 0.01, unpaired t test. (C) Infected mice were killed on the indicated days for TCID₅₀ measurement of virus titers in the lung. Data points collected for days 5 and 10 were from 2 independent experiments. (D) ELISA quantification of IL-6 in lungs collected on the indicated days postinfection. In C and D, each point represents an individual mouse, and bars represent mean values. Error bars represent SD of the mean. *P < 0.001, unpaired t test.

Fig. 3.

IFITM3 KO mice show uncontrolled influenza virus replication in the heart. WT and IFITM3 KO mice were intranasally infected with IAV strain PR8 (10 TCID₅₀). (A) Infected mice were killed on day 5, 7, or 10 postinfection for TCID₅₀ measurement of virus titers in the heart. Data points collected for days 5 and 10 were from 2 independent experiments. Each point represents an individual mouse, and bars represent mean values. Error bars represent SD of the mean. *P < 0.05, ***P < 0.0001, unpaired t test. ND, not detected. (B) Representative images of heart sections from mice killed on day 10 postinfection. Green, anti-influenza virus nucleoprotein (NP) staining; blue, DAPI; gray, brightfield imaging. (Scale bar: 10 μm.) (C) Infected mice were killed on day 10 postinfection for TCID₅₀ measurement of virus titers in the spleen, liver, kidney, and brain. Each point represents an individual mouse, and bars represent mean values. Error bars represent SD of the mean. ***P < 0.0001, unpaired t test. ND, not detected.

Fig. 4.

IFITM3 KO mice suffer from cardiac electrical dysfunction following influenza virus infection. WT and IFITM3 KO mice were intranasally infected with IAV strain PR8 (10 TCID₅₀). (A) ECG measurements over the time course of infection. Except for day 25, data were collected over at least 3 independent experiments. Each point represents an individual mouse, and bars represent mean values. Error bars represent SD of the mean. *P < 0.05, unpaired t test. (B) Example ECG readings from WT and KO mice preinfection and postinfection with PR8. Selected RR intervals of the infected mice are highlighted by blue (WT) or green (KO) double arrows. (C) RR interval times plotted for a representative WT mouse and a representative IFITM3 KO mouse on day 10 postinfection. (D) RR ranges, defined as the difference between the longest and shortest RR intervals over an ECG measurement period of 5 min, were calculated for individual mice on day 10 postinfection. Each point represents an individual mouse, and bars represent mean values. Error bars represent SD of the mean. ***P < 0.0001, unpaired t test.

Fig. 5.

Cardiac fibrosis in influenza virus infection of IFITM3 KO mice. WT and IFITM3 KO mice were intranasally infected with influenza virus strain PR8 (10 TCID₅₀) or were mock-infected. (A) Hearts were collected on day 10 postinfection, and sections were stained with Masson’s trichrome stain, in which blue staining is indicative of fibrotic collagen deposition. A representative infected heart and a representative mock-infected sample are shown for each genotype. Boxed areas are regions magnified in the far-right images. (B) Blue pixel percentages from images as in A were quantified for mock-infected and infected WT and KO hearts using ImageJ software. (C and D) qRT-PCR on mRNA extracted from hearts collected at day 10 postinfection was performed to assess expression of Col1A2 (C) and Tgfb1 (D). (E) IL-6 ELISA was performed on heart homogenates collected on day 10 postinfection. In B–E, each point represents a heart from an individual mouse, and bars represent mean values. Error bars represent SD of the mean. *P < 0.05, ***P < 0.0001, unpaired t test.