Ifitm3 limits the severity of acute influenza in mice

Abstract

Interferon-induced transmembrane (IFITM) proteins are a family of viral restriction factors that inhibit the entry processes of several pathogenic viruses, including influenza A virus (IAV), in vitro. Here we report that IAV-infected knockout mice lacking the Ifitm locus on chromosome 7 exhibited accelerated disease progression, greater mortality, and higher pulmonary and systemic viral burdens as compared to wild type controls. We further observed that the phenotype of Ifitm3-specific knockout mice was indistinguishable from that of mice lacking the entire Ifitm locus. Ifitm3 was expressed by IAV target cells including alveolar type II pneumocytes and tracheal/bronchial respiratory epithelial cells. Robust Ifitm3 expression was also observed in several tissues in the absence of infection. Among murine Ifitm promoters, only that of Ifitm3 could be induced by type I and II interferons. Ifitm3 could also be upregulated by the gp130 cytokines IL-6 and oncostatin M on cells expressing appropriate receptors, suggesting that multiple cytokine signals could contribute to Ifitm3 expression in a cell or tissue-specific manner. Collectively, these findings establish a central role for Ifitm3 in limiting acute influenza in vivo, and provide further insight into Ifitm3 expression and regulation.

Figure 1. An Ifitm locus deletion renders mice more susceptible to influenza.

IfitmDel knockout mice challenged intranasally with either 1000 or 500 PFU of influenza A/PR/8/34 (H1N1) (PR8) exhibited more rapid disease progression and higher mortality than wild type and heterozygous controls. (A) At 1000 PFU, a survival analysis between wild type and knockout animals showed significant differences (p = 0.0067, Mantel-Cox test) in survival time as did a comparison of wild type and heterozygous mice (p = 0.0266). With a 500 PFU challenge, survival differences between all pairs of groups were statistically significant: wild type and knockout (p = 0.0011), wild type and heterozygous (p = 0.0366), heterozygous and knockout (p = 0.0012). At both challenge doses, heterozygous animals exhibited an intermediate phenotype consistent with a gene dosage requirement for the antiviral effects of Ifitm. (B) Weight loss measurements from all surviving study animals at 3 and 5 DPI are shown. Closed symbols represent animals challenged with 1000 PFU and open symbols represent animals challenged with 500 PFU. At both time points, weight loss differences between any two genotypes were statistically significant (Mann Whitney U test). Bars show mean ± SEM. (C) Pulmonary and systemic viral loads were measured at 3 DPI from animals challenged with 500 PFU of PR8. The relative abundance of IAV M2 transcript levels from total lung or spleen RNA, normalized to endogenous Gapdh transcript, is shown. Values represent fold changes relative to the mean viral burden of wild type mice. Pulmonary viral loads were 2.7-fold higher in knockout than in wild type controls (p = 0.0159, Mann Whitney U test). Error bars show mean ± SEM. Splenic viral loads were 15.9-fold greater in knockout than wild type animals (p = 0.0079), but splenic viral loads of wild type animals were barely above the detection threshold of the assay. Splenic viral loads of heterozygotes were not significantly different from those of wild type or knockout animals but a non-significant trend was evident across all three genotypes.

Figure 2. IfitmDel and Ifitm3-specific knockout mice exhibit indistinguishable phenotypes.

IfitmDel knockout, Ifitm3egfp^−/− (Ifitm3-specific knockout), Ifitm3egfp^+/−, and wild type mice were challenged with 500 PFU of intranasal PR8. No differences were observed in disease progression between IfitmDel and Ifitm3egfp knockouts. Ifitm3egfp heterozygotes exhibit an intermediate phenotype similar to that of IfitmDel heterozygotes in Figure 1. (A) Survival times of Ifitm3egfp and IfitmDel knockouts were significantly different from those of wild type animals (p = 0.0038 and p = 0.0067 respectively, Mantel-Cox test) but not significantly different from one another (p = 0.5380). (B) Mean+SD weight loss over time is shown for mice in Figure 2A.

Figure 3. Both interferons and gp130-mediated cytokines induce Ifitm3 expression.

Ifitm3 protein expression can also be induced by gp130-family cytokines in cells expressing appropriate receptors. The Ifitm3 promoter alone among murine Ifitm promoters is interferon responsive. (A) Expression of Ifitm3 was determined by western blot in NIH 3T3 and RAW 264.7 cells treated for 2 days with the indicated cytokines. IFNλ2 failed to induce Ifitm3 expression at similar concentrations, likely due to lack of the type III IFN receptor. The gp130-family cytokine oncostatin M (OSM) was a potent inducer of Ifitm3 expression in 3T3 cells whereas RAW264.7 cells were responsive to IL-6, but not OSM. (B) Surface expression of the OSM receptor, IL-6 receptor, and gp130 by NIH 3T3 and RAW264.7 cells was determined by flow cytometry. Solid blue lines correspond to the fluorescence signal of the indicated receptor-binding antibodies and red lines show fluorescence of the isotype control. The responsiveness of 3T3 and RAW cells to OSM and IL-6 respectively (as shown in panel A) correlates with their expression of their corresponding receptors. (C) Activities of the six murine Ifitm promoters were assayed in mock-treated, IFNα2-treated, and IFNγ-treated NIH 3T3 cells transfected with plasmids encoding luciferase under the control of the indicated promoters. Ifitm1, 2, and 6 promoters appear constitutively active, and in contrast to human orthologs, insensitive to interferon stimulation. The Ifitm5 promoter shows almost no activity under any condition, consistent with its tissue-restricted expression in vivo . The Ifitm3 promoter alone is interferon responsive (4.4-fold induction with IFNα2; 2.2-fold with IFNγ). Bars show mean of triplicates ± SEM. Asterisk indicates p<0.0001 (two-factor ANOVA Bonferroni post test). (D) Cytokine regulation of Ifitm1, 2, and 3 was confirmed at the mRNA level by qRT-PCR in both 3T3 and RAW cells. Bars depict fold upregulation of the indicated transcripts following a 24-hour incubation with the indicated cytokines relative to transcript levels in untreated cells. Ifitm3 transcript levels were significantly upregulated by IFNα2 (500 ng/ml) and IFNγ (100 ng/ml) in both cells lines. As in panel (A), OSM (250 ng/ml) upregulated Ifitm3 in 3T3 cells and IL-6 (250 ng/ml) upregulated Ifitm3 in RAW cells. Ifitm1 and Ifitm2 transcript levels were not increased by interferons. Mean Ifitm1 expression increased in response to OSM treatment but the difference was not significant due to the high variance of these samples. Error bars show mean ± range of triplicates. Asterisk indicates p<0.05 and double asterisk indicates p<0.01 (1-tail t-test assuming heteroscedastic samples).

Figure 4. Ifitm3 is constitutively expressed by many respiratory tissues and induced in lower airway epithelium by influenza.

Many lung tissues constitutively express Ifitm3. Panels show Ifitm3 expression (stained red/brown) in uninfected wild type (top) and infected wild type (6 DPI, middle) animals. Comparable tissues from knockout mice (bottom) are included as negative controls. (A–C) Ifitm3 expression was induced on lower airway (*) epithelium following influenza infection. Constitutive expression was observed on the endothelium of blood vessels (arrowheads). The endothelial cells in (A) and (B) provide a reference for staining intensity between the two slides. (D–E) In contrast to lower airway epithelial cells, upper airway epithelial cells constitutively express Ifitm3. Ifitm3 localizes to the apical borders of ciliated epithelial cells (arrows, inset shows magnified view of indicated cell in which the cilia can be visualized). In the infected animal in (E), the lesioned epithelium has become dysplastic. The flattened, non-ciliated epithelial cells show punctate cytoplasmic, rather than apical, staining. Interspersed goblet cells (arrowhead) do not stain. (G–I) Ifitm3 is constitutively and robustly expressed by the visceral pleura. (J–L) Leukocytes (arrows) often strongly express Ifitm3. The arrow in (J) indicates a circulating leukocyte. In (K), Ifitm3 expression can be seen on inflammatory cells recruited to a pneumonia lesion.

Figure 5. Ifitm3 is expressed by the target cells of influenza A virus.

Ifitm3 is expressed by the target cells of IAV in infected wild type animals. Laser-scanning confocal micrographs show Ifitm3 (red), influenza A virus antigen (blue), nuclear chromatin (cyan), and either type II pneumocyte marker DC-LAMP (left, green) or lysosomal marker MAC-3 (right, green), against a differential interference contrast background. Tissues shown were harvested from an infected wild type animal at 6 DPI. (A) Ifitm3 is expressed on alveolar type II pneumocytes (arrows) marked by DC-LAMP (green), many of which are Ifitm3 positive (yellow). Occasional infected cells (arrowheads) including an infected Ifitm3 positive cell (magenta) are scattered throughout the parenchyma. Inset: Ifitm3 colocalizes with the lysosome-related organelle marker of type II pneumocytes, DC-LAMP (colocalization depicted in yellow). (B) Ifitm3 is localized to the apical membranes of ciliated respiratory epithelial cells (arrows) lining a bronchiole filled with IAV-infected (blue), necrotic, and Ifitm3-positive cellular debris (*). Ifitm3 does not colocalize with lysosomal marker MAC-3 (green) in these cells. Inset: magnified view of ciliated cell.