IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses

Abstract

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

Fig. 1. Ifitm3 enhances IL-6 downstream of TLRs and MyD88.

Female mixed bone marrow chimeras with 50:50 of Ifitm3^−/− and wt-zDC-DTR were generated and treated (n = 6) or not (n = 5) with DT. a Mice were infected with 5 × 10⁵ PFU MCMV and weight loss was assessed over time. Mean and SEM are shown. b 4 days p.i. spleens and livers were harvested from MCMV-infected mice, homogenised, and IL-6 was assayed. Mean and SEM are shown. c Replicating virus in liver and spleen was quantified 4 days p.i. by plaque assay. Individual data points, median and inter-quartile range are shown. d BM-DCs from wt and Ifitm3^−/− mice were stimulated with TLR ligands and IL-6 in supernatants was assayed 6 and 24 h post stimulation. e BM-DCs from wt, Tlr3^−/−, Tlr7^−/− and Tlr9^−/− mice were infected with MCMV (MOI 1 or 0.1), and IL-6 in supernatants was assayed 6 and 24 h post infection. f BM-DCs from wt and Ifitm3^−/− mice were pre-incubated with or without TLR7 synthetic blocking peptide or g ODN 2088 for 1 h prior to infection with MCMV (MOI 1 or 0.1). IL-6 in supernatants was assayed 24 h post infection. d–g Data are shown as mean ± SEM. h Ifitm3^wtMyD88^wt (n = 8), Ifitm3^−/−MyD88^wt (n = 7), Ifitm3^wtMyD88^−/− (n = 7), Ifitm3^−/−MyD88^−/− (n = 6) male and female mice (mixed genders in all groups) were infected with 5 × 10⁵ PFU MCMV and weight loss was assessed over time. Data are shown as mean ± SEM. i BM-DCs from Ifitm3^wtMyD88^wt, Ifitm3^−/−MyD88^wt, Ifitm3^wtMyD88^−/−, Ifitm3^−/−MyD88^−/− mice were infected with MCMV (MOI 1), and IL-6 in supernatants was assayed 6 and 24 h post infection. Data are shown as mean ± SEM. Statistical significance was assessed using Student’s t test (a, b, d), Mann–Whitney U (c), one-way ANOVA analysis with Tukey’s multiple comparisons test (e–g, i) or two-way ANOVA analysis (h). p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01; ***, ≤0.001; and ****, ≤0.0001. Source data are provided as a Source Data file.

Fig. 2. IFITM3 regulates HCMV- and TLR-induced IL-6 in human DCs.

ELISA data presented from assays performed in triplicate or more, for at least two independent technical replicates per assay. HCMV (merlin strain) was used for all iPS-DC experiments at MOI 5 unless otherwise stated. a IFITM3 or GAPDH protein levels after stimulation of control and IFITM3^−/− iPS-DCs for 24 h with HCMV were measured by Western blot. Lysate from THP-1 cells was used as a positive control. b iPS-DCs (n = 3–6 separate cell cultures in different wells) were stimulated with HCMV, and IL-6 in supernatants were assayed 24 h later. c iPS-DCs (n = 3–6 separate cell cultures in different wells) were stimulated with TLR ligands and IL-6 was measured. d iPS-DCs (n = 4–8 separate cell cultures in different wells) were pre-treated with or without IKK16 for 1 h and then stimulated with HCMV and IL-6 measured in supernatants. e iPS-DCs (n = 6 separate cell cultures in different wells) were pre-treated for 1 h with or without neutralising antibody to TLR2 and stimulated with HCMV or TLR2 ligand Pam3CSK4, and IL-6 was assayed after 24 h. f iPS-DCs (n = 4 separate cell cultures in different wells) were pre-treated with Cytotect CP Biotest, or left untreated, and stimulated with HCMV, with IL-6 in supernatant assayed 24 h later. g Monocyte-derived DCs isolated from human donors genotyped for SNP rs12252 (n = 7–8 different donors) were stimulated with HCMV (MOI 1 or 10), and IL-6 and TNF in supernatants were assayed 24 h post infection. h iPS-DCs (n = 6 separate cell cultures in different wells) were stimulated with IAV A/X31 (H3N2), PR8 (H1N1), or gamma-irradiated A/X31 (MOI 1) and IL-6 was measured 6 and 24 h later. i BM-DCs from wt and Ifitm3^−/− mice (n = 2 separate cell cultures in different wells) were infected with IAV A/X31 (H3N2) (MOI 1 or 0.1), and IL-6 in supernatants was assayed 6 and 24 h later. Mean ± SEM are shown, and statistical significance was assessed using one-way ANOVA analysis with Tukey’s multiple comparisons test (b, d–f, h) or Student’s t test (c, g, i). p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01. Source data are provided as a Source Data file.

Fig. 3. Ifitm3 interacts with the reticulon protein Nogo-B.

a, b GM-CSF differentiated BM-DCs from wt and Ifitm3^−/− mice were grown in ‘Medium’ or ‘Heavy’ SILAC medium respectively. Cells were either mock infected (a) or infected with MCMV (MOI 1) (b) for 3 h, lysed and IP for anti-fragilis (Ifitm3) was performed. The fold enrichment of each protein is shown. p values were estimated using significance A values (two-tailed), then corrected for multiple hypothesis testing using the Benjamini–Hochberg correction (82). c BM-DCs from wt and Ifitm3^−/− mice were infected with MCMV (MOI 1) for 3 h, lysed and IP for anti-fragilis was performed. Ifitm3, Nogo-B and ACTIN (input samples only) levels were detected by Western blot. Data represent two separate experiments. d Wt and Nogo-A/B^−/− male and female mice were infected with MCMV and weight loss was assessed over time. Data are shown as mean ± SEM from of 25 (wt) and 26 (Nogo-A/B^−/−) mice. e Replicating virus from harvested spleens and livers was measured by plaque assay at d2 and d4 p.i. Individual data points (n = 5/group), median and inter-quartile range are shown. Harvested spleens and liver tissue supernatant from either naïve, or from d2 and d4 p.i. was assayed for (f) IL-6 and (g) TNF. Data are shown as mean ± SEM (n = 6–10 mice/group) and represent three replicate experiments. BM-DCs from wt and Nogo-A/B^−/− mice were stimulated with or without (h) TLR ligands or (i) MCMV or IAV A/X31 (MOI 1), for 6 and 24 h and IL-6 was assayed in supernatants. Data are shown as mean ± SEM of two biologically independent cultures. Statistical significance was assessed using Student’s t test (d, f–i) or Mann–Whitney U (e). p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01; ***, ≤0.001; and ****, ≤0.0001. Source data are provided as a Source Data file.

Fig. 4. Nogo-B/IFITM3 interaction regulates CMV-induced IL-6.

a Nogo-B and GAPDH was detected by Western blot, after stimulation of Kolf2 and IFITM3^−/− H12 iPS-DCs for 1, 3 or 24 h with HCMV (MOI 5), or mock treatment, and preparation of whole-cell extracts. Data represent three experiments. b Relative expression of Nogo-B to GAPDH from a was assessed using ImageJ software. Mean ± SEM of three biological replicates are shown. c THP-1s were treated for 72 h with RTN4/Nogo, Non-targeting (NT) and/or IFITM3 siRNAs and assayed for anti-IFITM3 and anti-Nogo-B by Western blot 72 h after siRNA addition and preparation of whole-cell extracts. Data represent two separate experiments. d THP-1s (n = 6 separate cell cultures in different wells) treated for 72 h with RTN4/Nogo, Non-targeting (NT) and/or IFITM3 siRNAs were stimulated with HCMV (MOI 5) for 24 h, with IL-6 in supernatant measured. e BM-DCs (n = 2, n = 4 separate cell cultures in different wells) from wt and Ifitm3^−/− mice targeted with either Rtn4/Nogo targeting siRNA or AllStars control siRNA, and were infected with MCMV (MOI 1), and IL-6 in supernatants was assayed 6 and 24 h post infection. f Nogo-B expression was detected in Rtn4/Nogo siRNA-treated wt and Ifitm3^−/− BM-DCs by Western blot at 3 h post infection with MCMV. Data represent two experiments. g BM-DCs (n = 4 separate cell cultures in different wells) from Nogo-A/B^wt, Ifitm3^−/−, Nogo-A/B^−/− and Ifitm3^−/−Nogo-A/B^−/− mice were infected with MCMV (MOI 1) for 24 h with IL-6 assayed in supernatants. Data are shown as mean ± SEM and statistical significance was assessed using Student’s t test for relevant comparisons (b) and one-way ANOVA with Tukey’s multiple comparisons test (d, e, g). p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01; ***, ≤0.001; and ****, ≤0.0001. Source data are provided as a Source Data file.

Fig. 5. Nogo-B drives viral pathogenesis in Ifitm3^−/− mice during MCMV infection.

Wt (n = 13), Ifitm3^−/− (n = 9), Nogo-A/B^−/− (n = 14), and Nogo-A/B^−/−Ifitm3^−/− (n = 9) male and female mice were infected with MCMV for 4 days. Data are merged from two independent experiments. a Weight changes were measured over time and data are shown as mean ± SEM. Statistical significance between weight curves of Ifitm3^−/− and either wt (black) or Nogo-A/B^−/−Ifitm3^−/− (blue) mice is shown. After 4 days, IL-6 (b) and virus load (c) in spleen homogenates were measured in wt (n = 7), Nogo-A/B (n = 8), Ifitm3^−/− (n = 5) and Nogo-A/B^−/−Ifitm3^−/− (n = 3) mice. Data are representative of two experiments and are shown as mean ± SEM (b) or individual mice + median and inter-quartile range (c). Statistical significance was assessed using two-way ANOVA (a) or one-way ANOVA analysis with Tukey’s multiple comparisons test (b, c). p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01; ***, ≤0.001. Source data are provided as a Source Data file.

Fig. 6. IFITM3 regulates Nogo-B expression via the ubiquitin-proteasome pathway.

a, b Kolf2, IFITM3^−/− iPS-DCs or c, d BM-DCs (from two male donor mice/group) were pre-treated with Bafilomycin A1, NH₄Cl or MG132 for 1 h, then stimulated with (a, b) HCMV (iPS-DCs) (MOI 5) for 1 h or infected with (c, d) MCMV (MOI 1) for 1 h (BM-DCs). Nogo-B levels were assayed by Western blot (a iPS-DCs, c BM-DCs), with results from three independent replicates quantified relative to GAPDH (b iPS-DCs) or relative to ACTIN (d BM-DCs). Data are shown as mean ± SEM (b, d). Statistical significance was assessed using Student’s t test for relevant comparisons. p values are reported as follows: n.s., >0.05 and *, ≤0.05. Source data are provided as a Source Data file.

Fig. 7. TLR dynamics are altered in IFITM3-deficient DCs.

a Immunostaining for anti-Nogo-B (Red) and anti-TLR2 (Green) in iPS-DCs mock treated or stimulated with HCMV (MOI 5), (Blue; DAPI). b Surface TLR2 was assessed by flow cytometry in Kolf2 and IFITM3^−/− iPS-DCs either mock treated or stimulated for 24 h with HCMV. Data are shown as mean values from biological separate cell cultures (n = 9) ± SEM. c iPS-DCs (n = 3 separate cell cultures in different wells) were stimulated for 0–6 h with TLR2 ligand Pam3CSK4 and % TLR2 was quantified. Data are represented as mean ± SEM. d, e Surface TLR2 was assessed by flow cytometry in HCMV-stimulated THP-1s treated for 72 h with RTN4/Nogo, Non-targeting (NT) and/or IFITM3 siRNAs. Individual cell cultures (n = 3) + mean (d) and representative histograms (e) are shown. Data are expressed as mean MFI ± SEM (d). f, g IL-6 production by healthy control Kolf2 or IFITM3^−/− H12 iPS-DCs pre-treated for 1 h with (f) endocytosis inhibitor (ES9-17) and/or NFκB inhibitor (IKK-16), or (g) Bafilomycin A1 and NH₄Cl, followed by stimulation with HCMV (MOI 5) for 24 h. Data presented are from at least two independent experiments, with samples run in at least triplicate for each ELISA. Mean ± SEM are shown from 11 (f) or 4 (g) biologically independent cell cultures. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (b, d) or Dunnett’s repeated measures test comparing experimental groups with HCMV-alone group in either Kolf2 or IFITM3 H12 cells (f). g Statistical significance was assessed using one-way ANOVA analysis with Tukey’s multiple comparisons test. p values are reported as follows: n.s., >0.05; *, ≤0.05; **, ≤0.01; ***, ≤0.001; and ****, ≤0.0001. Source data are provided as a Source Data file.