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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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Dendritic Cell

SUBMITTER: Robin Antrobus

LAB HEAD: Professor Ian Humphreys

PROVIDER: PXD035254 | Pride | 2022-07-28

REPOSITORIES: Pride

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Project description:Interferon-induced transmembrane protein (IFITM1) plays a dual role in restriction of RNA viruses and in metastatic cancer cell growth. The signal transduction events that are orchestrated by IFITM1 are not well defined. We set out to identify IFITM1 interacting proteins to begin to define its mechanism of action. Affinity purification of SBP-tagged IFITM1, coupled to SWATH-mass spectrometry, identified significantly higher confidence interacting proteins from IFN- treated cells, relative to non-treated cells. This promoted an examination of the protein synthetic machinery in order to determine whether IFITM1 can impact on ribosomal proteome from IFN- treated cells. Isogenic ifitm1 null and ifitm1-ifitm3 null-SiHa cell panels were generated using CRISPR gRNAs to define signaling events that are linked to IFN--dependent protein synthesis. Ultracentrifugation sedimentation of cell lysates from interferon gamma treated wt and ifitm1-ifitm3 null-SiHa cells was carried out to isolate ribosomal constituents. Although SWATH-mass spectrometry of the 40S, 60S, and 80S fractions demonstrated changes in selected protein content, a significant reduction in A254 (RNA) in the 80S ribosomal fractions suggested a relatively select defect in 80S ribosomal biogenesis in ifitm1-ifitm3 null cells. The localization of IFITM1 to ribosomal protein components prompted an analysis of IFN--dependent protein synthesis using pulse SILAC. STAT1 and B2M were two dominant proteins whose synthesis increased equivalently in wt or ifitm1-ifitm3 null cells. However, MHC class I molecules and ISG15 were the most highly suppressed IFN--responsive proteins in the ifitm1-ifitm3 double null cells and this was confirmed using ifitm1 single null cells. Transient depletion of IFITM1 using targeted siRNA also depleted MHC class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1 in mediating IFN--stimulated protein synthesis and associated antigen presentation during either oncogenic or anti-viral signalling.

Project description:Ifitm3 (interferon-inducible transmembrane protein 3) was previously identified as an endosomal antiviral effector protein that blocks viral infection1-4. In analyses of gene expression data from patients with B-cell leukemia and lymphoma, we identified Ifitm3 as one of the strongest predictors of poor clinical outcome. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, engagement of the B-cell receptor (BCR) and PI3K-activation strongly induced expression of Ifitm3 and phosphorylation of its N-terminus at Y20, resulting in accumulation at the cell surface. In B-cell leukemia, oncogenic kinases induced phosphorylation at Y20, resulting in constitutive plasma membrane localization of Ifitm3. Ifitm3¯ / ¯ naïve B-cells developed at normal numbers, however, B-cell activation upon antigen encounter, germinal center formation and production of antigen-specific antibodies were compromised. Likewise, oncogenes that typically induce development of leukemia and lymphoma failed to transform Ifitm3¯ / ¯ B-cells. Conversely, a phosphomimetic mutation of Y20 induced oncogenic PI3K-signaling and initiated transformation of premalignant B-cells into overt leukemia. Functional experiments revealed a previously unrecognized function of Ifitm3 as PIP3-scaffold and central amplifier of PI3K signaling downstream of the BCR and adhesion receptors. PI3K signal-amplification depends on binding of Ifitm3 to PIP3 via two lysine residues (K83 and K104) in its conserved intracellular loop. In Ifitm3¯ / ¯ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors, which impaired BCR-signaling and cellular adhesion. We conclude that phosphorylation of IFITM3 upon Bcell antigen-encounter induces a dynamic switch from antiviral effector functions in endosomes to a PI3K-amplification loop at the cell surface. IFITM3-dependent amplification of PI3K-signaling downstream of the BCR and adhesion receptors is critical to enable rapid expansion of B-cells with high affinity to antigen. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3- dependent signaling complexes and amplify PI3K-signaling for malignant transformation.

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

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