A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection

Abstract

The interferon (IFN)-induced transmembrane (IFITM) proteins are critical mediators of the host antiviral response. Here, we expand the role of IFITM proteins to host defense against intracellular bacterial infection by demonstrating that they restrict Mycobacterium tuberculosis (MTb) intracellular growth. Simultaneous knockdown of IFITM1, IFITM2, and IFITM3 by RNAi significantly enhances MTb growth in human monocytic and alveolar/epithelial cells, whereas individual overexpression of each IFITM impairs MTb growth in these cell types. Furthermore, MTb infection, Toll-like receptor 2 and 4 ligands, and several proinflammatory cytokines induce IFITM1-3 gene expression in human myeloid cells. We find that IFITM3 co-localizes with early and, in particular, late MTb phagosomes, and overexpression of IFITM3 enhances endosomal acidification in MTb-infected monocytic cells. These findings provide evidence that the antiviral IFITMs participate in the restriction of mycobacterial growth, and they implicate IFITM-mediated endosomal maturation in its antimycobacterial activity.

Figure 1. IFITM1, IFITM2, and IFITM3 Gene Expression Is Induced in Human Macrophages and Monocytic Cells in Response to MTb Infection via TLR2/4- and MyD88-Dependent Signaling Pathways

(A) MDMs were left uninfected (mock) or infected with MTb strains CDC1551 or HN878 as indicated for 3, 24, and 48 hr and IFITM1–3 transcripts were measured. (B) THP-1 cells were left uninfected (mock) or infected with MTb strain H37Rv for 3, 6, 24, and 48 hr and IFITM1–3 transcripts were measured. (C) THP-1 cells were left untreated (mock) or treated with the TLR2 agonist Pam3Cys (100 ng/ml) or the TLR4 agonist LPS (100 ng/ml) for 3, 6, 24, and 48 hr and IFITM1–3 transcripts were measured. (D) THP-1 cells in which MyD88 is constitutively ablated by shRNA, or THP-1 cells expressing a control shRNA, were infected with MTb strain H37Rv or left uninfected (mock) for 48 hr and IFITM1–3 transcripts were measured. (E) THP-1 cells were left untreated (mock) or treated with recombinant IL-1β (100 ng/ml), IL-6 (50 ng/ml), TNF (10 ng/ml), or IFN-β (100 ng/ml) for 24 and 48 hr. IFITM1–3 and cyclophilin B mRNA levels were assayed using qPCR. Results are the mean ± SEM from three independent experiments (*p ≤ 0.05, **p ≤ 0.01, ^***p ≤ 0.005).

Figure 2. Ablation of Endogenous IFITM1–3 Increases MTb Replication

(A) IFITM1, IFITM2, and IFITM3 mRNA levels were measured in THP-1 transduced with a lentiviral vector encoding a single shRNA targeting IFITM1, 2, and 3, or a vector encoding a control shRNA. (B) IFITM1–3 shRNA KD or control THP-1 cells were infected with MTb strain H37Rv-mCherry for 24 or 48 hr and analyzed by flow cytometry. Gated area and numerical values show the percentage of the cells infected with bacilli. Dot plots are representative of three independent experiments. (C) CFU assay of IFITM-KD or control THP-1 cells at days 0, 2, and 3 post-infection with H37Rv-mCherry. Results are the mean ± SEM from two experiments (**p < 0.01).

Figure 3. IFITM1, IFITM2, or IFITM3 Overexpression Inhibits MTb Replication in Monocytic Cells

(A) THP-1 cells transduced with an empty control vector or vectors encoding IFITM1, IFITM2, or IFITM3 were infected with MTb H37Rv-mCherry for 24 hr. Gated area and numerical values show the percentage of the cells infected with bacilli. Dot plots are representative of three independent experiments. (B and C) Representative images of intracellular mycobacteria that are associated with individual cells indicated after acquisition on an ImageStream X Mark II instrument and IDEAS software analysis. Bright-field microscopy indicates cells magnified 603 and infected with MTb H37Rv-mCherry for 24 hr. THP-1 cells were transduced with (B) control or IFITM3 overexpression vectors or (C) vectors encoding IFITM1–3 shRNA or control shRNA. Images shown from left to right are as follows: bright field (gray) indicating cellular outline, MTb-mCherry fluorescence (red) indicating MTb, and merged image of MTb-mCherry fluorescence and bright field showing intracellular MTb. Dot plot graphs on the right-hand side show the pixel intensity of intracellular mCherry in the infected cells. Results are from one of two independent experiments, acquiring 2,500 cells per sample each time (**p < 0.01, ^***p < 0.005).

Figure 4. IFITM3 Co-localizes with MTb as Phagosome Maturation Proceeds and IFITM3 Overexpression Enhances Endosomal Acidification in the Presence of MTb Infection

(A) A549 cells transduced with IFITM3-V5 tag (IFITM3) or control vectors were infected with MTb H37Rv-mCherry as described in the Experimental Procedures. (Top) MTb-mCherry- (red) infected cells were immunostained with anti-V5 (IFITM3, green), anti-Rab5 (white), and DAPI (nuclei, blue). The graph shows quantitative measurement of MTb co-localization with IFITM3 and Rab5. (Bottom) MTb-mCherry-(red) infected cells were immunostained with anti-V5 (IFITM3, green), anti-Rab7 (white), and DAPI (nuclei, blue). The graph shows quantitative measurement of MTb co-localization with IFITM3 and Rab7. Scale bar, 10 mM. Enlarged images on the right-hand side of the merged images show the MTb-containing phagosomes. Data are representative of four independent experiments. Results are the mean ± SEM (**p < 0.01, ^***p < 0.005). (B) THP-1 cells transduced with a control vector or a vector overexpressing IFITM3 were left uninfected (mock) or infected with MTb H37Rv for 48 hr, after which they were incubated with LysoTracker Red for 2 hr. Histograms show LysoTracker Red signal in the control and IFITM3-overexpressing cells in the absence (left) or presence (right) of MTb infection.