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TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection

Nature Microbiology volume 9, pages 949–963 (2024)Cite this article

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Abstract

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip−/− mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip−/− alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip−/− AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip−/− AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

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Fig. 1: TOLLIP is required for Mtb control in mice.
Fig. 2: Tollip−/− AM exhibit diminished Mtb intracellular carriage in a cell-autonomous manner.
Fig. 3: Tollip−/− AM undergo increased necrosis in a cell-autonomous manner during prolonged Mtb infection.
Fig. 4: Tollip−/− AM autonomously develop increased EIF2 phosphorylation after prolonged Mtb infection.
Fig. 5: Mycolic acid-treated Tollip−/− macrophages accumulate lipids and permit increased Mtb replication.
Fig. 6: ISRIB treatment restores immune control in Tollip−/− mice.

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Data availability

Datasets generated in this study are available as Source data. Image data are available on Zenodo at https://doi.org/10.5281/zenodo.10475042. All sequencing data that support the findings of this study have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession number GSE243818. Any other relevant data are available from the corresponding author on request. Source data are provided with this paper.

Code availability

All custom scripts have been made available at https://github.com/altman-lab/Shah_mouse_collab. Additional modified scripts can be accessed upon request.

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Acknowledgements

We thank the University of Washington Center for Lung Biology Histology and Imaging Core for helpful advice on pathology staining and analysis; the Seattle Children’s Research Institute Cell Sorting Core for assistance and technical support; L. Ramakrishnan for helpful conversations and M. Sabo for copy editing. Flow cytometry data were acquired through the University of Washington, Cell Analysis Facility Shared Resource Lab, with NIH award 1S10 OD024979-01A1. This work was supported by the NIH (R01 AI136912 to J.A.S.; R01 DK108921 to S.A.S.) and the Department of Veterans Affairs (I01 BX004444 to S.A.S.). G.L.P. was supported by the American Diabetes Association (19-PDF-063).

Author information

Authors and Affiliations

  1. Department of Medicine, University of Washington, Seattle, WA, USA

    Sambasivan Venkatasubramanian, Kimberly A. Dill-McFarland, Divya A. Rane, Mackenzie K. Meyer, Aparajita Saha, Sarah A. Hinderstein, Yu-Hua Chow, Chi F. Hung, Matthew Altman & Javeed A. Shah

  2. Seattle Children’s Research Institute, Seattle, WA, USA

    Courtney R. Plumlee, Sara B. Cohen, Benjamin H. Gern & Kevin B. Urdahl

  3. Department of Pediatrics, University of Washington, Seattle, WA, USA

    Benjamin H. Gern

  4. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA

    Gemma L. Pearson, Anne C. Lietzke, Amanda Pacheco & Scott A. Soleimanpour

  5. VA Ann Arbor Healthcare System, Ann Arbor, MI, USA

    Scott A. Soleimanpour

  6. Departments of Pediatrics and Immunology, University of Washington, Seattle, WA, USA

    Kevin B. Urdahl

  7. VA Puget Sound Healthcare System, Seattle, WA, USA

    Javeed A. Shah

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  1. Sambasivan Venkatasubramanian
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Contributions

S.V., K.B.U., S.A.S. and J.A.S. conceptualized the project. S.V., S.A.S., K.B.U., M.A. and J.A.S. developed the methodology. J.A.S. and S.A.S. procured resources. K.A.D.-M. and M.A. developed software. S.V., C.R.P, S.B.C, J.A.S. and A.S. performed validation. M.A. K.A.D.-M. and B.H.G. conducted formal analysis. S.V., S.A.H., C.R.P., S.B.C, G.L.P., A.C.L., A.P., J.A.S., B.H.G., D.A.R., M.K.M., A.S., Y.-H.C. and C.F.H. conducted investigations. S.V. and J.A.S. wrote the original draft. K.B.U., S.A.S., M.A., D.A.R., C.R.P. and J.A.S. reviewed and edited the paper. J.A.S., K.B.U., M.A., S.A.S., B.H.G. and C.F.H. supervised the project. S.V. and J.A.S. administered the project. J.A.S., K.B.U., S.A.S., M.A. and B.H.G. acquired funding.

Corresponding author

Correspondence to Javeed A. Shah.

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Nature Microbiology thanks Maziar Divangahi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Tollip−/− macrophages are hyperinflammatory.

a, b) Peritoneal extract macrophages (PEM) were isolated from Tollip−/− and B6 mice, plated, stimulated with control (Uns), LPS (10 ng/ml), PAM3 (250 ng/ml), or Mtb whole cell lysate (1 µg/ml) overnight and a) TNF and b) IL-10 were measured from cellular supernatants by ELISA. c–e) PEM were infected with Mtb H37Rv strain (MOI 2.5) overnight ex vivo, then c) TNF, d) IL-1β, and e) IL-10 were measured from cellular supernatants by ELISA. f–h) Baseline immune cell number in Tollip−/− mice. Total number of f) splenocytes, g) CD3+, C4 + CD8+, F4/80+, and NK1.1+ splenocytes, and the h) proportion of alveolar macrophages (AM) in the lungs of healthy 8-week-old mice, measured by flow cytometry. N = 3 measurements per experiment; each experiment was performed twice. * p < 0.05, student’s two sided t-test.

Source data

Extended Data Fig. 2 Related to Fig. 1. Characteristics of Mtb-infected Tollip−/− lungs.

a–g) Gating strategy used to identify Mtb-infected lung-resident myeloid cell subsets. a) Live/Dead Fixability dye was used to exclude dead cells, b) AM were identified by coexpression of SiglecF and CD11c. c) SiglecF- cells were gated; CD11b + Ly6G+ cells were classified as neutrophils, and CD11b + Ly6G- cells were considered macrophages. d) MHC-II+CD11c+ macrophages were subclassified as monocyte-derived macrophage cells (MDM) and MHC-II+CD11c- as interstitial macrophages (IM). e) CD11b-Ly6G- cells were gated and MHC-II+CD11c+ cells were classified as conventional DC (cDC) or f) CD103+ cDC) was measured. g) Representative flow cytometry images of the proportion of Mtb-infected myeloid subsets 28 and 56 days post infection (dpi). h–i) Proportion of AM, MDM, IM, PMN, and DC in the lungs h) 28 and i) 56 dpi. j) Representative images of Mtb-mCherry+ AM and MDM in Rag1−/− and Tollip−/−Rag1−/− mice.

Source data

Extended Data Fig. 3 Related to Fig. 2. Gating strategy used to identify lung-resident myeloid cells in mixed bone marrow chimeric mice.

a) Mixed bone marrow chimeric mice were infected with Mtb H37Rv expressing mCherry reporter plasmid (Mtb-mCherry) (50-100 CFU) via aerosol. At selected time points flow analysis was performed to identify populations infected with Mtb. Myeloid populations were identified as in Extended Data Fig. 2. a) Alveolar macrophages (AM), neutrophils (PMN), monocyte-derived macrophages (MDM), andinterstitial macrophages (IM) were subclassified as B6 or Tollip−/− based on CD45.1/CD45.2 or CD45.2 expression, respectively, and the proportion of mCherry-Mtb cells measured. Representative images from 28 days post infection are shown. b) Representative image of CD45.2+ Tollip−/− AM adoptively transferred into CD45.1+ mice 56 days after AM depletion.

Extended Data Fig. 4 Related to Fig. 3. Gating strategy used for sorting Mtb-infected AM.

Mixed bone marrow mice were infected with Mtb H37Rv (50-100 CFU) via aerosol and AM were sorted for RNA seq analysis 28 days post infection, from left to right. a) Lymphocyte gating, followed by b) Singlet identification. c) Live/Dead Fixability dye was used to exclude dead/dying cells, and d) SiglecF+ CD11c+ cells were classified as AM. e) Uninfected and Mtb-infected AM were identified by mCherry expression f) Uninfected AM and g) Mtb-infected AM genetic lineage was defined based on CD45.1/CD45.2 (F1 B6) or CD45.2 (Tollip−/−) expression, respectively.

Extended Data Fig. 5 Related to Fig. 4. Tollip is dispensable for bulk autophagy in macrophages.

Bone marrow from B6 (white bars) or Tollip−/− (black bars) were differentiated ex vivo to macrophages using 40ng/mL M-CSF for 7-9 days. Following differentiation, BMDMs were treated for 6 hr with or without 250 nM Bafilomycin A (BafA). a) Representative western blot results showing protein levels of LC3I/II and p62. b) Quantification of protein levels (by densitometry) of LC3II using total LC3 (I + II) as a loading control. ***p < 0.001 2-way ANOVA for an effect of BafA. c) Quantification of p62 levels (by densitometry) using vinculin as a loading control. p = 0.056 2-way ANOVA effect of BafA. d–f) TNF concentrations in the supernatants of TOLLIP-deficient d) BMDM, e) alveolar macrophages (p = 0.02 between genotypes in Mtb and p = 0.03 in MA+Mtb groups; AM), and f) THP-1 cells 24 hours after Mtb infection (MOI 5) and mycolic acid (MA; 10 µg/ml) treatment by ELISA (p = 0.0002 between genotypes in Mtb and p < 0.0001 in MA+Mtb groups; AM)* p < 0.05, ** p < 0.01, *** p < 0.001, two-sided t-test. Experiment was performed in BMDM twice and all other cell types three times. g) Expression of EIF2AK1, EIF2AK2, EIF2AK4, and TOLLIP in human whole blood in heathy controls (control) or patients with latent tuberculosis infection (LTBI), active symptomatic pulmonary TB disease (Mtb). EIF2AK3 was not detected in this dataset. Data are shown as violin plots with lines indicating 25th, 50th, and 75th percentile, extending to minimum and maximum value. **** p < 0.0001, two-sided ANOVA. Obtained from GSE 1949152.

Source data

Extended Data Fig. 6 Related to Fig. 6. Evaluation of integrated stress responses in Tollip−/− mice and macrophages.

a–c) B6 and Tollip−/− peritoneal extract macrophages (PEM) were incubated with media or mycolic acid (MA; 10 µg/ml) for 72 hours, then infected with Mtb (MOI 1) overnight. mRNA transcripts of key regulatory genes of the cellular stress response were measured before and after Mtb infection. a) Ern1 (IRE1a; p = 0.001 for media, p = 0.049 for MA), b) Eif2ak3 (PERK; p = 0.002 for media, p = 0.007 for MA), and c) Atf6 (ATF6; p = 0.02 for media) were measured and displayed as their fold change from baseline after Mtb infection. FC = (Normalized mRNA expression after Mtb infection) / (Normalized mRNA expression after media control stimulation). N = 2/group and are representative of at least two independent experiments. d) Western blot of PEM incubated as above measuring pEIF2 and tubulin expression. e) Optical density at 600 nm (OD600) of Mtb H37Rv in 7H9 broth culture in the presence of raphin-1 (10 µM), ISRIB (250 nM), or vehicle control over time. N = 2 over two independent experiments; error bars – SEM. f–h) TNF concentrations in cellular supernatants from TOLLIP-deficient f) bone marrow-derived macrophages (BMDM), g) alveolar macrophages (AM; p = 0.04 for Mtb+MA, p = 0.003 for Mtb+ISRIB) and h) THP-1 cells (p = 0.002 for Mtb+MA, p = 0.005 for Mtb+ISRIB, p = 0.004 for Mtb+MA + ISRIB), after 24 hours of Mtb infection (MOI 5), mycolic acid (MA, 10 µg/ml), and ISRIB (250 nM), measured by ELISA. This experiment was performed twice, each with three technical replicates. * p < 0.05, ** p < 0.01, *** p < 0.001 two-sided t-test.

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Extended Data Fig. 7 Images related to Fig. 6 histocytometry studies.

a) Representative confocal microscopy image of the lungs of a B6 mouse infected with Mtb for 56 days. Yellow – SiglecF; green – CD11b; red – pEIF2; blue – LipidTox; white — PPD. b) Histocytometry positional mapping of AM and MDM within the Mtb-infected lung. c) Proportion of pEIF2+ PPD + AM (p = 0.017 for overall ANOVA effect) and MDM (p = 0.016 for overall ANOVA effect) in B6 and Tollip−/− mice at baseline and after ISRIB treatment. p = 0.02 for overall ANOVA effect. * p < 0.05, two-sided ANOVA. N = 4 B6 control mice, N = 5 B6 ISRIB mice, N = 6 Tollip−/− control, and N = 6 Tollip−/− ISRIB mice. d) Representative images staining from the lungs of Mtb-infected B6 and Tollip−/− mice with and without ISRIB treatment. Red – pEIF2; green – CD68; blue – SiglecF; white – PPD. e) Spatial correlation analysis of cell types within 20-mm-radius neighborhoods in B6 and Tollip−/− mice. Red shades indicate positive correlation, and blue shades indicate negative correlation.

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Extended Data Fig. 8 Overall experimental model.

a) Basal homeostasis. During chronic Mtb infection, lipid products are released in Mtb-infected AM. TOLLIP prevents lipid accumulation and controls inflammation, which maintains EIF2 signaling at a basal level. b) Tollip−/− AM. Tollip−/− mice develop excess TNF and IFNg responses from macrophages and T cells. Mtb-infected Tollip−/− AM undergo lipid accumulation, which increases EIF2 phosphorylation. Excess pEIF2 induces sensitivity to inflammation in AM. c) Chronic infection in Tollip−/− AM. During prolonged infection, increased and prolonged EIF2 phosphorylation from lipids and inflammation leads to cellular necrosis, decreasing the Mtb burden within individual AM and releasing extracellular Mtb. d) ISRIB treatment. ISRIB improves AM host defense, which improves Mtb control in both B6 and Tollip−/− mouse models, making it an effective therapeutic across genetic backgrounds.

Supplementary information

Supplementary Information

Extended Data figure legends 1–8, Source Data legends 1–7, Extended Data Source Data figure legends 1–5.

Reporting Summary

Supplementary Table 1

List of study resources.

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Source data related to RNA-seq experiments, including differentially expressed genes, measures of effect size and statistical significance.

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Statistical source data, with uncropped western blots.

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Statistical source data, with uncropped western blots.

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Venkatasubramanian, S., Plumlee, C.R., Dill-McFarland, K.A. et al. TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection. Nat Microbiol 9, 949–963 (2024). https://doi.org/10.1038/s41564-024-01641-w

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