Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism

Nature Immunology volume 17, pages 677–686 (2016)Cite this article

Subjects

Abstract

Mycobacterium tuberculosis (Mtb) survives in macrophages by evading delivery to the lysosome and promoting the accumulation of lipid bodies, which serve as a bacterial source of nutrients. We found that by inducing the microRNA (miRNA) miR-33 and its passenger strand miR-33*, Mtb inhibited integrated pathways involved in autophagy, lysosomal function and fatty acid oxidation to support bacterial replication. Silencing of miR-33 and miR-33* by genetic or pharmacological means promoted autophagy flux through derepression of key autophagy effectors (such as ATG5, ATG12, LC3B and LAMP1) and AMPK-dependent activation of the transcription factors FOXO3 and TFEB, which enhanced lipid catabolism and Mtb xenophagy. These data define a mammalian miRNA circuit used by Mtb to coordinately inhibit autophagy and reprogram host lipid metabolism to enable intracellular survival and persistence in the host.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Mtb infection upregulates the miR-33 locus in macrophages.
Figure 2: miR-33 and miR-33* diminish fatty-acid oxidation and promote lipid-body formation in Mtb-infected macrophages.
Figure 3: Regulation of human and mouse autophagy-related gene targets by miR-33 and miR-33*.
Figure 4: miR-33 and miR-33* repress AMPKα and downstream transcription factors that control autophagy and lysosomal gene programs.
Figure 5: miR-33 and miR-33* cooperatively inhibit macrophage autophagy.
Figure 6: Silencing of miR-33 and miR-33* enhances targeting of Mtb by the autophagy machinery.
Figure 7: Inhibition of miR-33 and miR-33* enhances the killing of Mtb.
Figure 8: Hematopoietic miR-33 deficiency enhances autophagy-related gene expression in the lungs and diminishes the Mtb burden in mice.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Stanley, S.A. & Cox, J.S. Host-pathogen interactions during Mycobacterium tuberculosis infections. Curr. Top. Microbiol. Immunol. 374, 211–241 (2013).

    CAS  PubMed  Google Scholar 

  2. Huang, J. & Brumell, J.H. Bacteria-autophagy interplay: a battle for survival. Nat. Rev. Microbiol. 12, 101–114 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Gutierrez, M.G. et al. Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753–766 (2004).

    CAS  PubMed  Google Scholar 

  4. Sakowski, E.T. et al. Ubiquilin 1 promotes IFN-γ–induced xenophagy of Mycobacterium tuberculosis. PLoS Pathog. 11, e1005076 (2015).

    PubMed  PubMed Central  Google Scholar 

  5. Stanley, S.A. et al. Identification of host-targeted small molecules that restrict intracellular Mycobacterium tuberculosis growth. PLoS Pathog. 10, e1003946 (2014).

    PubMed  PubMed Central  Google Scholar 

  6. Watson, R.O., Manzanillo, P.S. & Cox, J.S. Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway. Cell 150, 803–815 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Castillo, E.F. et al. Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation. Proc. Natl. Acad. Sci. USA 109, E3168–E3176 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Kimmey, J.M. et al. Unique role for ATG5 in neutrophil-mediated immunopathology during M. tuberculosis infection. Nature 528, 565–569 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Collins, A.C. et al. Cyclic GMP-AMP synthase is an innate immune DNA sensor for Mycobacterium tuberculosis. Cell Host Microbe 17, 820–828 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Majlessi, L. & Brosch, R. Mycobacterium tuberculosis meets the cytosol: the role of cGAS in anti-mycobacterial immunity. Cell Host Microbe 17, 733–735 (2015).

    CAS  PubMed  Google Scholar 

  11. Watson, R.O. et al. The cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy. Cell Host Microbe 17, 811–819 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Singh, R. & Cuervo, A.M. Autophagy in the cellular energetic balance. Cell Metab. 13, 495–504 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. He, C. & Klionsky, D.J. Regulation mechanisms and signaling pathways of autophagy. Annu. Rev. Genet. 43, 67–93 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Sardiello, M. et al. A gene network regulating lysosomal biogenesis and function. Science 325, 473–477 (2009).

    CAS  PubMed  Google Scholar 

  16. Rubinsztein, D.C., Codogno, P. & Levine, B. Autophagy modulation as a potential therapeutic target for diverse diseases. Nat. Rev. Drug Discov. 11, 709–730 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Sanchez, A.M. et al. The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis. Am. J. Physiol. Cell Physiol. 303, C475–C485 (2012).

    CAS  PubMed  Google Scholar 

  18. Russell, D.G., Cardona, P.J., Kim, M.J., Allain, S. & Altare, F. Foamy macrophages and the progression of the human tuberculosis granuloma. Nat. Immunol. 10, 943–948 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Daniel, J., Maamar, H., Deb, C., Sirakova, T.D. & Kolattukudy, P.E. Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages. PLoS Pathog. 7, e1002093 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Singh, V. et al. Mycobacterium tuberculosis–driven targeted recalibration of macrophage lipid homeostasis promotes the foamy phenotype. Cell Host Microbe 12, 669–681 (2012).

    CAS  PubMed  Google Scholar 

  21. D'Avila, H. et al. Mycobacterium bovis bacillus Calmette-Guérin induces TLR2-mediated formation of lipid bodies: intracellular domains for eicosanoid synthesis in vivo. J. Immunol. 176, 3087–3097 (2006).

    CAS  PubMed  Google Scholar 

  22. Dávalos, A. et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc. Natl. Acad. Sci. USA 108, 9232–9237 (2011).

    PubMed  PubMed Central  Google Scholar 

  23. Najafi-Shoushtari, S.H. et al. MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 328, 1566–1569 (2010).

    CAS  PubMed  Google Scholar 

  24. Rayner, K.J. et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 328, 1570–1573 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Rottiers, V. et al. MicroRNAs in metabolism and metabolic diseases. Cold Spring Harb. Symp. Quant. Biol. 76, 225–233 (2011).

    CAS  PubMed  Google Scholar 

  26. Goedeke, L. et al. A regulatory role for microRNA 33* in controlling lipid metabolism gene expression. Mol. Cell. Biol. 33, 2339–2352 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Ouimet, M. et al. Autophagy regulates cholesterol efflux from macrophage foam cells via lysosomal acid lipase. Cell Metab. 13, 655–667 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhao, G.J. et al. NF-kappaB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. Int. J. Cardiol. 171, e93–95 (2014).

    PubMed  Google Scholar 

  29. Singh, R. et al. Autophagy regulates lipid metabolism. Nature 458, 1131–1135 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Settembre, C. et al. TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Tresse, E. et al. VCP/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD. Autophagy 6, 217–227 (2010).

    CAS  PubMed  Google Scholar 

  33. Martin, C.J. et al. Efferocytosis is an innate antibacterial mechanism. Cell Host Microbe 12, 289–300 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Murphy, J., Summer, R., Wilson, A.A., Kotton, D.N. & Fine, A. The prolonged life-span of alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 38, 380–385 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Okamura, K. et al. The regulatory activity of microRNA* species has substantial influence on microRNA and 3′ UTR evolution. Nat. Struct. Mol. Biol. 15, 354–363 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Voellenkle, C. et al. Deep-sequencing of endothelial cells exposed to hypoxia reveals the complexity of known and novel microRNAs. RNA 18, 472–484 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Schober, A. et al. MicroRNA-126-5p promotes endothelial proliferation and limits atherosclerosis by suppressing Dlk1. Nat. Med. 20, 368–376 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Mattos, K.A. et al. Lipid droplet formation in leprosy: Toll-like receptor-regulated organelles involved in eicosanoid formation and Mycobacterium leprae pathogenesis. J. Leukoc. Biol. 87, 371–384 (2010).

    CAS  PubMed  Google Scholar 

  39. Zhao, G.J. et al. NF-κB suppresses the expression of ATP-binding cassette transporter A1/G1 by regulating SREBP-2 and miR-33a in mice. Int. J. Cardiol. 171, e93–e95 (2014).

    PubMed  Google Scholar 

  40. Brzostek, A., Pawelczyk, J., Rumijowska-Galewicz, A., Dziadek, B. & Dziadek, J. Mycobacterium tuberculosis is able to accumulate and utilize cholesterol. J. Bacteriol. 191, 6584–6591 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Marrero, J., Rhee, K.Y., Schnappinger, D., Pethe, K. & Ehrt, S. Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc. Natl. Acad. Sci. USA 107, 9819–9824 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Muñoz-Elías, E.J. & McKinney, J.D. Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat. Med. 11, 638–644 (2005).

    PubMed  PubMed Central  Google Scholar 

  43. Pandey, A.K. & Sassetti, C.M. Mycobacterial persistence requires the utilization of host cholesterol. Proc. Natl. Acad. Sci. USA 105, 4376–4380 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Rodríguez, J.G. et al. Global adaptation to a lipid environment triggers the dormancy-related phenotype of Mycobacterium tuberculosis. MBio 5, e01125–14 (2014).

    PubMed  PubMed Central  Google Scholar 

  45. van der Wel, N. et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287–1298 (2007).

    CAS  PubMed  Google Scholar 

  46. Simeone, R. et al. Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog. 8, e1002507 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Wong, K.W. & Jacobs, W.R. Jr. Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis. Cell. Microbiol. 13, 1371–1384 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Pandey, A.K. et al. NOD2, RIP2 and IRF5 play a critical role in the type I interferon response to Mycobacterium tuberculosis. PLoS Pathog. 5, e1000500 (2009).

    PubMed  PubMed Central  Google Scholar 

  49. Galic, S. et al. Hematopoietic AMPK β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity. J. Clin. Invest. 121, 4903–4915 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Horie, T. et al. MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo. Proc. Natl. Acad. Sci. USA 107, 17321–17326 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Marchiando, A.M. et al. A deficiency in the autophagy gene Atg16L1 enhances resistance to enteric bacterial infection. Cell Host Microbe 14, 216–224 (2013).

    CAS  PubMed  Google Scholar 

  52. Schoggins, J.W. et al. Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity. Nature 505, 691–695 (2014).

    CAS  PubMed  Google Scholar 

  53. Moore, K.J., Rayner, K.J., Suárez, Y. & Fernández-Hernando, C. microRNAs and cholesterol metabolism. Trends Endocrinol. Metab. 21, 699–706 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Wolf, A.J. et al. Mycobacterium tuberculosis infects dendritic cells with high frequency and impairs their function in vivo. J. Immunol. 179, 2509–2519 (2007).

    CAS  PubMed  Google Scholar 

  55. Listenberger, L.L. B.D. Fluorescent detection of lipid droplets and associated proteins. Curr. Protoc. Cell Biol. 24, 24.2 (2007).

    Google Scholar 

  56. Mehra, A. et al. Mycobacterium tuberculosis type VII secreted effector EsxH targets host ESCRT to impair trafficking. PLoS Pathog. 9, e1003734 (2013).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank H. Virgin (Washington University School of Medicine) and D. MacDuff (Washington University School of Medicine) for Cgas−/− mice; K. Caldwell (New York University) for Atg16l1flox/flox mice; B. Norris for help with flow cytometry; C. O'Shaughnessy for help with mouse harvests; New York University Langone Medical Center Immune Monitoring Core for use of the XFe24 Extracellular Flux Analyzer (supported by the NYU-HHC CTSI grant UL1 TR000038 and the NYU Cancer Institute's Cancer Center Support grant P30CA016087). Supported by the US National Institutes of Health (R01 HL108182 and HL119047 to K.J.M.; R01 AI087682 and R21 AI105298 to J.A.P.), the American Heart Association (13POST14490016 to B.R., 14POST20180018 to C.v.S.), the NYU Physician-Scientist Training Program (C.P.-C.), the Potts Memorial Foundation (S.K.), Edward J. Mallinckrodt, Jr. Foundation (J.A.P.), Science Foundation Ireland (13/SIRG/2136 to F.J.S.), and the Canadian Institutes of Health Research (postdoctoral fellowship to M.O.; MOP130365 and MSH130157 to K.J.R.).

Author information

Author notes

  1. Jennifer A Philips

    Present address: Present address: Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri, USA.,

  2. Jennifer A Philips and Kathryn J Moore: These authors contributed equally to this work.

Authors and Affiliations

  1. Leon H. Charney Division of Cardiology, Department of Medicine, Marc and Ruti Bell Vascular Biology and Disease Program, New York University Medical Center, New York, New York, USA

    Mireille Ouimet, Bhama Ramkhelawon, Coen van Solingen, Scott Oldebeken, Tathagat Dutta Ray & Kathryn J Moore

  2. Division of Infectious Diseases and Immunology, Department of Medicine, New York University Medical Center, New York, New York, USA

    Stefan Koster, Erik Sakowski, Cynthia Portal-Celhay & Jennifer A Philips

  3. University of Ottawa Heart Institute and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada

    Denuja Karunakaran, Katey J Rayner & Yves L Marcel

  4. Department of Clinical Medicine, School of Medicine, Trinity College, Dublin, Ireland

    Frederick J Sheedy

  5. and Department of Biochemistry & Molecular Pharmacology, RNA Therapeutics Institute, Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Katharine Cecchini & Philip D Zamore

Authors
  1. Mireille Ouimet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Koster
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erik Sakowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bhama Ramkhelawon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Coen van Solingen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Scott Oldebeken
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Denuja Karunakaran
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Cynthia Portal-Celhay
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Frederick J Sheedy
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tathagat Dutta Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Katharine Cecchini
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Philip D Zamore
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Katey J Rayner
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yves L Marcel
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Jennifer A Philips
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Kathryn J Moore
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.O., S.K., J.A.P. and K.J.M. designed and analyzed the experiments. T.D.R., S.O., D.K. and K.J.R. assisted with gene expression analyses. B.R. performed immunostaining. C.v.S. performed immunoprecipitation assays. S.K., E.S., C.P.-C. and F.J.S. performed M. tuberculosis studies. K.C. and P.D.Z. provided Mir33−/− bone marrow cells. Y.L.M., K.J.M. and J.A.P. supervised the experiments. M.O., J.A.P. and K.J.M. wrote the manuscript.

Corresponding authors

Correspondence to Jennifer A Philips or Kathryn J Moore.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Induction of the miR-33 locus following Mtb infection in human THP-1 macrophages.

qPCR quantification of hsa-miR-33a, hsa-miR-33a*, and SREBF2 from RNA collected from human THP-1 macrophages infected with Mtb for 24 hours.

Supplementary Figure 2 miR-33 and miR-33* targets in the autophagy gene network.

Putative miR-33 and miR-33* targets are indicated by stars in the diagram depicting autophagy-mediated cholesterol efflux.

Supplementary Figure 3 miR-33 and miR-33* repress protein expression of autophagy gene targets.

Relative protein quantification levels for (a) THP-1 cells treated with miR-33a as compared to control (ctrl) mimic, (b) THP-1 cells treated with miR-33a* as compared to ctrl mimic, (c) mouse macrophages treated with miR-33 as compared to ctrl mimic, and (d) mouse macrophages treated with miR-33* as compared to ctrl mimic. Band intensities for each protein were quantified and normalized to their respective loading control (HSP90 or GAPDH) and data is presented as fold-change relative to the control mimic. Data are the mean ± s.e.m. of 2-3 independent experiments. #P≤0.1, *P≤0.05, **P≤0.005 (Student’s t-test).

Supplementary Figure 4 miR-33 and miR-33* control the expression of multiple genes in the autophagy pathway

(a, b) mRNA expression profiling of a panel of mouse autophagy genes in peritoneal macrophages treated with miR-33 mimic or anti-miR33 (a) or miR-33* mimic or anti-mR33* (b). Additional genes analysed that were not included on the mouse autophagy PCR array are shown below the red dotted line. Data are expressed as fold change relative to control mimic or anti-miR and are representative of 2 independent experiments. Red stars indicate genes containing predicted miR-33 or miR-33* binding sites.

Supplementary Figure 5 AMPK-dependant activation of FOXO3 and TFEB following inhibition of miR-33 and miR-33*.

(a, b) Immunofluorescence (IF) imaging of FOXO3a and TFEB in green, F-actin (red) and Dapi (blue) in peritoneal macrophages treated with (a) anti-miR-33 or (b) anti-miR-33* and ctrl anti-miR in the presence or absence of compound C (Ampki, 5μM). Scale bar = 50μm. Arrows indicate nuclear localization of transcription factors.

Supplementary Figure 6 Inhibition of miR-33 and miR-33* promotes bacterial killing in vitro, partially through xenophagy.

Quantification of bacterial viability in WT vs cGAS-/- macrophages treated with control anti-miR, anti-miR-33, or anti-miR-33* and infected with an Mtb H37Rv strain co-expressing mCherry and anhydrotetracycline-inducible GFP. Data are from one experiment (mean ± s.e.m) representative of 2 independent experiments. #P≤0.1, *P≤0.05 (One-way ANOVA).

Supplementary Figure 7 Proposed model.

M. tuberculosis induces the miR-33 locus to enhance its survival by repressing autophagy and fatty acid oxidation, resulting in a nutrient-rich lipid niche for bacteria that escape xenophagy.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1733 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ouimet, M., Koster, S., Sakowski, E. et al. Mycobacterium tuberculosis induces the miR-33 locus to reprogram autophagy and host lipid metabolism. Nat Immunol 17, 677–686 (2016). https://doi.org/10.1038/ni.3434

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3434

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing