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Inflammatory signaling in human tuberculosis granulomas is spatially organized

Nature Medicine volume 22, pages 531–538 (2016)Cite this article

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Abstract

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

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Figure 1: High-resolution mass-spectrometry profiling of granuloma composition.
Figure 2: Quantitative proteome analysis reveals spatially distinct protein signatures in granulomas.
Figure 3: AA is abundant and synthesized diffusely within all granulomas.
Figure 4: Leukotriene biosynthesis is enriched in the caseum and in the cellular layer directly adjacent to the caseum.
Figure 5: TNF-α and LTA4H are more abundant in the caseum and its margins.
Figure 6: COX1 and COX2 are diffusely expressed throughout all granuloma regions.

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Acknowledgements

We thank the patients and staff of National Masan TB Hospital, as well as the technical staff from the International Tuberculosis Research Center, for their participation in our studies. We acknowledge A. Martinot for generating some of the H&E-stained images and for useful advice on the pathology studies, and thank members of the Rubin and Fortune laboratories for useful discussions. This work was supported by a Visiting Science Award from HHMI–K-RITH (E.J.R.), the US National Institutes of Health (NIH) grants 1S10OD018072-01A1 (shared instrumentation grant for the MALDI orbitrap; V.D.) and R01AI098637 (S.M.B.), the National Institute of Mental Health grant MH096625 (E.A.E.), the US Bill and Melinda Gates Foundation TB Drug Accelerator grant OPP 1066499 (V.D.), a US Public Health Research Institute (New Jersey Medical School) central grant (E.A.E.), the Division of Intramural Research, National Institute of Allergy and Infectious Disease, NIH (C.E.B.) and the Korean Centers of Disease Control, Ministry of Health, Welfare and Family Affairs (C.E.B.). We would like to acknowledge the PRIDE Team for upload of proteomics raw data.

Author information

Author notes

  1. Mohlopheni J Marakalala, Ravikiran M Raju, Kirti Sharma and Yanjia J Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA

    Mohlopheni J Marakalala, Ravikiran M Raju, Yanjia J Zhang & Eric J Rubin

  2. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany

    Kirti Sharma & Matthias Mann

  3. Public Health Research Institute, New Jersey Medical School, Newark, New Jersey, USA

    Eliseo A Eugenin, Brendan Prideaux, Isaac B Daudelin, Pei-Yu Chen & Véronique Dartois

  4. Department of Microbiology and Molecular Genetics, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA

    Eliseo A Eugenin & Véronique Dartois

  5. Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Matthew G Booty & Samuel M Behar

  6. Division of Medical Sciences, Program in Immunology, Harvard Medical School, Boston, Massachusetts, USA

    Matthew G Booty

  7. National Masan TB Hospital, Changwon, Republic of Korea

    Jin Hee Kim

  8. International Tuberculosis Research Center, Changwon, Republic of Korea

    Seok Yong Eum

  9. Tuberculosis Research Section, Laboratory of Clinical Infectious Disease, National Institute of Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland

    Laura E Via & Clifton E Barry III

  10. Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Rondebosch, Republic of South Africa

    Laura E Via & Clifton E Barry III

  11. Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Rondebosch, Republic of South Africa

    Laura E Via & Clifton E Barry III

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  1. Mohlopheni J Marakalala
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Contributions

M.J.M., R.M.R., K.S., Y.J.Z., E.A.E., B.P., M.M., V.D. and E.J.R. designed experiments; M.J.M., R.M.R., K.S., Y.J.Z., E.A.E., B.P., I.B.D., P.-Y.C. and L.E.V. performed experiments and analyzed data; M.J.M., K.S., Y.J.Z., E.A.E. and B.P. prepared the figures; J.H.K., S.Y.E., L.E.V. and C.E.B. contributed clinical samples and intellectual expertise; M.G.B. and S.M.B. contributed intellectual expertise; M.J.M., R.M.R., Y.J.Z., V.D. and E.J.R. wrote the manuscript; and all authors read the manuscript and approved the submission. Authors who contributed equally to the work are listed alphabetically.

Corresponding authors

Correspondence to Véronique Dartois or Eric J Rubin.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Tables 1 and 4–6 (PDF 27468 kb)

Supplementary Table 2

Excel file of the raw data of all the proteomes; interior of a solid granuloma, the cellular borders of the caseous and cavitary granulomas, as well as the necrotic caseum of the caseous and cavitary granulomas. This includes imputed values as a separate sheet (XLSX 1589 kb)

Supplementary Table 3

Excel file of PCA GO and KEGG enrichment table (XLSX 166 kb)

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Marakalala, M., Raju, R., Sharma, K. et al. Inflammatory signaling in human tuberculosis granulomas is spatially organized. Nat Med 22, 531–538 (2016). https://doi.org/10.1038/nm.4073

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