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:

Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis

Nature Microbiology volume 2, Article number: 17072 (2017) Cite this article

Subjects

Abstract

Nitric oxide contributes to protection from tuberculosis. It is generally assumed that this protection is due to direct inhibition of Mycobacterium tuberculosis growth, which prevents subsequent pathological inflammation. In contrast, we report that nitric oxide primarily protects mice by repressing an interleukin-1- and 12/15-lipoxygenase-dependent neutrophil recruitment cascade that promotes bacterial replication. Using M. tuberculosis mutants as indicators of the pathogen's environment, we inferred that granulocytic inflammation generates a nutrient-replete niche that supports M. tuberculosis growth. Parallel clinical studies indicate that a similar inflammatory pathway promotes tuberculosis in patients. The human 12/15-lipoxygenase orthologue, ALOX12, is expressed in cavitary tuberculosis lesions; the abundance of its products correlates with the number of airway neutrophils and bacterial burden and a genetic polymorphism that increases ALOX12 expression is associated with tuberculosis risk. These data suggest that M. tuberculosis exploits neutrophilic inflammation to preferentially replicate at sites of tissue damage that promote contagion.

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: Anti-inflammatory activity of Nos2 protects mice from TB disease.
Figure 2: Neutrophilic inflammation produces a growth-permissive environment for Mtb.
Figure 3: IL-1-dependent 12/15-LOX products contribute to TB susceptibility in Nos2−/− mice.
Figure 4: Increased expression and activity of 12-LOX are associated with active TB in humans.
Figure 5: ALOX12 expressed in inflammatory areas of cavitary TB lesions.

Similar content being viewed by others

References

  1. Canetti, G. The tubercle bacillus in the pulmonary lesion of man. Am. J. Med. Sci. 231, 480 (1956).

    Article  Google Scholar 

  2. Mishra, B. B. et al. Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1β. Nat. Immunol. 14, 52–60 (2013).

    Article  CAS  Google Scholar 

  3. Niazi, M. K. K. et al. Lung necrosis and neutrophils reflect common pathways of susceptibility to Mycobacterium tuberculosis in genetically diverse, immune-competent mice. Dis. Model. Mech. 8, 1141–1153 (2015).

    Article  CAS  Google Scholar 

  4. Mattila, J. T., Maiello, P., Sun, T., Via, L. E. & Flynn, J. L. Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques. Cell. Microbiol. 17, 1085–1097 (2015).

    Article  CAS  Google Scholar 

  5. Berry, M. P. R. et al. An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature 466, 973–977 (2010).

    Article  CAS  Google Scholar 

  6. Scanga, C. A. et al. The inducible nitric oxide synthase locus confers protection against aerogenic challenge of both clinical and laboratory strains of Mycobacterium tuberculosis in mice. Infect. Immun. 69, 7711–7717 (2001).

    Article  CAS  Google Scholar 

  7. Flynn, J. L., Scanga, C. A., Tanaka, K. E. & Chan, J. Effects of aminoguanidine on latent murine tuberculosis. J. Immunol. 160, 1796–1803 (1998).

    CAS  PubMed  Google Scholar 

  8. Tsiganov, E. N. et al. Gr-1dimCD11b+ immature myeloid-derived suppressor cells but not neutrophils are markers of lethal tuberculosis infection in mice. J. Immunol. 192, 4718–4727 (2014).

    Article  CAS  Google Scholar 

  9. Knaul, J. K. et al. Lung-residing myeloid-derived suppressors display dual functionality in murine pulmonary tuberculosis. Am. J. Respir. Crit. Care Med. 190, 1053–1066 (2014).

    Article  Google Scholar 

  10. Obregón-Henao, A., Henao-Tamayo, M., Orme, I. M. & Ordway, D. J. Gr1(int)CD11b+ myeloid-derived suppressor cells in Mycobacterium tuberculosis infection. PLoS ONE 8, e80669 (2013).

    Article  Google Scholar 

  11. MacMicking, J. D. Interferon-inducible effector mechanisms in cell-autonomous immunity. Nat. Rev. Immunol. 12, 367–382 (2012).

    Article  CAS  Google Scholar 

  12. Jayaraman, P. et al. IL-1β promotes antimicrobial immunity in macrophages by regulating TNFR signaling and caspase-3 activation. J. Immunol. 190, 4196–4204 (2013).

    Article  CAS  Google Scholar 

  13. Fremond, C. M. et al. IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection. J. Immunol. 179, 1178–1189 (2007).

    Article  CAS  Google Scholar 

  14. Zhang, G. et al. Allele-specific induction of IL-1β expression by C/EBPβ and PU.1 contributes to increased tuberculosis susceptibility. PLoS Pathogens 10, e1004426 (2014).

    Article  Google Scholar 

  15. Nouailles, G. et al. CXCL5-secreting pulmonary epithelial cells drive destructive neutrophilic inflammation in tuberculosis. J. Clin. Invest. 124, 1268–1282 (2014).

    Article  CAS  Google Scholar 

  16. Irwin, S. M. et al. Presence of multiple lesion types with vastly different microenvironments in C3HeB/FeJ mice following aerosol infection with Mycobacterium tuberculosis. Dis. Model. Mech. 8, 591–602 (2015).

    Article  Google Scholar 

  17. Padgett, E. L. & Pruett, S. B. Rat, mouse and human neutrophils stimulated by a variety of activating agents produce much less nitrite than rodent macrophages. Immunology 84, 135–141 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Denis, M. Human neutrophils, activated with cytokines or not, do not kill virulent Mycobacterium tuberculosis. J. Infect. Dis. 163, 919–920 (1991).

    Article  CAS  Google Scholar 

  19. Long, J. E. et al. Identifying essential genes in Mycobacterium tuberculosis by global phenotypic profiling. Methods Mol. Biol. 1279, 79–95 (2015).

    Article  CAS  Google Scholar 

  20. Padilla-Benavides, T., Long, J. E., Raimunda, D., Sassetti, C. M. & Argüello, J. M. A novel P(1B)-type Mn2+-transporting ATPase is required for secreted protein metallation in mycobacteria. J. Biol. Chem. 288, 11334–11347 (2013).

    Article  CAS  Google Scholar 

  21. Nambi, S. et al. The oxidative stress network of Mycobacterium tuberculosis reveals coordination between radical detoxification systems. Cell Host Microbe 17, 829–837 (2015).

    Article  CAS  Google Scholar 

  22. Darwin, K. H., Ehrt, S., Gutierrez-Ramos, J.-C., Weich, N. & Nathan, C. F. The proteasome of Mycobacterium tuberculosis is required for resistance to nitric oxide. Science 302, 1963–1966 (2003).

    Article  CAS  Google Scholar 

  23. Cambier, C. J. et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 505, 218–222 (2014).

    Article  CAS  Google Scholar 

  24. Quadri, L. E., Sello, J., Keating, T. A., Weinreb, P. H. & Walsh, C. T. Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin. Chem. Biol. 5, 631–645 (1998).

    Article  CAS  Google Scholar 

  25. Forrellad, M. A. et al. Role of the Mce1 transporter in the lipid homeostasis of Mycobacterium tuberculosis. Tuberculosis (Edinb.) 94, 170–177 (2014).

    Article  CAS  Google Scholar 

  26. Kendall, S. L. et al. A highly conserved transcriptional repressor controls a large regulon involved in lipid degradation in Mycobacterium smegmatis and Mycobacterium tuberculosis. Mol. Microbiol. 65, 684–699 (2007).

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. Dennis, E. A. & Norris, P. C. Eicosanoid storm in infection and inflammation. Nat. Rev. Immunol. 15, 511–523 (2015).

    Article  CAS  Google Scholar 

  29. Kashino, S. S., Ovendale, P., Izzo, A. & Campos-Neto, A. Unique model of dormant infection for tuberculosis vaccine development. Clin. Vaccine Immunol. 13, 1014–1021 (2006).

    Article  CAS  Google Scholar 

  30. Lämmermann, T. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498, 371–375 (2013).

    Article  Google Scholar 

  31. Van Leyen, K. et al. Baicalein and 12/15-lipoxygenase in the ischemic brain. Stroke 37, 3014–3018 (2006).

    Article  CAS  Google Scholar 

  32. Reynaud, D. & Pace-Asciak, C. R. 12-HETE and 12-HPETE potently stimulate intracellular release of calcium in intact human neutrophils. Prostaglandins Leukot. Essent. Fatty Acids 56, 9–12 (1997).

    Article  CAS  Google Scholar 

  33. Mrsny, R. J. et al. Identification of hepoxilin A3 in inflammatory events: a required role in neutrophil migration across intestinal epithelia. Proc. Natl Acad. Sci. USA 101, 7421–7426 (2004).

    Article  CAS  Google Scholar 

  34. Tobin, D. M. et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140, 717–730 (2010).

    Article  CAS  Google Scholar 

  35. Marakalala, M. J. et al. Inflammatory signaling in human tuberculosis granulomas is spatially organized. Nat. Med. 22, 531–538 (2016).

    Article  CAS  Google Scholar 

  36. McDonald, B. et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330, 362–366 (2010).

    Article  CAS  Google Scholar 

  37. Hurley, B. P., Pirzai, W., Mumy, K. L., Gronert, K. & McCormick, B. A. Selective eicosanoid-generating capacity of cytoplasmic phospholipase A2 in Pseudomonas aeruginosa-infected epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 300, L286–L294 (2011).

    Article  CAS  Google Scholar 

  38. Tamang, D. L. et al. Hepoxilin A(3) facilitates neutrophilic breach of lipoxygenase-expressing airway epithelial barriers. J. Immunol. 189, 4960–4969 (2012).

    Article  CAS  Google Scholar 

  39. Bhowmick, R. et al. Systemic disease during Streptococcus pneumoniae acute lung infection requires 12-lipoxygenase-dependent inflammation. J. Immunol. 191, 5115–5123 (2013).

    Article  CAS  Google Scholar 

  40. Mayer-Barber, K. D. et al. Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk. Nature 511, 99–103 (2014).

    Article  CAS  Google Scholar 

  41. Laskay, T., van Zandbergen, G. & Solbach, W. Neutrophil granulocytes—Trojan horses for Leishmania major and other intracellular microbes? Trends Microbiol. 11, 210–214 (2003).

    Article  CAS  Google Scholar 

  42. Herron, M. J. et al. Intracellular parasitism by the human granulocytic ehrlichiosis bacterium through the P-selectin ligand, PSGL-1. Science 288, 1653–1656 (2000).

    Article  CAS  Google Scholar 

  43. Winter, S. E., Lopez, C. A. & Bäumler, A. J. The dynamics of gut-associated microbial communities during inflammation. EMBO Rep. 14, 319–327 (2013).

    Article  CAS  Google Scholar 

  44. Kim, M.-J. et al. Caseation of human tuberculosis granulomas correlates with elevated host lipid metabolism. EMBO Mol. Med. 2, 258–274 (2010).

    Article  CAS  Google Scholar 

  45. Owens, C. P. et al. The Mycobacterium tuberculosis secreted protein Rv0203 transfers heme to membrane proteins MmpL3 and MmpL11. J. Biol. Chem. 288, 21714–21728 (2013).

    Article  CAS  Google Scholar 

  46. Nandi, B. & Behar, S. M. Regulation of neutrophils by interferon-γ limits lung inflammation during tuberculosis infection. J. Exp. Med. 208, 2251–2262 (2011).

    Article  CAS  Google Scholar 

  47. Palace, S. G., Proulx, M. K., Lu, S., Baker, R. E. & Goguen, J. D. Genome-wide mutant fitness profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue. mBio 5, e01385–14 (2014).

    Article  Google Scholar 

  48. Zhang, G. et al. An SNP selection strategy identified IL-22 associating with susceptibility to tuberculosis in Chinese. Sci. Rep. 1, 20 (2011).

    Article  Google Scholar 

  49. Chen, X. et al. Diagnosis of active tuberculosis in China using an in-house gamma interferon enzyme-linked immunospot assay. Clin. Vaccine Immunol. 16, 879–884 (2009).

    Article  CAS  Google Scholar 

  50. Guan, Y. Q., Cai, Y. Y., Zhang, X., Lee, Y. T. & Opas, M. Adaptive correction technique for 3D reconstruction of fluorescence microscopy images. Microsc. Res. Tech. 71, 146–157 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the Natural Science Foundation of China (81525016, 81501714 and 81500004 to X.C.), the National Institutes of Health (NIH; AI064282 and AI107774 to C.M.S., AI120556 to R.R.L., AI003749 to S.G.P. and MH096625 to E.E.) and the Arnold and Mabel Beckman Foundation (to A.J.O.). The authors thank C. Moss for providing technical help, S.M. Behar for critical review of the manuscript and the UMMS Department of Animal Medicine for Animal Care.

Author information

Authors and Affiliations

  1. Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, 01655, Massachusetts, USA

    Bibhuti B. Mishra, Rustin R. Lovewell, Andrew J. Olive, Clare M. Smith, Jia Yao Phuah, Jarukit E. Long, Samantha G. Palace, Jon D. Goguen, Richard E. Baker, Subhalaxmi Nambi, Matthew G. Booty, Christina E. Baer, Beth A. McCormick & Christopher M. Sassetti

  2. Guangdong Key Lab of Emerging Infectious Diseases, Shenzhen Third People's Hospital, Guangdong Medical College, Shenzhen, 518112, China

    Guoliang Zhang, Wenfei Wang & Xinchun Chen

  3. Public Health Research Institute Center at the International Center for Public Health, New Jersey Medical School – Rutgers, New Jersey, 07103, USA

    Eliseo Eugenin & Veronique Dartois

  4. Department of Biochemistry and Molecular Pharmacology, Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School, Worcester, 01605, Massachusetts, USA

    Michelle L. Dubuke & Scott A. Shaffer

  5. Department of Pathology, University of Massachusetts Medical School, Worcester, 01655, Massachusetts, USA

    Rabinarayan Mishra

  6. Department of Pathogen Biology, Shenzhen University School of Medicine, Shenzhen, 518060, China

    Xinchun Chen

Authors
  1. Bibhuti B. Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rustin R. Lovewell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andrew J. Olive
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Guoliang Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wenfei Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eliseo Eugenin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Clare M. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jia Yao Phuah
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jarukit E. Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michelle L. Dubuke
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Samantha G. Palace
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Jon D. Goguen
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Richard E. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Subhalaxmi Nambi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Rabinarayan Mishra
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Matthew G. Booty
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Christina E. Baer
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Scott A. Shaffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Veronique Dartois
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Beth A. McCormick
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Xinchun Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Christopher M. Sassetti
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.B.M. and C.M.S. conceived and designed the study. B.B.M., R.R.L. and A.J.O. performed mouse experiments. G.Z., W.W. and X.C. designed and performed the SNP study in Chinese cohorts. E.E. and V.D. designed and performed the IHC study of the lung biopsies from TB patients, and analysed data. S.G.P., S.N., C.M.S., M.G.B., R.N., C.E.B. and J.P.Y. provided technical help during various experiments. M.L.D. and S.A.S. performed LC–MS analysis. B.B.M., R.R.L. and C.M.S. analysed the data. B.B.M. and C.M.S. wrote the manuscript and C.M.S. supervised the overall study.

Corresponding authors

Correspondence to Xinchun Chen or Christopher M. Sassetti.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 4 and 5, Supplementary References. (PDF 12347 kb)

Supplementary Table 1

Genome-wide fitness profiling of Mtb mutants in C57BL/6, C3HeB and NOS2-deficient mice. (XLSX 1239 kb)

Supplementary Table 2

Mutants significantly altered in C3HeB and Nos2 KO mice compared to C57BL/6 mice. (XLSX 47 kb)

Supplementary Table 3

Polymorphisms used for genotyping human cohorts. (XLSX 59 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mishra, B., Lovewell, R., Olive, A. et al. Nitric oxide prevents a pathogen-permissive granulocytic inflammation during tuberculosis. Nat Microbiol 2, 17072 (2017). https://doi.org/10.1038/nmicrobiol.2017.72

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.72

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