Abstract

Invasive pulmonary aspergillosis causes substantial mortality in immunocompromised individuals. Recognition of Aspergillus fumigatus by the host immune system leads to activation of the inflammasome, which provides protection against infection. However, regulation of inflammasome activation at the molecular level is poorly understood. Here, we describe two distinct pathways that coordinately control inflammasome activation during A.fumigatus infection. The C-type lectin receptor pathway activates both MAPK and NF-κB signalling, which leads to induction of downstream mediators, such as the transcription factor IRF1, and also primes the inflammasomes. Toll-like receptor signalling through the adaptor molecules MyD88 and TRIF in turn mediates efficient activation of IRF1, which induces IRGB10 expression. IRGB10 targets the fungal cell wall, and the antifungal activity of IRGB10 causes hyphae damage, modifies the A.fumigatus surface and inhibits fungal growth. We also demonstrate that one of the major fungal pathogen-associated molecular patterns, β-glucan, directly triggers inflammasome assembly. Thus, the concerted activation of both Toll-like receptors and C-type lectin receptors is required for IRF1-mediated IRGB10 regulation, which is a key event governing ligand release and inflammasome activation upon A.fumigatus infection.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Dagenais, T. R. T. & Keller, N. P. Pathogenesis of Aspergillus fumigatus in invasive aspergillosis. Clin. Microbiol. Rev. 22, 447–465 (2009).

  2. 2.

    Latgé, J.-P. Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12, 310–350 (1999).

  3. 3.

    Segal, B. H. Aspergillosis. N. Engl. J. Med. 360, 1870–1884 (2009).

  4. 4.

    Carvalho, A. et al. TLR3 essentially promotes protective class I-restricted memory CD8+ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients. Blood 119, 967–977 (2012).

  5. 5.

    Chai, L. Y. A. et al. Modulation of Toll-like receptor 2 (TLR2) and TLR4 responses by Aspergillus fumigatus. Infect. Immun. 77, 2184–2192 (2009).

  6. 6.

    Kasperkovitz, P. V., Cardenas, M. L. & Vyas, J. M. TLR9 is actively recruited to Aspergillus fumigatus phagosomes and requires the N-terminal proteolytic cleavage domain for proper intracellular trafficking. J. Immunol. 185, 7614–7622 (2010).

  7. 7.

    Loures, F. V. et al. Recognition of Aspergillus fumigatus hyphae by human plasmacytoid dendritic cells is mediated by Dectin-2 and results in formation of extracellular traps. PLoS Pathog. 11, e1004643 (2015).

  8. 8.

    Meier, A. et al. Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages. Cell. Microbiol. 5, 561–570 (2003).

  9. 9.

    Netea, M. G. et al. Aspergillus fumigatus evades immune recognition during germination through loss of Toll-like receptor-4-mediated signal transduction. J. Infect. Dis. 188, 320–326 (2003).

  10. 10.

    Ramaprakash, H., Ito, T., Standiford, T. J., Kunkel, S. L. & Hogaboam, C. M. Toll-like receptor 9 modulates immune responses to Aspergillus fumigatus conidia in immunodeficient and allergic mice. Infect. Immun. 77, 108–119 (2009).

  11. 11.

    Serrano-Gómez, D., Leal, J. A. & Corbí, A. L. DC-SIGN mediates the binding of Aspergillus fumigatus and keratinophylic fungi by human dendritic cells. Immunobiology 210, 175–183 (2005).

  12. 12.

    Balloy, V. et al. Involvement of Toll-like receptor 2 in experimental invasive pulmonary aspergillosis. Infect. Immun. 73, 5420–5425 (2005).

  13. 13.

    Bellocchio, S. et al. The contribution of the Toll-like/IL-1 receptor superfamily to innate and adaptive immunity to fungal pathogens in vivo. J. Immunol. 172, 3059–3069 (2004).

  14. 14.

    Gringhuis, S. I. et al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1β via a noncanonical caspase-8 inflammasome. Nat. Immunol. 13, 246–254 (2012).

  15. 15.

    Gross, O. et al. Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence. Nature 459, 433–436 (2009).

  16. 16.

    Hise, A. G. et al. An essential role for the NLRP3 inflammasome in host defense against the human fungal pathogen Candida albicans. Cell Host Microbe 5, 487–497 (2009).

  17. 17.

    Karki, R. et al. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17, 357–368 (2015).

  18. 18.

    Lei, G. et al. Biofilm from a clinical strain of Cryptococcus neoformans activates the NLRP3 inflammasome. Cell Res. 23, 965–968 (2013).

  19. 19.

    Saïd-Sadier, N., Padilla, E., Langsley, G. & Ojcius, D. M. Aspergillus fumigatus stimulates the NLRP3 inflammasome through a pathway requiring ROS production and the Syk tyrosine kinase. PLoS ONE 5, e10008 (2010).

  20. 20.

    Tavares, A. H. et al. NLRP3 inflammasome activation by Paracoccidioides brasiliensis. PLoS Negl. Trop. Dis. 7, e2595 (2013).

  21. 21.

    Triantafilou, M., Hughes, T. R., Morgan, B. P. & Triantafilou, K. Complementing the inflammasome. Immunology 147, 152–164 (2015).

  22. 22.

    Werner, J. L. et al. Requisite role for the Dectin-1 β-glucan receptor in pulmonary defense against Aspergillus fumigatus. J. Immunol. 182, 4938–4946 (2009).

  23. 23.

    Man, S. M., Karki, R. & Kanneganti, T.-D. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol. Rev. 277, 61–75 (2017).

  24. 24.

    Man, S. M. et al. The transcription factor IRF1 and guanylate-binding proteins target activation of the AIM2 inflammasome by Francisella infection. Nat. Immunol. 16, 467–475 (2015).

  25. 25.

    Man, S. M. et al. IRGB10 liberates bacterial ligands for sensing by the AIM2 and caspase-11–NLRP3 inflammasomes. Cell 167, 382–396.e17 (2016).

  26. 26.

    Meunier, E. et al. Guanylate-binding proteins promote activation of the AIM2 inflammasome during infection with Francisella novicida. Nat. Immunol. 16, 476–484 (2015).

  27. 27.

    Rathinam, V. A. K. et al. TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by Gram-negative bacteria. Cell 150, 606–619 (2012).

  28. 28.

    Mambula, S. S., Sau, K., Henneke, P., Golenbock, D. T. & Levitz, S. M. Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus. J. Biol. Chem. 277, 39320–39326 (2002).

  29. 29.

    Wang, J. E. et al. Involvement of CD14 and Toll-like receptors in activation of human monocytes by Aspergillus fumigatus hyphae. Infect. Immun. 69, 2402–2406 (2001).

  30. 30.

    Ramirez-Ortiz, Z. G. et al. Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA. Infect. Immun. 76, 2123–2129 (2008).

  31. 31.

    Hohl, T. M. et al. Aspergillus fumigatus triggers inflammatory responses by stage-specific β-glucan display. PLoS Pathog. 1, e30 (2005).

  32. 32.

    Steele, C. et al. The β-glucan receptor Dectin-1 recognizes specific morphologies of Aspergillus fumigatus. PLoS Pathog. 1, e42 (2005).

  33. 33.

    Plato, A., Hardison, S. E. & Brown, G. D. Pattern recognition receptors in antifungal immunity. Semin. Immunopathol. 37, 97–106 (2015).

  34. 34.

    Bretz, C. et al. MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus. Infect. Immun. 76, 952–958 (2008).

  35. 35.

    Wevers, B. A. et al. Fungal engagement of the C-type lectin mincle suppresses Dectin-1-induced antifungal immunity. Cell Host Microbe 15, 494–505 (2014).

  36. 36.

    Feng, H. et al. NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR. Nat. Immunol. 18, 1299–1309 (2017).

  37. 37.

    Negishi, H. et al. Evidence for licensing of IFN-γ-induced IFN regulatory factor 1 transcription factor by MyD88 in Toll-like receptor-dependent gene induction program. Proc. Natl Acad. Sci. USA 103, 15136–15141 (2006).

  38. 38.

    Man, S. M., Place, D. E., Kuriakose, T. & Kanneganti, T.-D. Interferon-inducible guanylate-binding proteins at the interface of cell-autonomous immunity and inflammasome activation. J. Leukoc. Biol. 101, 143–150 (2017).

  39. 39.

    Kim, B.-H., Shenoy, A. R., Kumar, P., Bradfield, C. J. & MacMicking, J. D. IFN-inducible GTPases in host defense. Cell Host Microbe 12, 432–444 (2012).

  40. 40.

    Kim, B.-H. et al. Interferon-induced guanylate-binding proteins in inflammasome activation and host defense. Nat. Immunol. 17, 481–489 (2016).

  41. 41.

    Heinekamp, T. et al. Interference of Aspergillus fumigatus with the immune response. Semin. Immunopathol. 37, 141–152 (2015).

  42. 42.

    Caffrey, A. K. et al. IL-1α signaling is critical for leukocyte recruitment after pulmonary Aspergillus fumigatus challenge. PLoS Pathog. 11, e1004625 (2015).

  43. 43.

    Chang, T.-H. et al. Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic cell response to Histoplasma capsulatum. PLoS Pathog. 13, e1006485 (2017).

  44. 44.

    Drummond, R. A. & Brown, G. D. The role of Dectin-1 in the host defence against fungal infections. Curr. Opin. Microbiol. 14, 392–399 (2011).

  45. 45.

    Geijtenbeek, T. B. H. & Gringhuis, S. I. Signalling through C-type lectin receptors: shaping immune responses. Nat. Rev. Immunol. 9, 465–479 (2009).

  46. 46.

    Slack, E. C. et al. Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan. Eur. J. Immunol. 37, 1600–1612 (2007).

  47. 47.

    Kuriakose, T., Zheng, M., Neale, G. & Kanneganti, T.-D. IRF1 is a transcriptional regulator of ZBP1 promoting NLRP3 inflammasome activation and cell death during influenza virus infection. J. Immunol. 200, 1489–1495 (2018).

  48. 48.

    Takeuchi, O., Hoshino, K. & Akira, S. Cutting edge: TLR2-deficient and MyD88-deficient mice are highly susceptible to Staphylococcus aureus infection. J. Immunol. 165, 5392–5396 (2000).

  49. 49.

    Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

  50. 50.

    Hoshino, K. et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749–3752 (1999).

  51. 51.

    Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

  52. 52.

    Kawai, T., Adachi, O., Ogawa, T., Takeda, K. & Akira, S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11, 115–122 (1999).

  53. 53.

    Yamamoto, M. et al. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301, 640–643 (2003).

  54. 54.

    Marakalala, M. J. et al. Differential adaptation of Candida albicans in vivo modulates immune recognition by Dectin-1. PLoS Pathog. 9, e1003315 (2013).

  55. 55.

    Saijo, K. et al. Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development. Nat. Immunol. 4, 274–279 (2003).

  56. 56.

    Gurung, P. et al. Tyrosine kinase SYK licenses MyD88 adaptor protein to instigate IL-1α-mediated inflammatory disease. Immunity 46, 635–648 (2017).

  57. 57.

    Gross, O. et al. Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature 442, 651–656 (2006).

  58. 58.

    Ruland, J., Duncan, G. S., Wakeham, A. & Mak, T. W. Differential requirement for Malt1 in T and B cell antigen receptor signaling. Immunity 19, 749–758 (2003).

  59. 59.

    Matsuyama, T. et al. Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development. Cell 75, 83–97 (1993).

  60. 60.

    Yamamoto, M. et al. A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii. Immunity 37, 302–313 (2012).

  61. 61.

    da Silva Ferreira, M. E. et al. The akuB KU80 mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5, 207–211 (2006).

  62. 62.

    Lamkanfi, M., Malireddi, R. K. S. & Kanneganti, T.-D. Fungal zymosan and mannan activate the cryopyrin inflammasome. J. Biol. Chem. 284, 20574–20581 (2009).

  63. 63.

    Tzeng, T.-C. et al. A fluorescent reporter mouse for inflammasome assembly demonstrates an important role for cell-bound and free ASC specks during in vivo Infection. Cell Rep. 16, 571–582 (2016).

  64. 64.

    Man, S. M. et al. Differential roles of caspase-1 and caspase-11 in infection and inflammation. Sci. Rep. 7, 45126 (2017).

  65. 65.

    Coers, J. et al. Chlamydia muridarum evades growth restriction by the IFN-γ-inducible host resistance factor Irgb10. J. Immunol. 180, 6237–6245 (2008).

  66. 66.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  67. 67.

    Briard, B. et al. Dirhamnolipids secreted from Pseudomonas aeruginosa modify anjpegungal susceptibility of Aspergillus fumigatus by inhibiting β1,3 glucan synthase activity. ISME J. 11, 1578–1591 (2017).

  68. 68.

    Shepardson, K. M. et al. Hypoxia enhances innate immune activation to Aspergillus fumigatus through cell wall modulation. Microbes Infect. 15, 259–269 (2013).

  69. 69.

    Loures, F. V. & Levitz, S. M. XTT assay of antifungal activity. Bio Protoc. 5, e1543 (2015).

  70. 70.

    Torrent, M. et al. AMPA: an automated web server for prediction of protein antimicrobial regions. Bioinformatics 28, 130–131 (2012).

  71. 71.

    Meher, P. K., Sahu, T. K., Saini, V. & Rao, A. R. Predicting antimicrobial peptides with improved accuracy by incorporating the compositional, physico-chemical and structural features into Chou’s general PseAAC. Sci. Rep. 7, 42362 (2017).

  72. 72.

    Crooks, G. E., Hon, G., Chandonia, J.-M. & Brenner, S. E. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).

Download references

Acknowledgements

We thank members of the Kanneganti lab, the St. Jude Children’s Hospital Veterinary Pathology Core, Macromolecular Synthesis Core and Electron Microscopy Core. Images were acquired at the SJCRH Cell & Tissue Imaging Center, which is supported by SJCRH and NCI P30 CA021765–35. Work from our laboratories is supported by the US NIH (AI101935, AI124346, AR056296 and CA163507 to T.-D.K.) and the American Lebanese Syrian Associated Charities (to T.-D.K.). M.Y. is supported by the Research Program on Emerging and Re-emerging Infectious Diseases (18fk0108047h0002) and Japanese Initiative for Progress of Research on Infectious Diseases for Global Epidemic (18fm0208018h0002) from the Agency for Medical Research and Development.

Author information

Affiliations

  1. Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN, USA

    • Benoit Briard
    • , Rajendra Karki
    • , R. K. Subbarao Malireddi
    • , Anannya Bhattacharya
    • , David E. Place
    • , Jayadev Mavuluri
    •  & Thirumala-Devi Kanneganti
  2. Cellular Imaging Shared Resource, St. Jude Children’s Research Hospital, Memphis, TN, USA

    • Jennifer L. Peters
  3. Animal Resources Center and the Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN, USA

    • Peter Vogel
  4. Department of Immunoparasitology, Research Institute for Microbial Diseases, Laboratory of Immunoparasitology, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan

    • Masahiro Yamamoto

Authors

  1. Search for Benoit Briard in:

  2. Search for Rajendra Karki in:

  3. Search for R. K. Subbarao Malireddi in:

  4. Search for Anannya Bhattacharya in:

  5. Search for David E. Place in:

  6. Search for Jayadev Mavuluri in:

  7. Search for Jennifer L. Peters in:

  8. Search for Peter Vogel in:

  9. Search for Masahiro Yamamoto in:

  10. Search for Thirumala-Devi Kanneganti in:

Contributions

B.B., R.K., R.K.S.M., J.M., A.B., D.E.P. and J.L.P. performed the experiments. B.B., R.K., R.K.S.M., P.V., M.Y. and T.-D.K. analysed the data. B.B., A.B. and R.K. wrote the paper. T.-D.K. oversaw the project and funding.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Thirumala-Devi Kanneganti.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–10, Supplementary Tables 1–3, uncropped western blots.

  2. Reporting Summary

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/s41564-018-0298-0