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Host NLRP6 exacerbates graft-versus-host disease independent of gut microbial composition

Abstract

Host NOD-like receptor family pyrin domain-containing 6 (NLRP6) regulates innate immune responses and gastrointestinal homeostasis. Its protective role in intestinal colitis and tumorigenesis is dependent on the host microbiome. Host innate immunity and microbial diversity also play a role in the severity of allogeneic immune-mediated gastrointestinal graft-versus-host disease (GVHD), the principal toxicity after allogeneic haematopoietic cell transplantation. Here, we examined the role of host NLRP6 in multiple murine models of allogeneic bone marrow transplantation. In contrast to its role in intestinal colitis, host NLRP6 aggravated gastrointestinal GVHD. The impact of host NLRP6 deficiency in mitigating GVHD was observed regardless of co-housing, antibiotic treatment or colonizing littermate germ-free wild-type and NLRP6-deficient hosts with faecal microbial transplantation from specific pathogen-free wild-type and Nlrp6−/− animals. Chimaera studies were performed to assess the role of NLRP6 expression on host haematopoietic and non-haematopoietic cells. The allogeneic [B6Ly5.2 → Nlrp6−/−] animals demonstrated significantly improved survival compared to the allogeneic [B6Ly5.2 → B6] animals, but did not alter the therapeutic graft-versus-tumour effects after haematopoietic cell transplantation. Our results unveil an unexpected, pathogenic role for host NLRP6 in gastrointestinal GVHD that is independent of variations in the intestinal microbiome and in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis.

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Fig. 1: The absence of NLRP6 in hosts ameliorates GVHD in experimental BMT models.
Fig. 2: Nlrp6−/− animals showed similar expansion of donor-derived myeloid cells with B6 WT animals.
Fig. 3: Nlrp6−/− animals showed less GVHD severity after co-housing or antibiotic treatments.
Fig. 4: Absence of NLRP6 in host haematopoietic-derived APCs is dispensable for reducing GVHD severity and mortality.
Fig. 5: Absence of NLRP6 on host non-haematopoietic cells plays an important role in ameliorating GVHD severity and mortality.
Fig. 6: Absence of NLRP6 in IECs enhances gut homeostasis after allo-BMT.

Data availability

The raw sequencing reads have been deposited at the NCBI Short Read Archive under BioProject ID PRJNA491725. Metabolomic data, mass spectral analytical parameters and spectral raw data from the study and metadata have been deposited in the NIH Common Fund’s Data Repository and Coordinating Center under Metabolomics Workbench Project ID PR000728.

References

  1. Staffas, A., Burgos da Silva, M. & van den Brink, M. R. The intestinal microbiota in allogeneic hematopoietic cell transplant and graft-versus-host disease. Blood 129, 927–933 (2017).

    Article  CAS  Google Scholar 

  2. Zeiser, R. & Blazar, B. R. Acute graft-versus-host disease—biologic process, prevention, and therapy. N. Engl. J. Med. 377, 2167–2179 (2017).

    Article  CAS  Google Scholar 

  3. Elinav, E. et al. NLRP6 inflammasome regulates colonic microbial ecology and risk for colitis. Cell 145, 745–757 (2011).

    Article  CAS  Google Scholar 

  4. Levy, M., Shapiro, H., Thaiss, C. A. & Elinav, E. NLRP6: a multifaceted innate immune sensor. Trends Immunol. 38, 248–260 (2017).

    Article  CAS  Google Scholar 

  5. Hu, B. et al. Microbiota-induced activation of epithelial IL-6 signaling links inflammasome-driven inflammation with transmissible cancer. Proc. Natl Acad. Sci. USA 110, 9862–9867 (2013).

    Article  CAS  Google Scholar 

  6. Wlodarska, M. et al. NLRP6 inflammasome orchestrates the colonic host–microbial interface by regulating goblet cell mucus secretion. Cell 156, 1045–1059 (2014).

    Article  CAS  Google Scholar 

  7. Anand, P. K. et al. NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens. Nature 488, 389–393 (2012).

    Article  CAS  Google Scholar 

  8. Ferrara, J. L., Levine, J. E., Reddy, P. & Holler, E. Graft-versus-host disease. Lancet 373, 1550–1561 (2009).

    Article  CAS  Google Scholar 

  9. Mathewson, N. D. et al. Gut microbiome-derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease. Nat. Immunol. 17, 505–513 (2016).

    Article  CAS  Google Scholar 

  10. Jenq, R. R. et al. Regulation of intestinal inflammation by microbiota following allogeneic bone marrow transplantation. J. Exp. Med. 209, 903–911 (2012).

    Article  CAS  Google Scholar 

  11. Chen, G. Y., Liu, M., Wang, F., Bertin, J. & Nunez, G. A functional role for Nlrp6 in intestinal inflammation and tumorigenesis. J. Immunol. 186, 7187–7194 (2011).

    Article  CAS  Google Scholar 

  12. Normand, S. et al. Nod-like receptor pyrin domain-containing protein 6 (NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury. Proc. Natl Acad. Sci. USA 108, 9601–9606 (2011).

    Article  CAS  Google Scholar 

  13. Jankovic, D. et al. The Nlrp3 inflammasome regulates acute graft-versus-host disease. J. Exp. Med. 210, 1899–1910 (2013).

    Article  CAS  Google Scholar 

  14. Chen, S. et al. MicroRNA-155-deficient dendritic cells cause less severe GVHD through reduced migration and defective inflammasome activation. Blood 126, 103–112 (2015).

    Article  CAS  Google Scholar 

  15. Koehn, B. H. et al. GVHD-associated, inflammasome-mediated loss of function in adoptively transferred myeloid-derived suppressor cells. Blood 126, 1621–1628 (2015).

    Article  CAS  Google Scholar 

  16. Mamantopoulos, M. et al. Nlrp6- and ASC-dependent inflammasomes do not shape the commensal gut microbiota composition. Immunity 47, 339–348 (2017).

    Article  CAS  Google Scholar 

  17. Seregin, S. S. et al. NLRP6 protects Il10 −/− mice from colitis by limiting colonization of Akkermansia muciniphila. Cell Rep. 19, 733–745 (2017).

    Article  CAS  Google Scholar 

  18. Shono, Y. et al. Increased GVHD-related mortality with broad-spectrum antibiotic use after allogeneic hematopoietic stem cell transplantation in human patients and mice. Sci. Transl Med. 8, 339ra371 (2016).

    Article  Google Scholar 

  19. Shan, M. et al. Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals. Science 342, 447–453 (2013).

    Article  CAS  Google Scholar 

  20. Adolph, T. E. et al. Paneth cells as a site of origin for intestinal inflammation. Nature 503, 272–276 (2013).

    Article  CAS  Google Scholar 

  21. Thiagarajah, J. R., Zhao, D. & Verkman, A. S. Impaired enterocyte proliferation in aquaporin-3 deficiency in mouse models of colitis. Gut 56, 1529–1535 (2007).

    Article  CAS  Google Scholar 

  22. Linden, S. K. et al. MUC1 limits Helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy. PLoS Pathog. 5, e1000617 (2009).

    Article  Google Scholar 

  23. Johansson, M. E. et al. Bacteria penetrate the inner mucus layer before inflammation in the dextran sulfate colitis model. PLoS ONE 5, e12238 (2010).

    Article  Google Scholar 

  24. Wang, F. et al. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. Gastroenterology 145, 383–395 (2013).

    Article  CAS  Google Scholar 

  25. Lindemans, C. A. et al. Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 528, 560–564 (2015).

    Article  CAS  Google Scholar 

  26. Wu, S. R. & Reddy, P. Tissue tolerance: a distinct concept to control acute GVHD severity. Blood 129, 1747–1752 (2017).

    Article  CAS  Google Scholar 

  27. Wu, S. R. & Reddy, P. Regulating damage from sterile inflammation: a tale of two tolerances. Trends Immunol. 38, 231–235 (2017).

    Article  CAS  Google Scholar 

  28. Eriguchi, Y. et al. Graft-versus-host disease disrupts intestinal microbial ecology by inhibiting Paneth cell production of α-defensins. Blood 120, 223–231 (2012).

    Article  CAS  Google Scholar 

  29. Schwab, L. et al. Neutrophil granulocytes recruited upon translocation of intestinal bacteria enhance graft-versus-host disease via tissue damage. Nat. Med. 20, 648–654 (2014).

    Article  CAS  Google Scholar 

  30. Heimesaat, M. M. et al. MyD88/TLR9 mediated immunopathology and gut microbiota dynamics in a novel murine model of intestinal graft-versus-host disease. Gut 59, 1079–1087 (2010).

    Article  CAS  Google Scholar 

  31. Gill, N., Wlodarska, M. & Finlay, B. B. Roadblocks in the gut: barriers to enteric infection. Cell. Microbiol. 13, 660–669 (2011).

    Article  CAS  Google Scholar 

  32. Reddy, P. et al. A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses. Nat. Med. 11, 1244–1249 (2005).

    Article  CAS  Google Scholar 

  33. Toubai, T. et al. Host-derived CD8+ dendritic cells are required for induction of optimal graft-versus-tumor responses after experimental allogeneic bone marrow transplantation. Blood 121, 4231–4241 (2013).

    Article  CAS  Google Scholar 

  34. Reddy, P. et al. Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase-dependent DC functions and regulates experimental graft-versus-host disease in mice. J. Clin. Invest. 118, 2562–2573 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Levy, M. et al. Microbiota-modulated metabolites shape the intestinal microenvironment by regulating NLRP6 inflammasome signaling. Cell 163, 1428–1443 (2015).

    Article  CAS  Google Scholar 

  36. Cooke, K. R. et al. Tumor necrosis factor-α production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. J. Clin. Invest. 102, 1882–1891 (1998).

    Article  CAS  Google Scholar 

  37. Hill, G. R. et al. Interleukin-11 promotes T cell polarization and prevents acute graft-versus-host disease after allogeneic bone marrow transplantation. J. Clin. Invest. 102, 115–123 (1998).

    Article  CAS  Google Scholar 

  38. Toubai, T. et al. Ikaros–Notch axis in host hematopoietic cells regulates experimental graft-versus-host disease. Blood 118, 192–204 (2011).

    Article  CAS  Google Scholar 

  39. Toubai, T. et al. Induction of acute GVHD by sex-mismatched H-Y antigens in the absence of functional radiosensitive host hematopoietic-derived antigen-presenting cells. Blood 119, 3844–3853 (2012).

    Article  CAS  Google Scholar 

  40. Kozich, J. J., Westcott, S. L., Baxter, N. T., Highlander, S. K. & Schloss, P. D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol. 79, 5112–5120 (2013).

    Article  CAS  Google Scholar 

  41. Schloss, P. D. et al. Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 75, 7537–7541 (2009).

    Article  CAS  Google Scholar 

  42. Schloss, P. D. A high-throughput DNA sequence aligner for microbial ecology studies. PLoS ONE 4, e8230 (2009).

    Article  Google Scholar 

  43. Pruesse, E. et al. SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res. 35, 7188–7196 (2007).

    Article  CAS  Google Scholar 

  44. Schloss, P. D. & Westcott, S. L. Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl. Environ. Microbiol. 77, 3219–3226 (2011).

    Article  CAS  Google Scholar 

  45. Westcott, S. L. & Schloss, P. D. De novo clustering methods outperform reference-based methods for assigning 16S rRNA gene sequences to operational taxonomic units. PeerJ 3, e1487 (2015).

    Article  Google Scholar 

  46. Yue, J. C. & Clayton, M. K. A similarity measure based on species proportions. Commun. Stat. Theory Methods 34, 2123–2131 (2005).

    Article  Google Scholar 

  47. Anderson, M. J. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26, 32–46 (2001).

    Google Scholar 

  48. Segata, N. et al. Metagenomic biomarker discovery and explanation. Genome Biol. 12, R60 (2011).

    Article  Google Scholar 

  49. Wang, Q., Garrity, G. M., Tiedje, J. M. & Cole, J. R. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ. Microbiol. 73, 5261–5267 (2007).

    Article  CAS  Google Scholar 

  50. Cole, J. R. et al. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res. 42, D633–D642 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the National Institutes of Health grants AI-075284 (P.R.) and HL-090775 (P.R.), the American Society of Blood and Marrow Transplantation New Investigator Award (T.T.), the JSPS Postdoctoral Fellowships for Research Abroad (H.F.) and the YASUDA Medical Foundation Grants for Research Abroad (H.F.). This research was supported by work performed by the University of Michigan Microbial Systems Molecular Biology Laboratory.

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T.T. designed and performed the experiments, analysed the data and wrote the paper. H.F., C.R. and M.R. performed the experiments, analysed the data and wrote the paper. H.T. and C.Z. performed the experiments and analysed the data. C.L. performed the experiments and histopathological analysis. A.V.M., J.B., K.O.-W., I.M., S.B., Y.S., D.P., J.W., I.H., S.K., J.C., S.S. and S.P. performed the experiments. G.C. analysed the data, contributed reagents and wrote the paper. P.R. designed the experiments, analysed the data and wrote the paper.

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Correspondence to Grace Chen or Pavan Reddy.

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Supplementary Table 1

A dataset showing the exact P values for all graphs in this manuscript.

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Toubai, T., Fujiwara, H., Rossi, C. et al. Host NLRP6 exacerbates graft-versus-host disease independent of gut microbial composition. Nat Microbiol 4, 800–812 (2019). https://doi.org/10.1038/s41564-019-0373-1

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