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The receptor NLRP3 is a transcriptional regulator of TH2 differentiation

Nature Immunology volume 16, pages 859–870 (2015)Cite this article

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A Corrigendum to this article was published on 01 December 2015

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Abstract

The receptor NLRP3 is involved in the formation of the NLRP3 inflammasome that activates caspase-1 and mediates the release of interleukin 1β (IL-1β) and IL-18. Whether NLRP3 can shape immunological function independently of inflammasomes is unclear. We found that NLRP3 expression in CD4+ T cells specifically supported a T helper type 2 (TH2) transcriptional program in a cell-intrinsic manner. NLRP3, but not the inflammasome adaptor ASC or caspase-1, positively regulated a TH2 program. In TH2 cells, NLRP3 bound the Il4 promoter and transactivated it in conjunction with the transcription factor IRF4. Nlrp3-deficient TH2 cells supported melanoma tumor growth in an IL-4-dependent manner and also promoted asthma-like symptoms. Our results demonstrate the ability of NLRP3 to act as a key transcription factor in TH2 differentiation.

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Figure 1: NLRP3 is expressed in CD4+ T cells and is essential for TH2 differentiation.
Figure 2: IL-2 and STAT5 control NLRP3 expression.
Figure 3: The NLRP3 inflammasome is not functional in TH2 cells.
Figure 4: NLRP3 is located in the nucleus of TH2 cells.
Figure 5: NLRP3 acts as a transcription factor in TH2 cells.
Figure 6: NLRP3 interacts with IRF4 and enhances its ability to bind to the Il4 promoter.
Figure 7: Effect of NLRP3 deficiency in TH2 cells in an asthma model in mice.
Figure 8: Effect of NLRP3 deficiency on TH2 polarization and tumor growth.

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Change history

  • 12 October 2015

    In the version of this article initially published, reference 44 (Farrell, G.A. Responding to aggression; the role of significant others for student psychiatric nurses, an ethnographic study. Nurse Educ. Today 9, 335–340 (1989)) was incorrect. The correct reference is as follows: Sica, A. & Mantovani, A. Macrophage plasticity and polarization: in vivo veritas. J. Clin. Invest. 122, 787–795 (2012). The error has been corrected in the HTML and PDF versions of the article.

  • 01 December 2015

    Nat. Immunol. 16, 859–870 (2015); published online 22 June 2015; corrected after print 12 October 2015 In the version of this article initially published, reference 44 (Farrell, G.A. Responding to aggression; the role of significant others for student psychiatric nurses, an ethnographic study. NurseEduc.

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Acknowledgements

We thank V. Petrilli (INSERM 1052) for BALB/c Nlrp3−/− mice. Supported by the French National Research Agency (“Investissements d'Avenir” program; ANR-11-LABX-0021), the Ligue nationale contre le cancer (F.G. and F.V.), the Fondation de France (L.A.), the Institut National du Cancer (F.G.), the Fondation ARC (L.A.), the Conseil Régional de Bourgogne/INSERM (H.B.), the Centre National de la Recherche Scientifique, Fonds Européen de Développement Économique et Régional, Le Stadium, Orléans and Fondation pour la Recherche Médicale (F.G. and B.R.), the French National Research Agency (ANR-13-JSV3-0001 to L.A.), the Fondation pour l'aide à la Recherche sur la Sclérose en Plaques (L.A. and M.D.), the Ligue Régionale contre le cancer Comité Grand-Est (L.A.) and the European Commission (PCIG10-GA-2011-303719 to L.A.).

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Author notes

  1. Frédérique Végran and François Ghiringhelli: These authors jointly directed this work.

Authors and Affiliations

  1. Inserm, U866, Dijon, France

    Mélanie Bruchard, Cédric Rebé, Valentin Derangère, Romain Boidot, Etienne Humblin, Arlette Hamman, Fanny Chalmin, Hélène Berger, Angélique Chevriaux, Emeric Limagne, Lionel Apetoh, Frédérique Végran & François Ghiringhelli

  2. Faculté de Médecine, Université de Bourgogne, Dijon, France

    Mélanie Bruchard, Cédric Rebé, Valentin Derangère, Romain Boidot, Etienne Humblin, Arlette Hamman, Fanny Chalmin, Hélène Berger, Angélique Chevriaux, Emeric Limagne, Lionel Apetoh, Frédérique Végran & François Ghiringhelli

  3. Centre Georges François Leclerc, Dijon, France

    Cédric Rebé, Valentin Derangère, Romain Boidot, Angélique Chevriaux, Lionel Apetoh, Frédérique Végran & François Ghiringhelli

  4. Artimmune, Orleans, France

    Dieudonnée Togbé

  5. Laboratory of Experimental and Molecular Immunology and Neurogenetics, UMR 7355 CNRS–University of Orleans, Orleans, France

    Bernhard Ryffel

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Contributions

M.B. did in vitro and in vivo experiments; C.R., V.D., A.H., F.C., H.B., A.C. and E.L. did in vitro experiments; D.T. and B.R. generated asthma experiments; R.B. performed and supervised the extraction, amplification and library preparation for RNA sequencing and ChIP-seq; E.H. did in vitro experiments; F.V. performed bioinformatics analysis; M.B., L.A., F.V. and F.G. supervised the study and wrote the manuscript; and F.G. conceived of and designed the study.

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Correspondence to François Ghiringhelli.

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Integrated supplementary information

Supplementary Figure 1 NLRP3 is expressed in CD4+ T cells and is essential for TH2 differentiation.

(a)Western blot analysis of NLRP3 expression in T CD4+ cells from WT or Nlrp3−/– mice activated for 24 hours with anti-CD3 and anti-CD28 mAb. (b) Nlrp3 and Nlrp6 mRNA are expressed in T CD4+ T cells after TCR triggering. mRNA expression analysis of NLR genes in TH0, TH2, and TH1 differentiated from WT CD4+ T cells after 12h hours of stimulation with anti-CD3 and anti-CD28 mAb and the appropriate cytokines. (c) Ifng mRNA analysis in WT or Nlrp3−/– CD4+ T cells differentiated into TH1 or TH2 cells for 6 days. (d) Il4 mRNA analysis in WT or Nlrp3−/– CD4+ T cells differentiated into TH1 or TH2 cells for 6 days. (e) IL-13 production from WT or Nlrp3−/– CD4+ T cells differentiated into TH2 cells for 6 days were assessed by Elisa. (f) IL-5 production from WT or Nlrp3−/– CD4+ T cells differentiated in TH2 cells for 6 days were assessed by Elisa. (g) (Upper panel) Western blot analysis of GATA-3 expression in WT or Nlrp3−/– naïve and TH2 cells differentiated for 1, 3, or 6 days. (Lower panel) Quantification of GATA-3 expression. (h) WT or Nlrp3−/– CD4+ T cells were marked with CFSE and differentiated in TH2 cells for 3 days. Percentage of proliferating cells was assessed using CFSE dilution on day 1, 2 or 3. (i) Il4ra mRNA analysis in WT or Nlrp3−/– CD4+ T cells differentiated into TH0 or TH2 cells for 24h. (j) Ifng mRNA analysis in WT or Nlrp3−/– Balb/c CD4+ T cells differentiated into TH1 or TH2 cells for 3 days. (k) Il4m RNA analysis in WT or Nlrp3−/– Balb/c CD4+ T cells differentiated into TH1 or TH2 cells for 3 days.

Data are representative of three independent experiments *Indicates p<0.05

Supplementary Figure 2 Role of NLRP3 in TH1 and TH2 cells.

(a)IL1b mRNA analysis in Naïve T cells TH0 or TH2 cells after 3 days of differentiation. (b)Il4 mRNA analysis in TH0, TH2 and TH2 cultured with the Il-1 inhibitor anakinra or Nlrp3−/– after 3 days of differentiation. (c)Gata3 mRNA analysis in TH0, TH2 and TH2 cultured with the Il-1 inhibitor anakinra or Nlrp3−/– after 3 days of differentiation. (d) Caspase-1 activation was assessed by flow cytometry using FLICA-1 in naive T cells differentiated ex vivo in TH1 or TH2 either untreated or treated with Nigericin (Nig.10µM/ml) during the last hour. (e) IL-1β and IL-18 production was measured by ELISA in the same condition.

Data are representative of three independent experiments *Indicates p<0.05

Supplementary Figure 3 Subcellular localization of NLRP3.

(a)Frequency of cell with NLRP3 protein located in the nuclei or in the cytoplasm determined using immunofluorescence. (b) mRNA Expression of importin Ipo9, Ipo11 and Kpna2 in TH1 and TH2 cells after 3 days of differentiation

Data are representative of three independent experiments *Indicates p<0.05

Supplementary Figure 4 Global gene expression in wild-type or Nlrp3−/– T cells.

(a)Scatter plot displaying the RNA sequencing data of WT and Nlrp3−/– naive T cells. (b) Genes with similar expression in both Nlrp3−/– and WT Th2 were analyzed by using ClueGO software. Circles represent significantly enriched gene groups with p value corrected with Benjamini-Hochberg method.

Supplementary Figure 5 NLRP3 interacts with IRF4 to dictate the TH2 program.

(a)Il4 promoter activity mesured in NIH-3T3 cells transfected with an il4 promoter driven luciferas reporter plasmid along with IRF4 and NLRP3 expression plasmids. (b) Western Blot analysis of IRF4 expression in TH0, TH1, TH2, Treg, TH17 after 24 hours differentiation. (c) Nlrp3 analysis by qPCR in EL4 T cell clone after 24 hours of treatment with PBS or with PMA/Ionomycin. (d) NLRP3 analysis by Western blotting in EL4 T cell clone after 24 hours of treatment with PBS or with PMA/Ionomycin. (e) Western blot analysis of GATA3 after an immunoprecipitation assay with anti-NLRP3 antibody in WT TH2 cells differentiated for two days. (f) Putative IRF4 binding sites on the il4 promoter found by Bioinformatic analysis. (g) ChIP assay of IRF4 binding on the IRF4 putative binding site 2 and 3 on the Il4 promoter in WT or Nlrp3−/–CD4+ T cells differentiated in TH0 or TH2 cells for one day. (h) ChIP assay of NLRP3 binding on the IRF4 putative binding site 2 and 3 on the Il4 promoter in WT or Nlrp3−/– CD4+ T cells differentiated in TH0 or TH2 cells for one day.

Data are representative of two independent experiments. *Indicates p<0.05

Supplementary Figure 6 NLRP3 deficiency impedes TH2 programs in an asthma model.

(a)OVA sensitized Rag−/– mice reconstituted with Nlrp3−/– or WT CD4 T cells were challenged three times with OVA in absence of aluminium adjuvant. Eosinophil (left panel) and lymphocyte (right panel) counts were determined in bronchoalveolar lavage fluid (BALF) 24h after the last intranasal OVA administration (mean +sd., 6 mice per group, 2 independent experiments).(b) Lung sections from Nlrp3−/–- or WT CD4 T cells were stained with periodic acid Schiff reagent (PAS) to visualize mucus. A semi-quantitative histological assessment of mucus hypersecretion (left panel) and inflammatory cell infiltration (right panel) was performed by two independent observers (mean +sd., 6 mice per group, 2 independent experiments).

Supplementary Figure 7 NLRP3 deficiency impedes TH2 programs in cancer models.

(a)mRNA analysis of Il4 and Il13 in mediastinal lymph nodes of WT or Nlrp3−/– mice naive or 12 days after intravenous injection of B16F10 melanoma cells. (b) mRNA analysis of Il4 and Il13 in mediastinal lymph nodes of WT or Asc−/– or Caspase-1−/– mice naive or 12 days after intravenous injection of B16F10 melanoma cells.

Data are representative of three independent experiments. *Indicates p<0.05

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1 and 2 (PDF 760 kb)

Supplementary Movie 1

NRLP3 localization in Th1 cells (AVI 64 kb)

Supplementary Movie 2

NRLP3 localization in Th2 cells (AVI 88 kb)

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Bruchard, M., Rebé, C., Derangère, V. et al. The receptor NLRP3 is a transcriptional regulator of TH2 differentiation. Nat Immunol 16, 859–870 (2015). https://doi.org/10.1038/ni.3202

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