Research ArticleInflammation

Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation

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Science Translational Medicine  30 Mar 2016:
Vol. 8, Issue 332, pp. 332ra45
DOI: 10.1126/scitranslmed.aaf1471
  • Fig. 1. Clinical and immunological features of PAAND.

    (A) Dominant inheritance of PAAND over three generations, with complete penetrance observed for the S242R mutation (MEFV c.C726G, denoted CG). Trios indicated by red boxing were subjected to exome sequencing. (B) Twenty-two family members were assessed for neutrophilic dermatosis, recurrent childhood-onset fever, acute-phase reactants, arthralgia, myalgia, cardiomyopathy, anemia, pyogenic arthritis, and serositis across three generations (G1, G2, and G3). ND, not determined; “*,” recent onset. (C) Typical macroscopic features (top: pyoderma lesions on the arm; bottom: pustular acne lesions on the face) (representative from patient II.7). (D) Histologic presentation of inflammatory infiltrate in the subcutis, involving subcutaneous vessels and extending into the surrounding panniculus and deep dermis. Scale bar, 500 μm. Higher magnification shows predominantly polymorphonuclear vascular and perivascular infiltrate (representative from patient II.7). Scale bar, 50 μm. (E) Serum levels of IL-1β, IL-1Ra, and IL-6 in patients during inflammatory episodes (n = 6) and healthy controls (n = 121), compared by t test. For healthy controls, box and whiskers represent ranges of 25 to 75% and 5 to 95%. For patients, mean and SD are shown. Patients with values >95th percentile of the healthy range are annotated as “elevated.”

  • Fig. 2. Spontaneous inflammasome activation by pyrin S242R.

    (A) Confocal microscopy showing a pyrin-driven increase in cytoplasmic specks of GFP-tagged ASC when cotransfected into HEK293T cells with wild-type (WT) or mutated pyrin (S242R). Scale bar, 50 μm. (B) Representative fluorescence-activated cell sorting analysis of GFP-ASC specks with pyrin and pyrin S242R. (C) Quantification of three biological replicates for GFP-ASC specks by flow cytometry, with different pyrin mutants. (D) Western blots for active caspase-1 (p20 subunit) after expression of caspase-1 and ASC in HEK293T cells, together with an increasing concentration of Myc-tagged pyrin or pyrin (S242R). cDNA, complementary DNA. (E and F) Monocytic THP-1 cells where caspase-1 or pyrin was deleted by CRISPR, then reconstituted by retroviral expression of pyrin or pyrin (S242R). IL-1β was measured by enzyme-linked immunosorbent assay (ELISA) (E), and cell death was quantified by propidium iodide (PI) staining and flow cytometry 24 hours after stimulation with LPS (F). All are representative of at least three biologically independent experiments. Error bars represent means + SD for three biological replicates. ****P < 0.0001. Ctrl, control.

  • Fig. 3. Guard-like mechanism of pyrin activation.

    (A) Pyrin with or without the S242R variant was overexpressed in HEK293T cells and immunoprecipitated by virtue of a GST tag. Eluates were examined by Western blotting, probing for endogenous (pan)14-3-3, with GST (pyrin) and (pan)14-3-3 also quantified in the starting lysates. (B) HEK293T cells expressing GST alone or GST-pyrin were treated with or without TcdB for 6 hours before lysis and analyzed as in (A). (C) HEK293T cells expressing GST-tagged pyrin or S242R pyrin were left untreated or treated with TcdB as in (B), then immunoprecipitated before Western blotting for pSer (14-3-3 motif) and GST (pyrin). All are representative of at least three biologically independent experiments. IP, immunoprecipitation.

  • Fig. 4. PAAND is driven by local inflammasome activation and IL-1β production.

    (A) Monocytes were purified from three PAAND patients and three healthy controls before stimulation for 1 hour with LPS ± adenosine triphosphate (ATP), or ± TcdB. Mature IL-1β was measured in the supernatant. Means + SD for n = 3. (B) Immunofluorescence staining for cleaved caspase-1, IL-1β, and pro–IL-1β in healthy control skin and an affected biopsy from patient III.3 (scale bars, 200 μm). DAPI, 4′,6-diamidino-2-phenylindole. (C) CRP levels were measured before and after treatment with anakinra (100 mg/day) (day 0) in UK patient G1. Dashed line indicates 3-year average CRP level before treatment, arrow indicates initiation of treatment, ticks indicate sample points, and gray zone indicates normal CRP range.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/332/332ra45/DC1

    Fig. S1. Inflammatory manifestations of PAAND.

    Fig. S2. Results from linkage analysis.

    Fig. S3. Dominant inheritance of PAAND caused by mutation in pyrin, S242R.

    Fig. S4. Inheritance of S242R mutations in additional pedigrees.

    Fig. S5. Fluorescent microscopy of pyrin/ASC specks.

    Fig. S6. Activation of caspase-1 and IL-1β by pyrin S242R.

    Fig. S7. Proteomic analysis of pyrin interactions and phosphorylation.

    Fig. S8. Functional effect of FMF mutations in context of pyrin S242R.

    Fig. S9. Full Western blots.

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  • Supplementary Material for:

    Familial autoinflammation with neutrophilic dermatosis reveals a regulatory mechanism of pyrin activation

    Seth L. Masters,* Vasiliki Lagou, Isabelle Jéru, Paul J. Baker, Lien Van Eyck, David A. Parry, Dylan Lawless, Dominic De Nardo, Josselyn E. Garcia-Perez, Laura F. Dagley, Caroline L. Holley, James Dooley, Fiona Moghaddas, Emanuela Pasciuto, Pierre-Yves Jeandel, Raf Sciot, Dena Lyras, Andrew I. Webb, Sandra E. Nicholson, Lien De Somer, Erika van Nieuwenhove, Julia Ruuth-Praz, Bruno Copin, Emmanuelle Cochet, Myrna Medlej-Hashim, Andre Megarbane, Kate Schroder, Sinisa Savic, An Goris, Serge Amselem, Carine Wouters,* Adrian Liston*

    *Corresponding author. E-mail: masters{at}wehi.edu.au (S.L.M.); carine.wouters{at}uzleuven.be (C.W.); adrian.liston{at}vib.be (A.L.)

    Published 30 March 2016, Sci. Transl. Med. 8, 332ra45 (2016)
    DOI: 10.1126/scitranslmed.aaf1471

    This PDF file includes:

    • Fig. S1. Inflammatory manifestations of PAAND.
    • Fig. S2. Results from linkage analysis.
    • Fig. S3. Dominant inheritance of PAAND caused by mutation in pyrin, S242R.
    • Fig. S4. Inheritance of S242R mutations in additional pedigrees.
    • Fig. S5. Fluorescent microscopy of pyrin/ASC specks.
    • Fig. S6. Activation of caspase-1 and IL-1β by pyrin S242R.
    • Fig. S7. Proteomic analysis of pyrin interactions and phosphorylation.
    • Fig. S8. Functional effect of FMF mutations in context of pyrin S242R.
    • Fig. S9. Full Western blots.

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    Other Supplementary Material for this manuscript includes the following:

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