Research ArticleIMMUNODEFICIENCIES

Chronic mucocutaneous candidiasis and connective tissue disorder in humans with impaired JNK1-dependent responses to IL-17A/F and TGF-β

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Science Immunology  29 Nov 2019:
Vol. 4, Issue 41, eaax7965
DOI: 10.1126/sciimmunol.aax7965
  • Fig. 1 Identification of a heterozygous MAPK8 mutation in a kindred with AD CMC and CTD.

    (A) Pedigree and segregation of the MAPK8 mutation. The patients, indicated with filled black symbols, are heterozygous for the mutation. “E?” indicates individuals whose genetic status could not be evaluated. (B) Electropherograms of partial sequences of MAPK8 corresponding to the mutation in a healthy control (C) and four members of the kindred (II.1, P1, P2, and P3). (C) Schematic illustration of the genomic locus and of the protein encoded by the MAPK8 gene extracted from the Ensembl database. It has 13 exons (I to XIII), 12 of which are coding exons (II to XIII), encoding four isoforms (JNK1α1, JNK1α2, JNK1β1, and JNK1β2), with alternative usage of exon VII or VIII and alternative splicing of exon XIII. The red arrow indicates the position of the mutation.

  • Fig. 2 The mutant MAPK8 allele is loss-of-expression.

    (A) MAPK8 mRNA levels in EBV-B cells and SV40-fibroblasts from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3). TA cloning and subsequent sequencing of the five bands generated by amplification from exon III to exon V identified three spliced transcripts: band 1 corresponding to the WT sequence together with intron IV retention and exon IV skipping; band 2 (376 bp) corresponding to intron IV retention; band 3 corresponding to the WT sequence together with exon IV skipping; band 4 (284 bp) corresponding to the WT sequence; band 5 (225 bp) corresponding to exon IV skipping. (B) Schematic diagram of the constructs used for exon trapping. pET01, exon-trapping vector; RSV, Rous sarcoma virus long terminal repeat promoter; pA, polyadenylation; E in black, exon of the pET01 vector; IV and V in blue, MAPK8 exons IV and V; in yellow, MAPK8 intron IV. The red arrow indicates the position of the mutation. Reverse transcription PCR and subsequent sequencing identified three spliced transcripts: band 1 corresponding to intron IV retention and exon IV skipping; band 2 (354 bp) corresponding to the WT sequence; band 3 (295 bp) corresponding to exon IV skipping. (C) Schematic illustration of the mutant proteins. JNK1ES (JNK1 exon skipping) represents exon IV skipping, whereas JNK1IR (JNK1 intron retention) denotes intron IV retention. Both transcripts are predicted to encode proteins of about 10 kDa in size. Red arrows indicate the positions of premature stop codons. (D) mRNA levels for MAPK8 isoforms in EBV-B cells (top) and SV40-fibroblasts (bottom) from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3). Quantitative reverse transcription PCR was performed with primers specific for JNK1α1/JNK1α2 and JNK1β1/JNK1β2 mRNAs. α/β, total mRNA corresponding to JNK1α1, JNK1α2, JNK1β1, and JNK1β2; α, total mRNA corresponding to JNK1α1 and JNK1α2; β, total mRNA corresponding to JNK1β1 and JNK1β2. The values shown are the means ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ****P < 0.0001, unpaired t tests. (E and F) Immunoblot of JNK1 in EBV-B cells and SV40-fibroblasts from healthy controls (C1, C2, and C3) and patients (P1, P2, and P3) (E), and in HEK293T cells transfected with plasmids encoding four WT JNK1 isoforms (α1, α2, β1, and β2) and two mutants (ES and IR) inserted into the pTRIP-SFFV vector or the pCMV6-AN-Myc-DDK vector (F). Endogenous JNK1 was detected with an anti-JNK1 antibody recognizing the N terminus of JNK1. Myc-tagged JNK1 was detected with an anti-Myc antibody. EV, empty vector. The data shown are representative of three independent experiments (A, B, E, and F).

  • Fig. 3 The MAPK8 variant impairs fibroblast responses to IL-17A/F.

    (A) Production of GRO-α (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2), patients (P2 and P3), and an IL-17RA–deficient (IL17RA−/−) patient (16) stimulated with IL-17A, IL-17F, or IL-17A/F (10, 100, or 1000 ng/ml) for 24 hours. (B) Production of GRO-α (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2), patients (P2 and P3), and a NEMO-deficient (NEMO−/−) patient (92) stimulated with TNF-α (20 ng/ml) or IL-1β (10 ng/ml) for 24 hours. (C) Production of GRO-α (top) and IL-6 (bottom) by SV40-fibroblasts from healthy controls (C1 and C2) and patients (P2 and P3) transfected with empty vector (EV) or plasmids encoding WT JNK1α1 (α1), JNK1α2 (α2), JNK1β1 (β1), JNK1β2 (β2), all four isoforms (α1/α2/β1/β2), JNK1ES (ES), or JNK1IR (IR) in the presence of IL-17A (100 ng/ml) for 24 hours. (D) Production of GRO-α (left) and IL-6 (right) by primary fibroblasts from healthy controls (C1, C2, and C3) transfected with control siRNA (50 nM) or MAPK8 siRNA (50 nM) for 24 hours and then stimulated with IL-17A (100 ng/ml) for an additional 24 hours. The values shown are the means ± SEM of three independent experiments (A to D). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, unpaired t tests (A to D).

  • Fig. 4 Compromised T cell differentiation in the patients.

    (A) Percentage of total, naïve (CCR7+CD45RA+), central memory (CM; CCR7+CD45RA), effector memory (EM; CCR7CD45RA), or CD45RA+ effector memory (EMRA; CCR7CD45RA+) CD4+ and CD8+ T cells from healthy controls (n = 40) and patients (P2 and P3). (B) Frequency of TH1 (CXCR5CXCR3+CCR6), TH2 (CXCR5CXCR3CCR6CCR4+), TH17 (CXCR5CXCR3CCR6+CCR4+), TH1* (CXCR5CXCR3+CCR6+CCR4+), TFH (CXCR5+), and Treg (CD25+FOXP3+) subsets among CD4+ T cells from healthy controls (TH1, TH2, TH17, TH1*, and TFH, n = 34; Treg, n = 17) and patients (P2 and P3). (C) Production of IL-17A and IL-22 by whole blood from healthy controls (n = 33) and patients (P2 and P3) after stimulation with PMA plus ionomycin for 24 hours. (D) Percentage of IL-17A+, IL-17F+, and IFN-γ+ cells among memory CD4+ T cells from healthy controls (n = 36) and patients (P2 and P3) activated by TAE beads or PMA plus ionomycin (P/I) for 12 hours. (E) Cytokine production by naïve CD4+ T cells from healthy controls (n = 8) and patients (P2 and P3) cultured under TH0-, TH17-, or TH1-polarizing conditions. (F and G) Frequency of IL-17A+ and IFN-γ+ cells among naïve (F) and memory (G) CD4+ T cells from healthy controls (n = 10) and patients (P2 and P3) cultured under TH0-, TH17-, or TH1-polarizing conditions. C, healthy controls; P, P2 and P3. Horizontal bars represent median values (A to G). *P < 0.05 and **P < 0.01, two-tailed Mann-Whitney tests (A to G).

  • Fig. 5 Impaired response to TGF-β in the patients’ fibroblasts.

    (A) Immunofluorescence of FN; type V collagen (COLLV); type III collagen (COLLIII); α2β1, α5β1, and αvβ3 integrins; α-SMA; and CAD-11 in primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). Scale bar, 10 μm. (B) In vitro scratch assay with primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). Images were captured at 0 and 48 hours after scratching. Scale bar, 100 μm. (C) Transwell assay with primary fibroblasts from a healthy control (C), P2, a patient with hEDS (56), and a patient with cEDS (93). (D) mRNA induction in primary fibroblasts from healthy controls (C1 and C2) and patients (P2 and P3) stimulated with TGF-β (10 ng/ml) for the indicated times. (E) Top 10 up-regulated or down-regulated genes in terms of absolute fold change, in primary fibroblasts from healthy controls (C1 and C2) stimulated with TGF-β (10 ng/ml) for 2, 6, and 24 hours, with a greater than 1.5-fold change relative to patients (P2 and P3) at each time point. (F and G) Expression of JNK1 protein (F) and production of FN (top) and IL-11 (bottom) (G) by primary fibroblasts from healthy controls (C1 and C2) transfected with control siRNA (50 nM) or MAPK8 siRNA (50 nM) for 48 hours and then stimulated with TGF-β (10 ng/ml) for an additional 24 hours. NS, nonstimulated conditions. The values shown are the means ± SEM of two (C) or three (D and G) independent experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, unpaired t tests (D and G).

  • Fig. 6 JNK1-dependent IL-17 and TGF-β signaling.

    The binding of IL-17A/F to the IL-17RA/IL-17RC receptor facilitates the recruitment of ACT1 to the receptor, which mediates the activation of JNK1, ERK, p38, and NF-κB (p65/p50) signaling, leading to the production of pro-inflammatory cytokines and chemokines (e.g., CXCL1 and IL6). Similarly, TGF-β binds to its receptor (TGFBR1/TGFBR2), leading to the activation of JNK1, ERK, p38, and SMAD (SMAD2/3/4) signaling. This pathway ultimately results in the production of ECM proteins and regulators (e.g., FN1 and IL11). The mutation (yellow star) in MAPK8 encoding JNK1 impairs the JNK1-dependent activation of downstream AP-1 (c-Jun/ATF-2), thereby reducing the JNK1-dependent cellular responses to IL-17 and TGF-β.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/41/eaax7965/DC1

    Case reports

    Fig. S1. Identification of a private MAPK8 variant in the patients.

    Fig. S2. Impaired IL-17A/F signaling in the patients’ fibroblasts.

    Fig. S3. Normal B cell differentiation in the patients.

    Fig. S4. Impaired TGF-β signaling in the patients’ fibroblasts.

    Table S1. Immunological parameters of P1, P2, and P3.

    Table S2. Careful WES analysis of rare (MAF < 1%) nonsynonymous coding variants in the known CMC-, EDS-, LDS-, and MS-causing genes.

    Table S3. Heterozygous nonsynonymous variants common to P1, P2, and P3.

    Table S4. Clinical presentations of disorders caused by mutations in JNK1-dependent TGF-β target genes or in genes encoding the corresponding receptors.

    Table S5. Primers used for Sanger sequencing, reverse transcription PCR, and exon trapping.

    Table S6. Raw data file (Excel spreadsheet).

    References (94, 95)

  • Supplementary Materials

    The PDF file includes:

    • Case reports
    • Fig. S1. Identification of a private MAPK8 variant in the patients.
    • Fig. S2. Impaired IL-17A/F signaling in the patients’ fibroblasts.
    • Fig. S3. Normal B cell differentiation in the patients.
    • Fig. S4. Impaired TGF-β signaling in the patients’ fibroblasts.
    • Table S1. Immunological parameters of P1, P2, and P3.
    • Table S2. Careful WES analysis of rare (MAF < 1%) nonsynonymous coding variants in the known CMC-, EDS-, LDS-, and MS-causing genes.
    • Table S3. Heterozygous nonsynonymous variants common to P1, P2, and P3.
    • Table S4. Clinical presentations of disorders caused by mutations in JNK1-dependent TGF-β target genes or in genes encoding the corresponding receptors.
    • Table S5. Primers used for Sanger sequencing, reverse transcription PCR, and exon trapping.
    • References (94, 95)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S6. Raw data file (Excel spreadsheet).

    Files in this Data Supplement:

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