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. 2013 Oct 17;39(4):676-86.
doi: 10.1016/j.immuni.2013.09.002. Epub 2013 Oct 10.

An ACT1 mutation selectively abolishes interleukin-17 responses in humans with chronic mucocutaneous candidiasis

Bertrand Boisson  1 , Chenhui Wang, Vincent Pedergnana, Ling Wu, Sophie Cypowyj, Michel Rybojad, Aziz Belkadi, Capucine Picard, Laurent Abel, Claire Fieschi, Anne Puel, Xiaoxia Li, Jean-Laurent Casanova
Affiliations

An ACT1 mutation selectively abolishes interleukin-17 responses in humans with chronic mucocutaneous candidiasis

Bertrand Boisson et al. Immunity. .

Abstract

Patients with inborn errors of interleukin-17F (IL-17F) or IL-17RA display chronic mucocutaneous candidiasis (CMC). We report a biallelic missense mutation (T536I) in the adaptor molecule ACT1 in two siblings with CMC. The mutation, located in the SEFIR domain, abolished the homotypic interaction of ACT1 with IL-17 receptors, with no effect on homodimerization. The patients' fibroblasts failed to respond to IL-17A and IL-17F, and their T cells to IL-17E. By contrast, healthy individuals homozygous for the common variant D10N, located in the ACT1 tumor necrosis factor receptor-associated factor-interacting domain and previously associated with psoriasis, had impaired, but not abolished, responses to IL-17 cytokines. SEFIR-independent interactions of ACT1 with other proteins, such as CD40, heat shock protein 70 (HSP70) and HSP90, were not affected by the T536I mutation. Overall, human IL-17A and IL-17F depend on ACT1 to mediate protective mucocutaneous immunity. Moreover, other ACT1-dependent IL-17 cytokines seem to be largely redundant in host defense.

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Figures

Figure 1
Figure 1. A kindred with CMCD and autosomal recessive ACT1 deficiency
(A) Pedigree of the kindred showing the segregation of the ACT1 missense allele T536I. Individuals for whom genetic status could not be evaluated are indicated by “?”. Generations are designated by Roman numerals and individuals by Arabic numerals. (B) Manhattan plot showing the results for linkage analyses. The Lodscore (log10) for each SNP is plotted against chromosomal position (x-axis) for this kindred. The three genes carrying an unreported missense mutation identified by WES in the positive regions are indicated. (see also Table S2) (C) ACT1 DNA sequence electropherograms, for a healthy control and the four members of the kindred, for the region corresponding to the missense mutation (D) Alignment of ACT1 orthologs from different species. (E) Schematic diagram of the ACT1 protein. The gray box indicates the TRAF-interacting domain. The helix-loop-helix (HLH), U3-box (U3-box) and similar expression to fibroblasts growth factor and IL-17R (SEFIR) domains are shown. The arrow indicates the position of the missense mutation.
Figure 2
Figure 2. The T536I mutation impairs ACT1 recruitment to the IL-17 receptors
(A) ACT1 expression in EBV-B cells and SV-40 fibroblasts from P2. HEK293T cells transfected with WT-hACT1 or empty vector are shown as a control for the specificity of anti-ACT1 antibody. GAPDH is used as a loading control. (B–D) Immunoprecipitation of HA-tagged hACT1 in Act1−/− cells reconstituted with WT-hACT1-HA or T536I-hACT1-HA. Cells are cotransfected with IL-17RA-, IL-17-RB- or IL-17RC-V5-tagged receptor chains. Immunoblotting analyses were performed with anti-HA or anti-V5 specific antibodies (n=3). (E) Homodimerization of ACT1. hACT1-HA was immunoprecipitated in HEK293T cells overproducing WT- or T536I-hACT1-HA and WT- or T536I-hACT1-FLAG. The immunoblotting analysis was performed with specific anti-HA, anti-FLAG or anti-HSP90 antibodies (n=2). (F) Heterodimerization of hACT1 and HSP90. WT- or T536I-hACT1-HA was immunoprecipitated in HEK293T cells overproducing WT- or T536I-hACT1-HA and HSP90-FLAG. Immunoblotting analyses were performed with specific anti-HA, anti-FLAG or anti-HSP70 antibodies (n=2). (see also Figure S1).
Figure 3
Figure 3. The T536I ACT1 mutation abolishes the IL-17-responsive pathway in fibroblasts
(A) IL-6 and GRO-α quantification in the supernatant of SV-40 fibroblasts from controls (C1, C2), P2 or IL-17RA-deficient patients, in response to IL-17A (10, 100 and 1,000 ng/ml) or IL-1β (10 ng/ml); errors bars, SEM (n=3). (B) Complementation of the fibroblasts of P2 with the WT ACT1 allele. IL-6 and GRO-α quantification in the supernatant of SV-40 fibroblasts from the control, P2 or IL-17RA-deficient patients complemented (+ACT1) or not complemented with the WT ACT1 allele, after stimulation with IL-17A (100 ng/ml), TNF-α (2 ng/ml) or both. Errors bars, SEM (n=3). Statistical analyses were performed by the unpaired t-test method (NS: not significant; *, P<0.05; **, P< 0.01). (see also Figure S2)
Figure 4
Figure 4. The D10N ACT1 polymorphism impairs but do not abolishes he IL-17-responsive pathway in fibroblasts
(A) GRO-α and (B) IL-6 quantification in the supernatant of SV-40 fibroblasts from controls, D10N individual, P2 and an IL-17RA-deficient patient, upon stimulation with IL-17A (500 ng/ml), TNF-α (2 ng/ml) or both (IL-17A+TNF-α), or IL-1β (10 ng/ml); errors bars, SEM (n=7). Statistical analyses were performed by the unpaired t-test method (NS: not significant; *, P<0.05; **, P< 0.01).
Figure 5
Figure 5. The T536I ACT1 mutation abolishes the IL-17E response in T cells
(A) PBMCs from controls, patients (P1, P2) and healthy relatives (II.2, II.3) and from an IL-17RA-deficient patient were cultured in TSLP for 24 h, harvested, and restimulated with IL-2 and IL-17E for an additional 72 hours. IL-5 concentrations in the culture supernatants were determined by ELISA. IL-5 concentrations were normalized to 100 pg/ml upon IL-2 stimulation. Errors bars, SEM (n=2). (B) Percentages of IL-17A+-, IL-22+- and IFN-γ+- producing CD3+ cells, as determined by flow cytometry, in nonadherent PBMCs activated by incubation for 12 h with PMA and ionomycin. Each symbol represents a value from a healthy control individual (black circles), P1 (black rectangle) or an IL-17RA-deficient patient (black upside-down triangles). (see also Figure S3).
Figure 6
Figure 6. The T536I-ACT1 mutation does not perturb the CD40-responsive pathway
(A) Immunoprecipitation of ACT1-HA in HEK293T cells reconstituted with WT-hACT1-HA or T536I-hACT1-HA. Cells were cotransfected with human CD40-FLAG. Immunoblotting analyses were performed with anti-HA, anti-TRAF3 or anti-FLAG specific antibodies. (B) Activation of CD40 pathways in EBV-B cells from P2. Immunoblotting analysis was performed using antibodies against phospho-IKKα and phospho-IKKβ (P-IKKs), IκBα and phospho-p42 and phospho-p44 (P-p42-p44) in CD40L-stimulated EBV-B cells from the control or P2 (n=2).
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