Conceived and designed the experiments: TSP FLvdV AH JAV MGN. Performed the experiments: SPS TSP BH AH. Analyzed the data: SPS TSP FLvdV BH AH LABJ BJK JAV DL JWMvdM MGN. Contributed reagents/materials/analysis tools: PDA AG DL MGN. Wrote the paper: SPS TSP FLvdV BH AH LABJ BJK JAV DL JWMvdM MGN.
¶ This author also contributed equally to this work.
The authors have declared that no competing interests exist.
We recently reported the genetic cause of autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) as a mutation in the
Chronic mucocutaneous candidiasis (CMC) is a hereditary primary immunodeficiency characterized by severe skin and mucosal
APECED is due to mutations in the gene
In the present study we assessed the presence and function of
In the present study we report that mutations in the CC-domain of STAT1, the genetic cause of AD-CMC, lead to hyperphosphorylation of STAT1 resulting in increased responsiveness to IFN-γ and impaired IL-12 and IL-23 signaling pathways. We strengthened our recent observation that STAT1 mutations are responsible for AD-CMC
Individual P4 from the Dutch family (#1) (
(A) Pedigree of a Dutch family with four patients affected from 3 generations. (B) Pedigree of a UK sample with 3 patients affected from 3 generations. (C) Pedigree of a UK sample with 3 patients affected from 2 generations. Patients with candidiasis = closed black symbols; male patients = squares; female patients = circles.
IL-12 induced no IFN-γ production in cells obtained from AD-CMC patients (
Defective IL-12R (A) and IL-23R (B) pathways in cells isolated from AD-CMC patients. Data represent mean + SEM from 3 healthy controls and 3 AD-CMC patients (P3 and P4 from family #1 and P3 from family 2). (C) Stimulation of the IFN-γ receptor pathway results in increased production of the proinflammatory cytokine TNFα. Data represent mean + SEM from 3 healthy controls and 3 AD-CMC patients. CD4+ T cells from healthy controls were transfected with wild-type or mutant STAT1 plasmid. (D) Decreased transcription of STAT4 upon stimulation with IL-1β (10 ng/mL) and IL-23 (50 ng/mL), (E) and decreased transcription of MCP1 upon IL-12 (10 ng/mL) and IL-18 (10 ng/mL) stimulation. (F) The CD4+ cells transfected with mutant STAT1 plasmid showed increased transcription of PD-L1 upon stimulation with IFN-γ (1 µg/mL). Data represent mean + SEM from 6 healthy controls from 3 separate experiments.
The transcription of several IFN-γ-regulated genes was measured in CD4+ T-cells from healthy controls transfected with either wild-type
Based on the results of these transfection studies, it can be concluded that the differences in gene expression observed in AD-CMC patients as compared to healthy controls can be fully ascribed to the presence of the Arg274Trp mutation in STAT1.
The consistent finding of increased responsiveness to IFN-γ in cells from patients and in cells transfected with the mutated STAT1 suggested a gain of function of STAT1. We therefore assessed whether the Arg274Trp mutation affects phosphorylation of STAT1. PBMCs were incubated with various stimuli and STAT1 phosphorylation was assessed by Western blotting. Phosphorylation of STAT1 after IFN-γ stimulation was higher in AD-CMC patients than in healthy controls (
(A) PBMC from two healthy controls and two AD-CMC patients were stimulated for two hours with culture medium, IFN-β (500 U/mL), IFN-γ (1 µg/mL), IL-23 (50 ng/mL) or IL-12 (10 ng/mL). Cells were lysed, and total STAT1, phosphorylated STAT1 and actin were assessed by Western blot. Figure is representative of two separate experiments. (B) Increased immunofluorescence of pSTAT1 in IFN-γ-stimulated CD4+ cells from AD-CMC patients. Figure is representative of three separate experiments. (C) PBMCs from three controls and two AD-CMC patients were stimulated with culture medium, IFN-β, or IFN-γ for two hours. The transcription of ICAM1 and MCP-1 was measured using RT-PCR. Bars represent means + SEM.
Upon IFN-γ stimulation through the IFN-γ receptor in physiological conditions, the downstream signaling molecules JAK1, JAK2 and STAT1 are activated
How do these molecular mechanisms explain the defective antifungal defense in AD-CMC patients? In the present study we show that these patients display significant deficiencies in the capacity to mount Th1 and Th17 responses, due to defective IL-12R and IL-23R pathways. The contributing role of a defective IL-12R signaling for CMC is strengthened by the observation that 23% of patients with isolated IL-12R deficiency have a mild form of CMC
In conclusion, we strengthen the very recent observations that
The clinical characteristics and pedigree of a Dutch Caucasian non-consanguinous family are represented in
The clinical characteristics and pedigree of a non-consanguinous British Caucasian family are presented in
The clinical characteristics and pedigree of a non-consanguinous British Caucasian family are presented in
The study was approved by the Ethics Committee of Radboud University Nijmegen Medical Centre, and the Newcastle and North Tyneside Local Research Ethics Committee. Informed consent was obtained from all family members and healthy controls.
To assess for the presence of
PBMCs were isolated by density gradient centrifugation of PBS-diluted blood (1∶1) over Ficoll-Paque, as previously described
For immunoblotting, 10×106 cells were lysed in 100 ml lysis buffer (50 mM Tris, 1 mM EDTA, 150 mM NaCL, 1% ND40, 5 mM NaF, 0.05% Sodium Deoxycholate, PhosSTOP and cOmplete proteinase inhibitor cocktail (Roche, Almere, the Netherlands)). The homogenate was frozen, thawed then centrifuged at 4 °C for 10 min at 14,000 rpm, and the supernatant was taken for Western blotting. Equal amounts of protein were subjected to SDS-PAGE using 10% and 15% polyacrylamide gels at a constant voltage of 100V. After SDS-PAGE, proteins were transferred to nitrocellulose membrane (0.2 mm). The membrane was blocked with 5% (w/v) milk powder in PBS for 1 hour at room temperature followed by incubation overnight at 4°C with a STAT1 or pSTAT1 (Tyr701) antibody (9172 and 9167 respectively, Cell Signaling, Leiden, the Netherlands) in 5% BSA/TBS/T (5% bovine serum albumin/Tris-buffered saline/Tween 20). After overnight incubation the blots were washed three times with TBS/T and incubated with HRP-conjugated goat anti-rabbit antibody at a dilution of 1∶10,000 in 5% (w/v) milk powder in PBS for 1 hour at room temperature. After washing the blots three times with TBS/T, the blots where developed with ECL according to manufacturer's instructions (GE Healthcare, Diegem, Belgium).
CD4+ cells were stimulated for 10 minutes with IFN-γ on cover slides. Subsequently, the cells were fixed with 10% PFA, and stained with α-pSTAT1 (Tyr701) or α-STAT1 and goat anti-rabbit Alexa 488 according to manufacturer instructions. The coverslides were mounted using Vectashield containing DAPI.
The Arg274Trp mutation was introduced into a plasmid containing the human STAT1α gene (pUNO-hSTAT1a, Invivogen, Toulouse, France), using the QuickChangeTM Site-Directed Mutagenesis Kit (200518, Stratagene, Eindhoven, the Netherlands). The following primers were used for the PCR reaction: 5′- GAG-AGT-CTG-CAG-CAA-GTT-TGG-CAG-CAG-CTT-AAA-AAG-3′ (forward) and 5′- CTT-TTT-AAG-CTG-CTG-CCA-AAC-TTG-CTG-CAG-ACT-CTC-3′ (reverse). Human CD4+ cells were transfected using HIPerfect reagent (Qiagen, Venlo, the Netherlands), according to manufacturer instructions.
One million freshly isolated PBMCs or (transfected) CD4+ cells were stimulated with IFN-γ, IL-β and IL-23 or IL-12 and IL-18. After two hours, total RNA was extracted in 400 µL TRIzol reagent (Invitrogen). Isolated RNA was reverse transcribed into complementary DNA using oligo(dT) primers and MMLV reverse transcriptase. PCR was performed using a 7300 realtime PCR system (Applied Biosystems, Lennik, Belgium). Primer sequences are presented in Supplemental Information. PCR conditions were as follows: two minutes at 50°C and 10 minutes at 95°C, followed by 40 cycles of PCR reaction at 95°C for 15 seconds, and 60°C for one minute. Primer sequences used for RT-PCR assessment were: STAT4:
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