Figure legends
Figure 1. Schematic representation of the cytokine pathways tested in the patient. Pathways evaluated in this manuscript include:
IFN-γ-JAK-STAT pathway (purple): binding of IFN-γ to its receptor leads to activation of Janus-kinase and phosphorylation (p) of STAT1. Dimers of pSTAT1 translocate to the nucleus to activate interferon stimulated genes (ISG). Patients with gain-of-function mutation in STAT1 are characterized by high levels of STAT1 and pSTAT1 resulting in enhanced ISG expression. IFN: interferon; JAK: Janus Kinase; STAT: signal transducer and activator of transcription;
NF-κB pathway: this pathway can be triggered by cytokines such as IL-17 (red), IL-1β (green), and TNF-α (pink), leading to the recruitment of TRAF proteins to trigger downstream activation of NF-κB and MAPK/AP-1 leading to the expression of pro-inflammatory molecules and chemokines (eg. CXCL1). Patients with defects of the adaptor molecule ACT1 (red) display impaired NF-κB activation upon IL-17. IL: interleukin; TNF: tumor necrosis factor; TRAF: tumor necrosis factor receptor-associated factor; NF-κB: nuclear factor κB; MAPK: mitogen-activated protein kinase; AP-1: activator protein-1.
Figure 2. Autosomal recessive ACT1 deficiency. A) Clinical features (from left to right) of the patient with oral thrush, angular cheilitis, Type 1 hypersensitivity allergic reaction post insect bites, fungal infection of the toe, and impetigo of the trunk. B)Pedigree of the kindred showing the familial segregation of STAT1and ACT1 alleles. C) Schematic representation of the ACT1 protein including the previously reported amino acid changes found in previously reported ACT1 deficiency cases (black) and in our patient (red).
Figure 3. IFN-γ-dependent STAT1 activation in patient’s CD14+ monocytes: Geometric mean fluorescence intensity (gMFI) of total STAT1 (A), and pSTAT1 (B),evaluated by flow cytometry in non-stimulated (black), or IFN-γ-stimulated (gray) CD14+ monocytes from healthy controls, patient’s relatives (father, mother and sister), and the patient.
Figure 4. Patient’s ACT1 allele molecular characterization and impact on IL-17A responses. A) HEK-293T cells were transfected with plasmids encoding WT or mutant (D451G, K454fs11*, or T536I) ACT1. Protein expression was analyzed western blot using antibodies against ACT1 and GAPDH, as a loading control. B)HEK-293T cells were co-transfected with plasmids encoding Flag-tagged IL-17RA, together with HA-tagged WT or mutant D451G, K454fs11*, or T536I ACT1. Cell lysates were immunoprecipitated with anti-Flag antibodies. Left panel shows the input, and right panel the immunoprecipitation. Immunoblotting analysis was performed with anti-HA or anti-Flag specific antibodies. Empty plasmid (EV) was used as negative control. C)HEK-293T cells were co-transfected with WT or mutant (D451G, K454fs11*, or T536I) ACT1-encoding plasmids along with NF-κB–dependent firefly luciferase plasmids and a Renilla luciferase reporter. An empty plasmid (EV) was used as negative control. Luciferase activity was evaluated after 24 hours of stimulation with IL-17A and normalized to Renilla signal using the Dual-Glo Stop & Glo system (Promega). D)GRO-α production, as measured by ELISA, after stimulation (IL-17A, TNF-α, IL-17A+TNF-α, or IL-1β) of SV40-fibroblasts from healthy individuals, patients with IL-17RA, IL-17RC, or ACT1 (ACT1T536I/T536I) deficiency, and the patient under study (D451G, K454fs11*).
Figure 5. Proportions of IL-17A-producing memory CD4+ T cells in the patient. Controls and patient’s PBMCs were stimulated with PMA/ionomycin for 5 hours. Levels of memory CD4+ T cells expressing IL-17, IL-22, IFN-γ, and IL-4 were evaluated by flow cytometry. White symbols are the healthy controls, with the square representing a travel control. The black symbols are the patient’s relatives, with the circle representing the father, the square the mother, and the sister by the diamond, and red symbols represent the patient.