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T. Shahin et al.
recently identified a singleton patient (herein referred to as PN404Y) carrying a homozygous loss-of-function mutation in IL6ST (p.N404Y) encoding the cytokine receptor subunit GP130 with remarkable clinical manifestations resembling that of STAT3 HIES, whereby PN404Y experienced recurrent infections, eczema, elevated IgE, eosinophilia, impaired acute-phase response, scoliosis and craniosynostosis.5
The GP130 subunit binds to several cytokine receptors including IL-6 receptor alpha (IL-6RA), IL-11RA, IL-27RA, leukemia inhibitory factor (LIF) receptor, oncostatin M (OSM) receptor and ciliary neurotrophic factor (CNTF) receptor. Upon stimulation with the respective cytokine, several JAK/STAT pathways are activated downstream of these receptors.6 The importance of GP130-mediated sig- naling was shown in Il6st-/- mice that die embryonically due to myocardial and hematopoietic defects.7 Moreover, postnatal conditional inactivation of gp130 in mice leads to neurological, hepatic and immunological defects with impaired acute-phase response, increased susceptibility to infections, and development of lung emphysema.8 With regards to cytokines, the contribution of IL-6 signaling to immune responses against pathogens has been empha- sized in Il-6-deficient mice as well as in children with autoantibodies against IL-6.9-12 The roles of other GP130- dependent cytokines in the context of disease have been studied, whereby the importance of IL-27 signaling has been shown in mediating T-cell responses, IL-11 in bone development, LIF in hematopoiesis and thymocyte devel- opment, and CTNF in the maintenance of motor neu- rons.13-17
Here, we identify a patient harboring a novel biallelic mutation in IL6ST (herein referred to as PP498L), presenting with elevated IgE levels, recurrent infections, severe atopic dermatitis, and characteristic skeletal abnormalities. We observe complete or partial disruption of selected cytokine signaling pathways in various cell types from PP498L. We extend our initial findings on human GP130 defi- ciency by comparatively dissecting signaling defects and perturbations in T-cell differentiation in the PP498L patient and our previously reported patient (PN404Y).5 Finally, we pinpoint selective roles of impaired IL-6, IL-11 and IL-27 signaling in the presence of other functional cytokine sig- naling pathways such as IL-21 and IL-10.
Methods
Subjects
Patients and healthy controls were included with informed written consent and approval from the Institutional Review Boards of the Medical University of Vienna, Hacettepe University Medical School in Ankara, Oxfordshire Research Ethics Committee B, the London Riverside Research Ethics Committee, and Oxford Gastrointestinal Illness Biobank.5
GP130 expression analysis
Patient and healthy donor fibroblasts were detached by incuba- tion with 50 mM EDTA in PBS for 30 minutes (min) on ice fol- lowed by gentle scraping with a silicon blade (CytoOne®). Cells were immediately washed with PBS and stained with GP130- BV421 or an isotype control, CD4-BV421 (both IgG1κ), for 35 min on ice. Cells were washed and resuspended in PBS for flow cytometry analysis.
p-STAT analysis by flow cytometry
Peripheral blood mononuclear cells (PBMCs). Frozen PBMCs from patient and healthy donor controls were thawed and allowed to recover for four hours (h) at 37°C in complete media (RPMI-1640 with 10% FBS). Cells were subsequently stained with CD3-FITC, CD4-BV605, CD8-V450 and CD19-PECy7 for 20 min at 37°C. Five minutes through the extracellular staining, cells were stimu- lated for 15 min at 37°C with IL-6, IL-21, IL-27 (all 100 ng/mL) or IL-10 (50 ng/mL). Cells were then immediately fixed for 10 min at 37°C, washed and permeabilized for 35 min on ice. Cells were then stained with p-STAT3-AF647 for 1 h at room temperature, washed again, and resuspended in FACS buffer for flow cytome- try analysis.
T lymphoblasts and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs). T lymphoblasts from patient and healthy donors were starved for 2 h in T-cell media (RPMI-1640 with 5% human- serum) without IL-2, while EBV-LCLs were starved for 2-3 h in serum-deprived media (RPMI-1640). Cells were then stimulated with the cytokines mentioned and p-STAT staining performed as described above. (See Online Supplementary Table S1 for all anti- bodies used.)
Fibroblasts and HEK293 GP130-KO cell line. Fibroblasts and HEK293 GP130-KO cells5 were grown in complete media (DMEM with 10% FBS) in 12-well and 96-well plates, respectively. Fibroblasts and HEK293 GP130-KO cells were serum-starved overnight or for 2-3 h, respectively, followed by stimulation with increasing concentrations (0.01, 0.1, 1, 10, 100 ng/mL) of IL-6, IL- 11, IL-27, LIF and OSM cytokines for 15 min, immediate detach- ment using Trypsin-EDTA, and fixation for 10 min at 37°C. The p-STAT staining was performed as described above.
HEK293 GP130-KO cells were transiently transfected with empty plasmid [pcDNA3.1(+)] or plasmid encoding WT or mutant GP130 using Lipofectamine 2000 (Thermo Fisher) before p-STAT3 analysis as previously described.5 Cells were co-trans- fected with either IL-6RA or IL-11RA to enhance the phosphory- lation signal. GFP-coding plasmid was co-transfected to allow gat- ing on successfully transfected cells.
Intracellular cytokine staining
T-cell cytokine production was analyzed in 0.5-1x106 total PBMCs that were stimulated for 5 h with Phorbol 12-myristate 13-acetate (PMA, 0.2 mM) and Ionomycin (1 mg/mL) with the addi- tion of Brefeldin A during the final 2.5 h. To identify T-cell subpop- ulations, surface staining was performed as indicated below. Subsequently, cells were fixed, permeabilized and stained for intracellular cytokines followed by flow cytometry analysis. The following antibodies were used: IL-4, IL-10, IL-13, IL-17A, IL-22, IFN-γ and TNF. Dead cells were excluded from analysis by staining with fixable viability dye eFluor780 (eBioscience).
Chemokine receptor profiling
For the analysis of surface marker expression by flow cytome- try, 0.5-1x106 total PBMCs were incubated with fluorochrome- conjugated antibodies for 15 min in PBS supplemented with 0.5% human serum at 37°C or room temperature, as assessed by titra- tion experiments at both temperatures. To exclude dead cells from the analysis, cells were stained with fixable viability dye eFluor780 (eBioscience). Antibodies used for cell surface immunophenotyp- ing included: CD3, TCRαb, CD4, CD8, CD25, CD45RA, CD127, CCR4, CCR6, CCR7, CCR9, CCR10, CXCR3, CXCR5, CRTh2. All samples were acquired using the Beckman Coulter CytoFlex, BD LSRII or BD LSR Fortessa. Other methods and details of the antibodies used can be found in Online Supplementary Table S1.
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haematologica | 2019; 104(3)