Introduction
Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity [
1‐
3]. Patients with MSMD are defined as individuals who are susceptible to infections by weakly virulent environmental mycobacteria and Bacillus Calmette-Guérin (BCG) disease post vaccination [
4]. To date, 19 genes are implicated in MSMD (
CYBB, IFNGR1, IFNGR2, IFNG, IL12RB1, IL12B, IL23R, IL12RB2, ISG15, IRF8, JAK1, NEMO, RORC, SPPL2A, STAT1, TBX21, TYK2, USP18, ZNFX1). The different genetic disorders are further defined by the nature of the causal alleles (null or hypomorphic), protein levels (normal, low, or absent), and the mode of inheritance (autosomal recessive: AR, autosomal dominant: AD, or X-linked recessive: XL) and whether they present as isolated MSMD or as a part of a broader spectrum (syndromic MSMD) involving infectious susceptibility to other pathogens and/or autoinflammation [
1,
5,
6]. The most frequent genetic defects are found in
IL12 or a subunit of its receptor (AR complete IL12p40 deficiency or AR IL12RB1 deficiency), followed by defects in the IFN-γ receptor (AD or AR complete/partial IFNGR1/2 deficiency) [
1]. For other genes such as
IL23R or
IL12RB2, only a single kindred with functional validation of the corresponding genetic defect has been described [
7]. This is not hypothesized to be due to increased rarity of loss of function (LOF) mutations in these genes, but due to the lower penetrance of these deficiencies [
7]. In case of IL23R deficiency, two affected pediatric-onset cases from the same kindred with a phenotype of disseminated BCG disease were found to have a pathogenic homozygous mutation (p.C115Y) in the extracellular domain of IL23R. Although the mutation did not impair mRNA or protein expression, reduced membrane-bound IL23R and impaired IL-23 signaling was observed. Another IL23R mutation (p.R381Q) has been reported as a partial LOF variant and associates with pulmonary tuberculosis severity [
8]. In this study, we describe a kindred with a homozygous mutation (c.1141C > T, R381X) in the intracellular domain of IL23R, presenting as adult-onset disseminated non-tuberculous mycobacterial disease. We used in silico prediction tools, immunophenotyping, and functional analysis of peripheral blood mononuclear cells (PBMCs) and cell lines to corroborate the genotype–phenotype relation in this patient.
Methods
gDNA Extraction and Whole Exome Sequencing
For gDNA extraction from whole blood, Purelink DNA Genomic DNA Mini kit (ThermoFisher Scientific) was used according to the manufacturers’ instructions. Whole exome sequencing was performed using SureSelect Human All Exon V7 (Agilent) for exome capture (Macrogen®, the Netherlands).
PBMC Isolation
Whole blood was diluted 1:1 with RPMI 1640 and layered over lymphocyte separation medium (LSM) (MP Biomedicals, 0,850,494-CF). Tubes were centrifuged at 400 G for 25 min and PBMCs were harvested and stored in liquid nitrogen until FACS staining or used immediately for functional assays.
mRNA Isolation and Synthesis of cDNA
mRNA was isolated from PBMCs using TRIzol reagent (Life Technologies). Five hundred microliters of TRIzol was added to PBMCs and frozen at − 80 °C. Later, samples were thawed and mRNA isolated according to manufacturer instructions. Complementary DNA (cDNA) was synthesized from RNA using the GoScript™ Reverse Transcription System (Promega) according to manufacturer instructions.
qPCR
Forward and reverse primers were purchased from Integrated DNA Technologies (IDT). A Taqman probe was designed on exon 2 (ccagacatgaatcaggtcactattcaatgg) with primers located on the span junction of exon1-2 (forward primer tcaaacaggttgaaagagggaaac) and exon 2–3 (reverse primer tcctccatgacaccagctga) and a Taqman probe on exon 8 (tgatcgtctttgctgttatgttgtcaattctttct) with primers on the span junction of exon 7–8 (forward primer acagggcaccttacttctgacaac) and on exon 8 (reverse primer agttcggaatgatctgttaaatatccc) HPRT1 or GAPDH was used as housekeeping gene for all performed qPCRs. The reaction was performed as followed: 0.6 µl of primers (0.30 µM), 0.4 µl of probe (0.25 µM), 6 ng of DNA (2 ng/µL) and 1 × Taqman Fast mastermix (ThermoFisher Scientific) were mixed and plated on a 96-well plate. The plate was run on the StepOneTM Real-Time PCR system (ThermoFisher Scientific) and analyzed with StepOne Software v2.3.
Stimulation
STAT3 and 4 phosphorylation was assessed by flow cytometry and Western blot after stimulation. For stimulation, fresh PBMCs or EBV-transformed B cells were plated overnight, and stimulated for 30 min with IL-23 (R&D 1290-IL, 10 or 100 ng/mL), IL-6 (R&D 206-IL, 10 ng/mL), IFN-α2b (Invivogen rcyc-hifna2b, 10 ng/mL), IL-12 (Biolegend 573,002, 40 ng/mL) in complete RPMI 1640 medium (cRMPI: supplemented with penicillin–streptomycin, FBS 5% + HEPES + MEM non-essential aminoacids) (GibcoTM). For STAT4-phosphorylation, PBMCs were seeded at 2 × 105 cells/mL in a 48-well plate and preactivated with plate-bound anti-CD3 antibody (16–0038-85, Invitrogen, 10 µg/ml) and soluble anti-CD28 antibody (16–0289-85, Invitrogen, 5 µg/ml) for 3 days, before stimulation with IL-12.
Western Blot
PBMCs or EBV-B cells were lysed in lysis buffer (50 mM Tris–HCl pH 7.5, 135 mM NaCl, 1.5 mM MgCl2, 1% Triton-X, 10% glycerol, 1 × protease inhibitor (Pierce TM Protease Inhibitor, ThermoFisher Scientific) and 1 × phosphatase inhibitor (PhosSTOP, Roche)). Protein concentrations were determined using a Bradford Protein Assay (Bio-Rad). Protein lysate was denaturized in LDS (NuPAGE LDS sample buffer, Novex) and DTT (Bio-Rad) at 70 °C for 10 min and was then loaded on a 4–12% Bis–Tris polyacrylamide gel (BoltTM Bis–Tris Plus, Thermo Fisher Scientific) in MOPS buffer (NuPAGE MOPS SDS running buffer, Novex). Separated proteins on the gel were transferred onto a methanol-activated PVDF membrane (GE Healthcare, Little Chalfont, UK) in transfer buffer (10% methanol, 1 × Tris/Glycine Buffer (Bio-Rad)) using the Tetra Blotting Module (Bio-Rad). The PVDF membrane was blocked in 5% milk Tris-buffered saline with Tween 0.1% (TBS-T) for 30 min at RT and then incubated with primary antibody O/N at 4 °C followed by a wash with TBS-T and incubation with a secondary antibody for 1 h at RT. Primary antibodies used for Western blot: STAT3 (9132, CST), pSTAT3 (Y705, 9145, CST) STAT4 (2653, CST), p-STAT4 (5267, CST) and mouse anti-GAPDH (Invitrogen). Secondary antibodies used were conjugated with HRP: goat anti-rabbit (abcam) and anti-mouse (Rockland). Secondary antibodies were visualized using ECLTM15 Prime Western Blotting Detection Reagent (AmershamTM) with the G:box Chemi-XRQ and quantified using ImageJ.
Cloning
pWPXL_EF1a_hIL23R-3HA_Ires_eGFP was generated using Gibson Assembly (NEB). First, hIL23R-3HA gene sequence was PCR amplified from the pCH_SFFV_hIL23R_3HA_tCD34 plasmid (from Leuven Viral Vector Core and Molecular Virology and Gene Therapy Laboratory at the KU Leuven, BE) using primers: FW: 5’- acgggatccaggcctaagcttacgcgtgccaccatgaatcaggtcact-3’, RV: 5’-agtcgactcatatatcggaattcctaggcataatcaggcacgtcata-3’. Next, the amplified hIL23R-3HA was inserted into pWPXL_EF1a_Ires_eGFP transfer plasmid, previously digested with EcoRI and MluI restriction enzymes. pWPXL_EF1a_hIL23R-3HA_IRES_eGFP plasmid was transformed into DH5a bacterial competent cells and plasmids extracted by midi-prep (Macherey–Nagel). For IL12RB1 and IL23R (F380-HA) a commercial plasmid (cat nr. SC1200 and OHU25802D) was bought from GeneScript in a pcDNA3.1 vector containing respectively a C-terminal Myc or HA tag.
Mutagenesis
pWPXL_EF1a_hIL23R-3HA_IRES_eGFP was mutagenized to insert the studied hIL23R variants (p.R381Q and p.R381X). pWPXL_EF1a_hIL23R-3HA_IRES_eGFP was PCR amplified by Q5 high fidelity DNA polymerase (NEB) using the following primers set: FW: 5’- atttaacagatcattccaaactgggattaaaag-3’, RV: 5’-cttttaatcccagtttggaatgatctgttaaat-3’ (p.R381Q). FW: 5’-atttaacagatcattctgaactgggattaaaag-3’, RV:5’- cttttaatcccagttcagaatgatctgttaaat-3’ (p.R381X). KLD enzyme Mix (NEB) was used to degrade the DNA template by DpnI. The mutagenized plasmids were transformed into DH5a bacterial competent cells and plasmids extracted by midi-prep (Macherey–Nagel).
Transfection of HeLa and HEK293T Cells
HeLa or HEK293T cells were seeded in a 6-well plate at a density of 1 × 105 cells/well in DMEM medium supplemented with FBS 5%, HEPES 25 mM and MEM non-essential amino acids) (GibcoTM). When reaching 70% confluency, transfection of C terminal HA tagged plasmids (empty vector, IL23R wild type (WT), IL23R-R381Q, IL23R-R381X, IL23R-F380) was performed using Lipofectamine 3000 (ThermoFisher Scientific) according to the manufacturer’s protocol. After 4 h, the medium was changed and cells were rested overnight. Subsequently, HeLa cells were stimulated with IL-23 (R&D 1290-IL, 100 ng/mL) or IFN-α2b (Invivogen rcyc-hifna2b, 10 ng/mL) for 30 min. Afterwards, cell lysis, protein extraction, quantification, and Western blot were performed as described before (section Western Blot for STAT3 phosphorylation).
Confocal Microscopy
HeLa cells were seeded in a 48-well plate at a density of 1 × 104 cells/well on a 12-mm coverslip precoated with poly-D-lysine (Sigma). Transfection was performed as described above. Coverslips were washed in PBS 1 × and fixed with paraformaldehyde 4% for 20 min. Afterwards, they were permeabilized (Triton 0.2% in PBS 1 ×), blocked (BSA 2%, Triton 0.2%), and stained with anti-HA (26,183, ThermoFisher Scientific), anti-Myc (71D10, CST), anti-GFP (A-21311 AF488 conjugated, ThermoFisher Scientific) in dilution buffer (BSA 1%, Triton 0.1%) overnight. Afterwards, coverslips were washed (Triton 0.05% in PBS 1 ×) and stained for 1 h with secondary antibodies (AF647 anti-rabbit (ab150083, Abcam) and AF568 anti-mouse (ab175473, Abcam)). Subsequently, coverslips were washed. Images were acquired using a Nikon AXR confocal unit, with a 60 × NA 1.4 oil objective at a 2048 × 2048 pixels resolution.
Lentiviral Vector Production
Lentiviral vectors LV.EF1a:hIL23Rwt-3HA-IRES-eGFP and LV.EF1a: eGFP were generated by co-transfection of HEK293T cells with p.sPAX2, p.MD2.G-VSV.G and the transfer plasmids pWPXL_EF1a_hIL23Rwt-3HA_Ires_eGFP and pWPXL_EF1a_ eGFP by using X-tremeGENE HP Transfection Reagent (Sigma-Aldrich). pMD2.G and psPAX2 were a gift from Didier Trono (Addgene plasmid #12,259 and #12,260).
Lentiviral Transduction
EBV-B cells were plated in a 96-well plate at a seeding density of 1 × 105 cells in 200 μL of cRPMI and transduced using LV vectors as described above. After adding the lentiviral vectors, cells were spinoculated for 90 min at 35 °C and 800 G. Afterwards, they were resuspended and medium was changed at day 1 and 3. At day 4, cells were centrifuged and resuspended in stimulation medium (IL-23 100 ng/mL, IFN-α 10 ng/mL) for 60 min. Subsequently, cells were lysed and Western blot was performed as described above (section Western Blot for STAT3 phosphorylation).
Staining
STAT3 and 4 Phosphorylation Assays
PBMCs were stained with fixable viability dye Zombie Aqua at room temperature for 10 min, washed twice in FACS buffer (3% FBS in PBS1 ×) followed by staining for the following surface markers before fixation and permeabilization: CD3 (11–0036-42, Invitrogen, FITC), CD4 (25–0047-42 or 47–0049-42, Invitrogen, PE-Cy7 or APC-Cy7), CD8 (17–0086-42 or 25–0088-42, Invitrogen, APC or PE-Cy7). Cells were fixed with BD cytofix for 10 min at 37 °C. Afterwards, permeabilization was performed using BD phosflow Perm III (558,050, BD Biosciences) on ice for 30 min followed by intracellular staining with p-STAT3 (560,312, BD Biosciences, Pacific Blue, pY705) or p-STAT4 (17,186,668, ThermoScientific, APC, pY693) for 60 min at 4 °C.
Th Compartment
Frozen PBMCs were thawed and counted, and cell concentration was adjusted to 5 × 106 for each sample. Cells were plated in a V-bottom 96-well plate, washed once with PBS (Fisher Scientific), and stained with live/dead marker and fluorochrome-conjugated antibodies recognizing surface markers: anti-CD8-BUV805 (clone SK1), anti-CD4-BUV496 (clone SK3), anti-CD95-BUV737 (clone DX2), anti-CD28-BB660-P (clone CD28.2), anti-ICOS-BB630 (clone DX29), anti-CXCR3-BV785 (clone 1C6), anti-PD-1-BV750-P (clone EH12.1), anti-CXCR5-BV650 (clone RF8B2), anti-CCR2-BV605 (clone 1D9) anti-CD31-BV480 (clone WM59), (all BD Biosciences); anti-CD3-PerCP-Vio700 (clone REA613) (Miltenyi Biotec); anti-CD45RA-FITC (clone HI100), anti-CD14-PE-Cy5.5 (clone TuK4), anti-CCR7-PE-Cy7 (clone 3D12) (all eBioscience); anti-CD25-BV711 (clone BC96), anti-HLA-DR-BV570 (clone L243), anti-CD127-BV421 (clone A019D5), anti-CCR4-PE/Dazzle 594 (clone L291H4) (all BioLegend). Samples were stained for 60 min at 4° C, washed twice in PBS/1% FBS (Tico Europe), and then fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (eBioscience), according to manufacturer’s instructions. Cells were stained overnight at 4° C with anti-Ki67-BUV615-P (clone B56), anti-CTLA-4-PE-Cy5 (clone BNI3), anti-RORγt-PE (clone Q21-559) (all BD Biosciences), and anti-FOXP3-AF647 (clone 206D) (BioLegend) anti-human intracellular antibody, and were then acquired on a Symphony flow cytometer with Diva software (BD Biosciences). A minimum of 5 × 105 events were acquired for each sample. Compensation Beads (ThermoFisher Scientific) were used to optimize Fluorescence compensation settings for multi-color flow cytometric analysis at a Symphony flow cytometer.
IL23R
PBMCs were isolated as described above and plated overnight. On the next day, cells were washed in PBS 1 × and stained for 30 min with anti-CD3 (17–0038-42, APC-Cy7), anti-CD4 (Pacific Blue, anti-CD8 (25–0088-42, PE-Cy7) (all Invitrogen), IL23R (ab222104, Abcam), and Zombie Aqua fixable viability dye. Cells were washed in FACS buffer (FBS 3% in PBS 1 ×) and stained for 30 min with secondary AF647 anti-rabbit (ab150083, Abcam). Data acquisition was performed using the BD FACSCanto II and analyzed using FlowJo software.
Th17 Differentiation
Naïve T cells were isolated from PBMCs by magnetic purification (MAGH115, R&D). They were cultured in Th0 conditions (anti-CD3 10 µg/mL, anti-CD28 5 µg/mL) or Th17 (anti-CD3 10 µg/mL, anti-CD28 5 µg/mL, TGF-β 10 ng/mL (Peprotech), IL-1β 10 ng/mL (Peprotech), IL-23 10 ng/mL (R&D 1290-IL), IL-6 10 ng/mL (Peprotech), anti-IL-4 µg/mL, anti-IFNγ 20 µg/mL) for 7 days. Supernatant was used for measurement of IL-17 by ELISA according to the manufacturer’s instructions (DY317-05, R&D).
IFN-γ Measurement
IFN-γ was assessed by flow cytometry and ELISA after stimulation. For alpha beta T cell subset and MAIT stimulation, fresh PBMCs were plated on an anti-CD3 (Invitrogen, 16–0038-85, 10 µg/mL) coated 48-well plate at a density of 2 × 105 cells/mL with addition of soluble anti-CD28 (Invitrogen, 16–0289-85, 5 µg/mL) in RPMI 1640 medium (supplemented with penicillin–streptomycin, FBS 5% + HEPES 25 mM + MEM non-essential amino acids) (GibcoTM). At day 1 of culture, IL-23 (R&D 1290-IL, 100 ng/mL) and IL-12 (Biolegend, 573,002, 40 ng/mL) were added. At day 7, Brefeldin A (Abcam, 5 µg/mL) was added for 4 h to the culture to assess intracellular cytokine production by flow cytometry. For NK and iNKT cells, fresh PBMCs were stimulated for 48 h with IL-23 or IL-12 and Brefeldin A was added during the last 8 h of stimulation. In brief, cells were washed with PBS 1 × , stained with fixable viability dye Zombie Aqua at room temperature for 10 min, washed twice in FACS buffer followed by staining for surface markers: anti-CD3 (11–0036-42, Invitrogen, FITC), anti-CD4 ( Pacific Blue), anti-CD8 (25–0088-42, Invitrogen, PE-Cy7), anti-Vα7.2 (351,708, Biolegend, APC), anti-CD161 (45–1619-42, Invitrogen, PerCP Cy5.5), anti-CD56 (35–0567-42, eBioscience, PE Cy5.5) for 30 min at 4 °C. Later, cells were permeabilized using eBio Fix/Perm, washed twice in Permeabilization buffer, and stained with anti-IFN-γ (47–7319-42, ThermoFisher Scientific, APC-e780) for 30 min at 4 °C. Data acquisition was performed using the BD FACSCanto II and analyzed using FlowJo software. For mycobacterial stimulation, cells were plated in the same medium, left unstimulated or co-cultured with Mycobacterium abscessus strain (ATCC19977) (MOI 2) in the presence or absence of IL-23 (R&D 1290-IL, 100 ng/mL) or IL-12 (Biolegend, 573,002, 40 ng/mL) for 3 or 7 days. As a positive control, PMA/ionomycin (25 ng/mL + 1 µg/mL) without mycobacteria was used. Supernatant was used to measure IFN-γ by ELISA according to the manufacturer’s instruction (DY285B, R&D).
Statistical Analysis
Statistical analysis was only performed if three biological replicates were available in each group. To compare between healthy controls and patient, an unpaired Student t-test (if normally distributed by Shapiro–Wilk) or Mann–Whitney U test (no normal distribution) was used. To compare the effects of stimulation in the healthy control or patient group, a paired Student T test, one-way ANOVA, or a Friedman test with post hoc tests was used depending on normality of the data. Significance levels were defined as followed: *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent the standard error of the mean (SEM).
Discussion
We report on a novel, homozygous
IL23R (p.R381X) mutation as a cause of MSMD. IL23R is a heterodimeric receptor consisting of an IL12RB1 and IL23R subunit. After binding to IL-23, it signals downstream through TYK2 (attached to IL12RB1) and JAK2 (attached to IL23Rα) to induce phosphorylation of STAT3. The role of IL-23 signaling is best studied in the context of inflammatory disease. A well-described example is inflammatory bowel disease, where both mouse models, human observational studies and population genetic studies underscore the importance of IL-23 [
11]. The connection of IL-23 signaling to mycobacterial disease is based on the observation that
IL23a−/− mice have impaired long-term control of pulmonary
Mycobacterium tuberculosis infection [
12], the fact that exogenous IL-23 can complement IL-12 deficiency for restoring mycobacterial immunity in mice [
13], the association of a partial LOF variant c.1142G > A (R381Q) with active pulmonary tuberculosis [
8] and the report of a kindred with MSMD harboring a homozygous complete LOF mutation in
IL23R [
7]. In addition, a second patient with MSMD and a splice homozygous splice site mutation, c.367 + 1G > A, at the exon 3 of
IL23R is reported but the effect of this variant on IL23R expression and signaling was not reported [
14].
In contrast to the first reported kindred by Martínez-Barricarte et al. where both patients had a homozygous missense mutation (p.C115Y) in the extracellular domain resulting in impaired IL-23 signaling, our patient has an adult-onset phenotype and a stop mutation (p.R381X) located in the intracellular domain before the JAK2 binding site. The mutation resides in a well-conserved residue and was predicted to be LOF using in silico prediction tools. Functional testing revealed that our patient had severely decreased total
IL23R mRNA levels and membrane-bound IL23R. The functional impact of this mutation was assessed by determining STAT3 phosphorylation following IL-23 stimulation in primary cells and in HeLa cells transfected with WT IL23R or the partial LOF variant R381Q [
15,
16]. The R381X mutation in our patient has a complete LOF effect in both overexpression and primary cells, which can be restored by lentiviral transduction of WT IL23R. The immunophenotype of the IL23R-deficient patient was characterized by a low number of MAIT cells, resembling the previously reported IL23R patient but also STAT3, IL12RB1 andTBX21 patients [
6,
17]. Furthermore, Th17 and Th1* cells were significantly reduced, differing from the previously reported patient where only a mild reduction was observed [
7]. In addition, the relative proportion of CD4
+ and CD8
+ T cells in our patient differed from healthy controls in contrast to the previously reported IL23R deficient patient [
7]. The effect of a homozygous stop mutation on Th17 cell development is consistent with previous data reporting reduced circulating Th17 cells in individuals carrying a heterozygous R381Q mutation [
16]. Moreover, the observation of reduced Th17 cells is supported by studies demonstrating a central function for IL-23 in TH17 differentiation in mice [
18]. In addition, we showed that the differentiation of naïve T cells to Th17 was impaired, confirming previous results [
7]. Remarkably, our patient did not have any susceptibility to fungal infections. This supports the hypothesis that—at least in humans—the contribution of IL-23- to IL-17-mediated anti-fungal immunity might be redundant [
7].
Finally, we assessed IFN-γ secretion by alpha–beta T cell subsets (CD4
+, CD8
+) and MAIT cells upon IL-23 stimulation. IL-23 is a less potent IFN-γ inducer in T cells compared to IL-12 as described by Martínez-Barricarte et al., characterizing IL-12- and IL-23-specific effects in different immune subsets [
7]. We observed a mild, but significant increase of IFN-γ production by alpha–beta T cell subsets and MAIT cells in healthy controls when stimulated with IL-23, while this was absent in the patient. IL-12 stimulation remained intact, confirming a selective IL-23 signaling defect. Furthermore, in the context of a non-tuberculous mycobacterial infection in vitro, the addition of IL-23 did not increase IFN-γ in the patient, in contrast to healthy controls. In addition, IL-17 secretion by stimulated PBMCs in the patient was low and did not increase upon IL-23 costimulation.
In conclusion, we identified a patient with MSMD and a novel stop mutation (p.R381X) located in the intracellular domain of IL23R. This report illustrates a mechanism of abolished membrane-bound IL23R expression. Functionally, this negatively affects the development of Th17 and MAIT cells and impacts IL-23-stimulated IL-17 and IFN-γ production by alpha–beta T cell subsets and MAIT cells. Our findings together with the report of a previously validated IL23R patient with MSMD and existing data on mycobacterial susceptibility of IL23a−/− mice link the genotype and phenotype in our patient. The absence of fungal infections in this adult patient and the previously reported pediatric onset patients are consistent with the hypothesis that the contribution of IL-23- to IL-17-mediated anti-fungal immunity might be redundant, despite the presence of decreased circulating Th17 cells and reduced IL-17 secretion in response to IL-23.
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