Homozygous IL37 mutation associated with infantile inflammatory bowel disease

Interleukin (IL)-37, an antiinflammatory IL-1 family cytokine, is a key suppressor of innate immunity. IL-37 signaling requires the heterodimeric IL-18R1 and IL-1R8 receptor, which is abundantly expressed in the gastrointestinal tract. Here we report a 4-mo-old male from a consanguineous family with a homozygous loss-of-function IL37 mutation. The patient presented with persistent diarrhea and was found to have infantile inflammatory bowel disease (I-IBD). Patient cells showed increased intracellular IL-37 expression and increased proinflammatory cytokine production. In cell lines, mutant IL-37 was not stably expressed or properly secreted and was thus unable to functionally suppress proinflammatory cytokine expression. Furthermore, induced pluripotent stem cell–derived macrophages from the patient revealed an activated macrophage phenotype, which is more prone to lipopolysaccharide and IL-1β stimulation, resulting in hyperinflammatory tumor necrosis factor production. Insights from this patient will not only shed light on monogenic contributions of I-IBD but may also reveal the significance of the IL-18 and IL-37 axis in colonic homeostasis.


Human Subjects
Written informed consent was provided by all human subjects or their legal guardians in accordance with the 1975 Helsinki principles for enrollment in research protocols that were approved by the Institutional Review Board of the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH). Patient and healthy control blood was obtained in Marmara University Faculty of Medicine in Istanbul, Turkey or at NIH under approved protocols. Members of the general public are involved in the institutional review board and therefore participate in the design of the study protocol used in this research.

Genetic Analysis
DNA was obtained from the proband and family members by isolation and purification from peripheral blood mononuclear cells (PBMCs) using Qiagen's DNeasy Blood and Tissue Kit. The DNA was then submitted for whole exome sequencing (WES) by Illumina sequencers in the United States. The reads were filtered for sequence quality and then mapped on to the h19 human genome reference by Burrows-Wheeler Aligner with default parameters. Alignment, variant calling, and annotation were performed by the in-house bioinformatics core using the Genome Analysis Toolkit version 3.4 (Broad Institute) and GEMINI (GEnome MINIng). The IL37 variant was confirmed by Sanger sequencing using following PCR primers: F: ttatagggctcaggtgggct and R: acagactcagccacttctgc (Sigma Aldrich) or F: gtaccaaggctgacacgtca and R: agcagaagccggtctctttc (Sigma Aldrich).

Cells and Media
Primary patient or control PBMCs were obtained from whole blood subjected to Histopaque/Ficoll density gradient separation. The PBMCs were then washed with PBS and frozen in complete RPMI with 10% DMSO in liquid nitrogen for later use or -80°C for transport. HEK293T, Jurkat, and THP-1 cells were obtained from the American Type Culture Collection and tested mycoplasma-free (ATCC). Human cells were cultured in RPMI or DMEM (Sigma Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (Sigma Aldrich), 1% penicillin/streptomycin (Gibco), and 1% Glutamax (Gibco). Primary human monocytes were isolated using magnetic isolation kit (Miltenyi) and cultured in complete RPMI supplemented with IL-4 (20 ng/mL, PeproTech) and GM-CSF (20 ng/mL, PeproTech). iPSCs and iPSCderived macrophages were cultured in specialized media described below.

Reprogramming of Induced Pluripotent Stem Cells (iPSCs)
Transgene-free iPSCs were generated from peripheral blood CD34+ hematopoietic stem/progenitor cells (HSPCs) as previously described (Merling et al. Blood 2013), from either healthy volunteers or the IL37-deficient patient. Briefly, CD34+ HSPCs were purified from peripheral blood mononuclear cells using magnetic-activated cell sorting with CD34 microbeads (Miltenyi), followed by transduction with non-integrating CytoTune-iPS 2.0 Sendai viruses (Invitrogen), and then cultured in StemFit Basic02 feeder-free culture medium (amsbio) supplemented with 10 ng/mL human basic fibroblast growth factor (PeproTech) on plates coated with ESC-qualified Matrigel (Corning) for formation of iPSC colonies during initial reprogramming. For routine maintenance, iPSCs were cultured on Matrigel-coated plates in mTeSR1 or mTeSR Plus medium (STEMCELL Technologies).

Intracellular Flow Cytometry
Cells were pelleted by centrifugation and incubated with Fixation and Permeabilization Solution (BD Biosciences) for 30 minutes on ice. The cells were then washed with 1X Permeabilization Wash Buffer (Biolegend) and stained with antibodies in FACS Buffer (1-2% FBS, 0.05% sodium azide, and 2-5 mM EDTA in PBS) at 4°C for 30-60 minutes. The stained cells were then washed with PBS or FACS buffer, pelleted, and resuspended at ~1x10 6 cells/ml in FACS Fix Buffer (FACS Buffer with or without 1% PFA) for flow cytometry analysis (Fortessa). The flow data was analyzed using FlowJo.

Western Blot
Cells were lysed with NuPage LDS sample buffer (Thermo Fisher Scientific) at the concentration of 10 5 cells per 15uL LDS supplemented with 10% BME and Benzonase Nuclease (Sigma Aldrich). iPSC macrophages were stimulated with IL-1b for 24 hours in cRPMI and then harvested for nuclear and cytoplasmic protein isolation (Abcam) according to the manufacturer's protocol. The samples were then denatured at 70°C. Protein lysates were separated by SDS-PAGE on 4-12% Bis-Tris pre-cast gels (Invitrogen) and transferred to a PVDF membrane (Invitrogen) by wet transfer. Membranes were then blocked in milk with 5% Tris-buffered saline with 0.01% Tween-20) TBST for an hour at room temperature and then incubated with primary antibody in milk or 5% BSA overnight at 4°C. The membrane was washed for 3 x 10 minutes with TBST at room temperature and then stained with HRP-linked secondary antibody in milk for 1 hour at room temperature. After 3 x 10 minute washes with TBST and 1 x 10 minute wash with PBS, the membrane was exposed to enhanced chemiluminescent (ECL) substrates (Thermo Fisher Scientific) and developed by film.

Cloning and Site-Directed Mutagenesis
The wild-type human IL37 gene (Genscript) was cloned into the pLV vector to produce pLV-WT-IL37 for subsequent transfection and transduction experiments using the In-Fusion HD Cloning Kit (Takara Clontech). IL37 with common variants was synthesized by IDT and also cloned into the pLV vector. Site-directed mutagenesis of T>C (p.I177T) was performed using the In-Fusion HD Cloning Kit (Takara Clontech) and following PCR primers (Sigma Aldrich): F: GGCGGCTCACCCCGGATGGTTCACCTGCAC and R: TTACAATTGCAGGAGGTGCAGGTGAACCAT. The PCR products were purified on a 1% agarose gel by gel electrophoresis and the desired mutagenized product band was cut out. The PCR product was purified using the NucleoSpin Gel and PCR Clean Up (Takara) from the InFusion Cloning Kit. The linearized, mutagenized product was ligated using the InFusion Enzyme (Takara) to generate the I177T mutant pLV-MUT-IL37 plasmid. Stellar cells (Takara) were transformed with the new plasmid by heat shock; the transformed cells were plated on ampicillin plates and incubated overnight at 37°C. Plasmid was extracted from individual colonies using the QIAprep Spin MiniPrep Kit (Qiagen). The mutation was confirmed by Sanger sequencing.

HEK293T Transfection and Jurkat and THP-1 Transduction
HEK293T cells were cultured in complete DMEM at 37°C in T75 flasks. 4x10 5 cells in 2mL media were seeded into 6 well plates and grown overnight at 37°C. At 40-50% confluence, the cells were transfected with either pLV-CV-IL37, pLV-WT-IL37 or pLV-MUT-IL37 alone or with pSPAX2, and pMD2.g for lentivirus production. Transfections were performed in OPTI-MEM (Gibco) and GeneJuice Transfection Reagent (VWR) or Lipofectamine 3000 (ThermoFisher Scientific). Lentivirus was harvested at 24, 48, and 72 hours post-transfection. The viral supernatants were pooled and concentrated using Lenti-X concentrator (Clontech). Jurkat and THP-1 cells were separately cultured in complete RPMI. The cells were transduced with a ratio of 100:1 WT-IL37 or MUT-IL37 lentivirus: Jurkat cells or 10:1 lentivirus: THP-1 cells on retronectin-coated plates by spinfection. The cells were transduced for 72 hours in complete RPMI. Transduced cells were then selected for using puromycin selection. Supernatants of transfected and transduced cells were taken for IL-37 ELISA measurements.

Cycloheximide Chase Assay
HEK293T cells transfected with WT-IL37 or MUT-IL37 were cultured in complete DMEM at 1x10 6 cells per mL and were treated with vehicle (complete DMEM) or cycloheximide (50 ug/mL, Sigma Aldrich) and MG132 (5 uM, Sigma Aldrich) for 10 or 20 hours. At each time point, the cells were harvested and subsequently analyzed by immunoblot.

IL-37 TNF Suppression Assay
THP-1 cells transduced with WT-IL37 or MUT-IL37 were cultured in complete RPMI at 3.5x10 5 cells per mL. The cells were then differentiated with PMA (100 ng/mL, Sigma Aldrich) for 24 hours. Then PMA was removed by washing the adherent differentiated THP-1 cells. Next the cells were stimulated with a titrated dose of LPS for 4 hours in complete RPMI. The supernatants were then taken for ELISA measurements. iPSC-derived macrophages were cultured in complete IMDM and stimulated with LPS (100 ng/mL) for 4 hours, IL-1beta (10 ng/mL, PeproTech) for 24 hours, or IL-18 (10 ng/mL, PeproTech) for 24 hours. The supernatants were taken for ELISA or multiplex FACS bead-based cytokine measurements.