Methods
Patients
Patient P1 was evaluated under protocols approved by the Insti-
tutional Review Board by Children’s Hospital of Fudan University.
Patients P2, P3, P4 and P5 and their unaffected family members were
evaluated at McMaster Children’s Hospital, and the Hospital for Sick
Children. Signed consent for their clinical information to be shared
and for research samples to be sent to Boston Children’s Hospital
was obtained. Ethics clearance was received from the Institutional
Review Board at Boston Children’s Hospital and from Western Insti-
tutional Review Board. All relevant ethical regulations were followed.
All patients and/or substitute decision markers provided written
informed consent.
Unaffected controls
We used unaffected controls for functional assays. Paediatric unaf-
fected controls are less than 10 years old and had no symptoms of
inflammation when sampling.
WES
DNA from whole blood was extracted using a Maxwell RSC Whole
Blood DNA Kit (Promega, AS1520). One microgram of DNA was used
for whole-exome sequencing. For the first family, WES and data analy-
sis were performed as previously described^23 –^25. Variants were anno-
tated by ANNOVAR (2018Apr16). Candidate variants were filtered to
remove those presenting in the gnomAD, Kaviar, dbSNP and an in-
house database. Variants were further filtered by de novo or dominant
inheritance. For the second family, WES was performed and analysed
concurrently for the proband, both parents and one affected son as
previously described^26. Other affected or unaffected family members
were tested by Sanger sequencing for the presence or absence of the
de novo variant identified in the proband.
Sanger sequencing
Sanger sequencing was used to confirm variants identified by exome
sequencing as previously described^23 –^25.
Cell preparation, culture and stimulation
The HEK293T cell line was from the American Type Culture Collec-
tion. Ripk1 gene knockout MEFs were established from Ripk1−/− mice.
MEFs derived from D325A knock-in mice were provided by J. Zhang.
PBMCs were separated by lymphocyte separation medium (LSM) and
SepMate tubes (Stemcell) according to the manufacturer’s instruc-
tions. Fibroblasts were derived from skin biopsies of patient and
control donors. HEK293T cells, MEFs and fibroblasts were grown
in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS)
(ExCell Bio) and penicillin/streptomycin (HyClone). PBMCs were
grown in RPMI-1640 (Gibco) supplemented with 10% FBS and peni-
cillin/streptomycin. All cell lines tested negative for mycoplasma
contamination.
Recombinant human TNF (Peprotech, 300-01A) was used to stimulate
PBMCs (50 ng ml−1, 100 ng ml−1), fibroblasts (20 ng ml−1, 50 ng ml−1) and
MEFs (50 ng ml−1) for the indicated amount of time. LPS (Sigma, L6529) was
used to stimulate PBMCs (1 μg ml−1), MEFs (1 μg ml−1) and fibroblasts (1 μg
ml−1) for the indicated amount of time. TRAIL (R&D, 1121-TL) was used to
stimulate MEFs (100 ng ml−1) for the indicated amount of time. Z-VAD-FMK
(100 μM) and SM-164 (50 nM) (from Selleck) and Nec-1s (10 μM) (made
by custom synthesis) were used to treat PBMCs, MEFs and fibroblasts.
Erastin and RSL3 were used to induce cell ferroptosis in PBMCs (10 μM,
1 μM), MEFs (10 μM, 0.5 μM) and fibroblasts (10 μM, 0.5 μM).
RNA sequencing
One microgram of RNA was used for library preparation. Libraries were
generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB)
following manufacturer’s recommendations and index codes were added
to attribute sequences to each sample. Library quality was assessed on the
Agilent Bioanalyzer 2100 system. The libraries were sequenced on Illu-
mina Novaseq and 150-bp paired-end reads were generated. Sequenced
reads were mapped against the human reference genome (GRCh38) or
mouse reference genome (GRCm38) using HISAT2. featureCounts was
used to count the reads numbers mapped to each gene. Differential
expression analysis was performed using the DESeq2 R package.Single-cell RNA sequencing
10X Genomics Chromium machine was used for 8,000–10,000 single-cell
capture and cDNA preparation. The machine divided thousands of cells
into nanolitre-scale Gel Bead-In-EMulsions for barcoding followed by clean
up using the silane magnetic beads and Solid Phase Reversible Immobili-
zation beads. Barcoded cDNA was then amplified by PCR. The library was
constructed according to the manufacturer’s instruction. Sequencing
was carried out on Illumina Novaseq. Sequence data were processed with
Cell Ranger V3.0.1 (10X Genomics). The resulting count matrices followed
the standard pipeline with default parameters. The UMAP plots were cal-
culated based on the first 20 components of the CCA, and clusters were
identified by Seurat R package (https://satijalab.org/seurat/).NanoString assay
One-hundred nanograms of total RNA was used for NanoString assay
and gene expression analysis was conducted using the nCounter Analy-
sis System (NanoString Technologies) with a codeset designed to target
594 immunologically related genes. NanoString assay and data analysis
were performed as previously described^25.Quantitative RT–PCR assay
Total RNA from fibroblasts, MEFs and PBMCs was extracted using the
RNeasy Mini kit (Qiagen, 74104). cDNA was generated by the Prime-
Script RT reagent kit with gDNA Eraser (Perfect Real Time) (Takara,
RR047A), and qPCR was performed using TB Green Premix Ex Taq II
(Tli RNaseH Plus) (Takara, RR820A). The reactions were run on Applied
Biosystems 7500 Real-Time PCR System (Life Technologies) and ROCHE
480II. Relative mRNA expression was normalized to ACTB or GAPDH
and analysed by the ΔΔCt method.Antibodies and expression plasmids
The following antibodies were purchased from Cell Signaling Technol-
ogy: β-actin (4970), β-tubulin (86298), GAPDH (5174), RIPK1 (3493),
p-RIPK1 (Ser166) (65746), MLKL (14993), p-MLKL (Ser358) (91689),
p-MLKL (Ser345) (37333), p65 (8242), p-p65 (Ser65) (3033), IKKα
(11930), IKKβ (2370), p-IKKα/β (Ser176/180) (2697), IκBα (4814), p-IκBα
(Ser32) (2859), p38 (8690), p-p38 (Thr180/Tyr182) (4511), TNFR1 (3736),
caspase-8 (4790), cleaved-caspase-8 (8592), caspase-3 (9662), cleaved-
caspase-3 (Asp175) (9661), HA-tag (3724), SLC7A11 (12691). Cyclophilin
A (ab41684), GPX4 (ab125066), LAMP2A (ab125068), COX2 (ab15191)
and p53 (ab32389) were purchased from Abcam. FADD (sc-6036) and
ACSL4 (sc-365230) were purchased from Santa Cruz Biotechnology.
p-RIPK1 (Ser166) (BX60008) was made by Biolyx. HSC70 (10654-1-AP)
was purchased from Proteintech Group. HSP90 (BF9107) was purchased
from Affinity. MLKL (reactivity for Mus musculus) was homemade^27.
Human wild-type RIPK1 plasmid (RC216024) was from Origene,
and the mutant RIPK1 plasmids (D324V, D324H and D324K) were con-
structed by site-directed mutagenesis. Mouse wild-type RIPK1 plasmid
was generated by PCR amplification from the cDNAs of MEFs, and then
cloned into the pMSCV vector made in-house, and the mutant mouse
RIPK1 plasmids (D325V and D325H) were constructed by site-directed
mutagenesis.Immunoprecipitation and western blotting
Cells were lysed in cold cell lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 0.5% NP-40, protease and phosphatase inhibitor mixture (Thermo