110 | Nature | Vol 577 | 2 January 2020
Article
Patients with RIPK1 non-cleavable variants
The first patient (P1) is a two-year-old Chinese boy. His symptoms began
at two months of age with periodic fever episodes occurring every eight
to ten days and lasting for three to five days (Fig. 1a, b, Extended Data
Table 1). His fevers were associated with increased levels of C-reactive
protein and white blood cell counts, but no other accompanying symp-
toms. He developed lymphadenopathy at two years of age (Fig. 1c).
The patient did not have a skin rash, arthritis, arthralgia or hepatos-
plenomegaly. Lymphocyte phenotyping revealed increased counts of
both double-negative T cells and naive B cells (Extended Data Table 2).
The second family is of European Canadian ancestry. The proband
(P2) is a 35-year-old female who experienced recurrent fevers from
six months of age, and developed intermittent lymphadenopathy,
hepatosplenomegaly and microcytic anaemia. Three of her four sons
are affected. Her eldest (P3, 14 years of age) and youngest (P5, 10 years
of age) sons have a similar history of recurrent fevers, intermittent
lymphadenopathy, splenomegaly and microcytic anaemia. Her sec-
ond son (P4, 12 years of age) has microcytic anaemia but no history of
recurrent fevers (Fig. 1a, c, Extended Data Table 1).
Whole-exome sequencing (WES) of P1 and his parents revealed that P1
has a heterozygous de novo D324V mutation in RIPK1 (Fig. 1a, Extended
Data Fig. 1a–c). For the second family, WES identified a single variant,
D324H in RIPK1, which is de novo in the mother and inherited by her
three affected sons (Fig. 1a, Extended Data Fig. 1c). No other muta-
tions—including rare variants of unknown importance in genes known
to cause periodic fever or autoinflammatory syndromes—were found
(Supplementary Tables 1, 2). Copy number variant analysis based on
WES data for the first family, and microarray analysis for the second
family, did not identify any copy number variants among affected indi-
viduals. Both variants affected the caspase-8 cleavage site, D324, which
is highly conserved in RIPK1 across species (Extended Data Fig. 1d, e).
These variants were not reported in any public database of human
exomes and were predicted to be deleterious (combined annotation-
dependent depletion (CADD) score > 20) for protein function by com-
putational in silico modelling (Extended Data Fig. 1f ).
Expression of wild-type and mutant RIPK1 in HEK293T cells indicated
that variants at residue D324, including D324V and D324H, blocked the
cleavage of RIPK1 by caspase-8 (Extended Data Fig. 1g). D325A mutation
in mouse RIPK1 (the equivalent residue for D324 in human RIPK1) did
not affect its turnover (Extended Data Fig. 1h), or block its interaction
with other proteins such as binding with caspase-8 into the FADDosome
complex (Extended Data Fig. 1i). The variants in D324 resulted in non-
cleavable RIPK1, which was directly demonstrated by incubating mutant
RIPK1 generated by TNT cell-free protein expression with recombinant
caspase-8 (Extended Data Fig. 1j). The inhibitory effect of the D324V
variant on the cleavage of RIPK1 by caspase-8 was further confirmed
in patient P1 fibroblasts after stimulation by TNF and cycloheximide
(CHX) (Extended Data Fig. 1k).Activation of inflammatory signalling in the patients
We detected markedly increased production of pro-inflammatory
cytokines and chemokines such as IL-6, TNF and IFNγ, and anti-inflam-
matory cytokines such as IL-10 in serum from patients by cytometric
bead array (Fig. 2a) or enzyme-linked immunosorbent assay (ELISA)
(Extended Data Table 3). Serial sampling from P1 showed that activation
of inflammatory responses was even more notable during fever epi-
sodes (Fig. 2a). Increased expression of IL-6, TNF and IL-8 in monocytes
and IL-6 in T cells from P1 after stimulation by lipopolysaccharide (LPS)
was detected by intracellular cytokine staining (Extended Data Fig. 2a,
b). Moreover, phosphorylation of STAT3, the downstream marker of IL-6
signalling, was upregulated during fever episodes in patient monocytes
at basal level when compared with unaffected controls (Extended Data
Fig. 2c). We also observed increased phosphorylation of MAPK p38 in
patient monocytes, B cells and T cells after LPS stimulation (Extended
Data Fig. 2c).
To study the transcriptional changes related to non-cleavable RIPK1
further, we performed single-cell RNA sequencing in patient peripheral
blood mononuclear cells (PBMCs). The patient had a higher percent-
age of monocytes compared with an age- and sex-matched unaffected
control (Fig. 2b, Extended Data Fig. 2d). We observed strong signals
in both NF-κB and type-I IFN inflammatory pathways in the patient
monocytes (Extended Data Fig. 2e, f ). The patient monocytes highly
expressed pro-inflammatory cytokines and chemokines, including
IL8, IL1B and CCL3 (Fig. 2c, Extended Data Figs. 2g, 3a, b). In addition,
RNA sequencing in PBMCs implicated different gene expression pat-
terns in cell death pathways that include increased expression of RIPK3
and MLKL, suggesting increased levels of necroptosis machinery in
the patient PBMCs (Fig. 2d). Quantitative PCR (qPCR) confirmed the
increased expression of IL6, TNF, IL8, IL1B, CXCL2 and CXCL3 in patient
PBMCs (Fig. 2e). Supporting a pathogenic role of excessive IL-6 produc-
tion, P1 experienced clinical improvement and the PBMCs displayed
normalized expression of inflammatory mediators after treatment
with tocilizumab (monoclonal antibody against IL-6R) (Extended Data
Fig. 4a).Increased cell death and inflammatory response
We examined the response of patient PBMCs to TNF by measuring cell
survival with the CellTiter-Glo assay, and quantified cell death by meas-
uring the plasma membrane permeability with ToxiLight assay (Fig. 3a,
Extended Data Fig. 4b). The PBMCs from patients P1, P2 and P3 showed
increased sensitivity to both apoptosis induced by co-treatment with
TNF and apoptosis-inducing SMAC mimetic SM-164, and necroptosis
induced by co-treatment with SM-164 and the pan-caspase inhibitor
Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluorome-
thylketone) compared with PBMCs from unaffected controls. Co-treat-
ment was required as treatment with these compounds individually
did not elicit cell death. Furthermore, both apoptosis and necroptosis
of patient PBMCs were effectively suppressed by the RIPK1 inhibitor
necrostatin-1s (Nec-1s) (Fig. 3a, Extended Data Fig. 4b). We found that
levels of RIPK1 phosphorylated at S166 (p-S166-RIPK1)—a marker for the
activation of RIPK1^7 ,^8 —were increased in the patient PBMCs treated with
various combinations of these compound known to activate apoptosis
or necroptosis, compared to that of controls (Fig. 3b, Extended Data
Fig. 4c), which suggests that blocking the cleavage of RIPK1 sensitizes
the activation of its kinase activity. We also found increased levels of
p-S358-MLKL—a biomarker for necroptosis^9 —in the patient PBMCs
treated with SM-164 plus Z-VAD-FMK compared with that of controlabcP1P314.2 cm363738394041Temperature (oC) Normal temperature Fever17/4/1827/4/187/5/1817/5/1827/5/186/6/1816/6/1826/6/186/7/1816/7/1826/7/185/8/1815/8/18P1Family 1
p.Asp324ValIIIIIIP2P3 P4 P5Family 2
p.Asp324HisFig. 1 | Heterozygous variants at the RIPK1 cleavage site cause
autoinf lammatory disease in humans. a, Pedigrees of two families with
variants in RIPK1 at the caspase-8 cleavage site. b, Timeline of recurrent fever
episodes in P1 over 4 months. Red dots denote increased temperatures during
fever episodes. Blue boxes denote normal temperatures between f lares.
c, Computerized tomography scans of P1 (top) and sonographic image of P3
(bottom) show lymphadenopathy (arrows) and splenomegaly, respectively.