112 | Nature | Vol 577 | 2 January 2020
Article
We also examined the effect of non-cleavable RIPK1 on ligands of
other death receptors such as TRAIL^13. We found that the cells express-
ing the non-cleavable mutant RIPK1 also showed increased sensitivity
to TRAIL-induced cell death, which could be rescued by the addition
of Nec-1s (Extended Data Fig. 5f ). Levels of p-S166-RIPK1 were also
increased after TRAIL stimulation in mutant RIPK1-complemented
MEFs compared to wild-type RIPK1-complemented MEFs (Extended
Data Fig. 5g). These data further illustrated that the non-cleavable
mutations in RIPK1 increase RIPK1 kinase activity and sensitize the
cells to cell death after stimulation by several stimuli.
We next characterized the effect of non-cleavable RIPK1 on
cytokine production. NanoString analysis of the patient PBMCs
stimulated with TNF alone exhibited upregulated gene expression
in the inflammatory pathway, including IL6, which was reduced by
Nec-1s (Extended Data Fig. 4d). Because patient P1 responded well to
IL-6 blockade, we also compared the effects of Il6 expression in Ripk1-
knockout MEFs expressing GFP alone, wild-type RIPK1 or mutant
RIPK1 (D325V or D325H) (Fig. 3k). We found that MEFs expressing the
D325V or D325H mutant showed distinctively enhanced transcription
of Il6 compared to that of wild-type-complemented Ripk1-knockout
MEFs in response to TNF alone. In addition, the transcriptional pro-
duction of Il6, Cxcl2 and Tnf were also enhanced in Ripk1D325A/D325A
Ripk3−/− MEFs after stimulation by TNF or both TNF and SM-164. The
enhancement was inhibited by the addition of Nec-1s (Fig. 3l, m).
Together, these results suggest that the augmented inflammatory
signals associated with the non-cleavable variants were dependent
on RIPK1 kinase activity. In keeping with the patient’s therapeutic
response to IL-6 blockade, these results demonstrate a pathogenic
mechanism that relies on the activation of RIPK1 to mediate the pro-
duction of IL-6.abcd efghijklmCtrl
T/S (2 h)T/S/N (2 h)Ctrl
T/S (2 h)T/S/N (2 h)Ctrl
T/S (2 h)T/S/N (2 h)Ripk1+/+Ripk3–/– ; Ripk1D325A/D325ARipk3–/–
Il6 Cxcl2 Tnf0204002040051015048Relative mRNA levels
Ctrl T (2 h)
T/N (2 h) T (4 h)T/N (4 h)Il6Relative mRNA levelsp-S166-RIPK1
(LE)
p-S166-RIPK1
(SE)RIPK1
Cl-CASP3(LE)
Cl-CASP3(SE)
TubulinNec-1sSM-164TNF (h)Ripk3–/–
MEFs–––+–+––––++–2 4422
–––+–+- –––++
–2 4422Ripk1+/+ Ripk1D325A/D325A75
75
75
20
20
50p-S166-RIPK1
HA
p-S345-MLKL
MLKL
Cl-CASP3
Tu bulinT/STNFRipk1–/–
MEFs
+++++++ +++++
70
70
55
55
15
55GFPHA WTHAD138NHAD325VHAD138ND325VHAD325HHAD138ND325Hp-S166-RIPK1
(LE)p-S166-RIPK1
(SE)
HA
p-S345-MLKL
MLKL
Cl-CASP3
β-actinT/SNec-1sTNF (h)Ripk1–/–
MEFs–––––+–––++––2 424 –
–––––+–––++––2 42 4–
–––––+–––++––2 424 –- ––––+
–––++–- 2424–
GFP HA-RIPK1-WT HA-RIPK1-D325V HA-RIPK1-D325H70
70
70
55
55
15
4020(^10075)
50
37
25
15
0
50
100
T T/N T/S
T/S/N T/S/Z T/S/Z/
N
Cell viability (%)
P = 0.0134
P = 0.012
P = 0.0023
P = 0.0003
P = 0.0006
P = 0.0002
P = 0.0017
P = 0.000074
Ripk1+/+Ripk3–/– ; Ripk1D325A/D325ARipk3–/–
0
5
10
15
0
5
10
15
Il6 Cxcl2
CtrlT (4 h)
T/N (4 h)
CtrlT (4 h)
T/N (4 h)
(^0) Relative mRNA levels
50
100
Cell viability (%)T
T/NT/ST/S/N
Ripk1+/+Ripk3–/–
Ripk1D325A/D325ARipk3–/–
P = 0.0134
P = 9 × 10–6
P = 0.0031
P = 0.0032 P = 9 × 10–5
P = 4 × 10–5
P = 0.0015
P = 0.0021
P = 0.0007
P = 0.001
P = 0.0043
P = 0.0004
0
5
10
15
Relative mRNA levels
CtrlT/ST/S/N
IL6
–2
–1
0
1
2
++–++–+
C1 C2
C3 P1
T/SNec-1s
PBMC
––+––+––+––
P1P1P1C1C2C3CtrlTSZ –++–
Urine Cell sup.
Cyclophilin ACoomassie staining
p-S166-RIPK1
RIPK1
p-S358-MLKL
MLKL
Cl-CASP3
Tu bulin
70
70
55
55
15
55
T/S
S/Z
Nec-1s
T/S/Z
PBMC
–––––+– ––––
––+–+ –––+–+
–––++ ––––++
–++ ––––++––
P1 Control
T/S T/S/N S/Z S/Z/N
0
50
100
Cell viability (%)
P1
C
P3
P2
WT
GFP
D325H
D325V
D138N/D325V
D138N D138N/D325H
WT
GFP
D325H
D325V
C
P1
Fig. 3 | RIPK1 cleavage site variants promote cell death and inf lammatory
response induced by TNF in patient PBMCs and MEFs. a, Cell viability (as
measured by CellTiter-Glo assay) of PBMCs from patients and eight paediatric
unaffected controls after treatment as indicated for 24 h. N, Nec-1s; S, SM-164;
T, TNF; Z, Z-VAD-FMK. Data are mean ± s.e.m. Circles indicate one sample from
each individual (P1 was sampled three times). Analysis of each sample was
performed in triplicate. b, Western blots of PBMCs from patient P1 and a
paediatric unaffected control after treatment as indicated for 24 h. −,
untreated; cl, cleaved. For gel source data, see Supplementary Fig. 1. Results are
representative of two independent experiments. c, Western blots of urine
samples from P1 during a fever episode (red) and remission (blue) and three
paediatric unaffected controls. Supernatant (sup.) of fibroblasts from an
unaffected control stimulated with TNF, SM-164 and Z-VAD-FMK (TSZ) served
as a positive control. For gel source data, see Supplementary Fig. 1. Results are
representative of three independent experiments. d, NanoString analysis of
PBMCs from patient P1 and three paediatric unaffected controls after
stimulation as indicated. For gene names, see Supplementary Fig. 2. e, qPCR
analysis of IL6 mRNA levels of PBMCs from patient P1 and four unaffected
controls treated as indicated. Data are mean ± s.e.m. Circles correspond to
each tested individual. Analysis of each sample was performed in triplicate.
The PBMCs in a, b, d and e were obtained during remission. f, Cell viability of
Ripk1-knockout MEFs complemented with: GFP; wild-type (WT) RIPK1; D325V,
D325H or D138N mutant; or D138N/D325H or D138N/D325V double mutants,
treated as indicated for 24 h. Data are mean ± s.e.m., n = 3. Circles correspond to
each independent experiment. P values determined by unpaired two-tailed
t-test (shown if P < 0.05). g, h, Western blots of Ripk1-knockout MEFs
complemented with: GFP; wild-type RIPK1; D325V, D325H or D138N mutant; or
D138N/D325H or D138N/D325V double mutants, treated as indicated. HA,
haemagglutinin; LE, long exposure; SE, short exposure. For gel source data, see
Supplementary Fig. 1. Results are representative of three independent
experiments. i, Cell viability of Ripk1D325A/D325ARipk3−/− and Ripk1+/+Ripk3−/− MEFs
treated as indicated for 24 h. Data are mean ± s.e.m., n = 3. Circles correspond to
each independent experiment. P values determined by unpaired two-tailed
t-test. j, Western blots of Ripk1D325A/D325ARipk3−/− and Ripk1+/+Ripk3−/− MEFs
treated as indicated. For gel source data, see Supplementary Fig. 1. Results are
representative of three independent experiments. k, Il6 mRNA expression of
Ripk1-knockout MEFs complemented with: GFP; wild-type RIPK1; or D325V or
D325H mutant, treated as indicated. Data are mean ± s.e.m., n = 3. Circles
correspond to each independent experiment. P values determined by unpaired
two-tailed t-test. l, m, qPCR analysis of Il6 and Cxcl2 (l) or Il6, Cxcl2 and Tn f (m)
expression in Ripk1D325A/D325ARipk3−/− and Ripk1+/+Ripk3−/− MEFs treated as
indicated for 2 or 4 h. Data are mean ± s.e.m., n = 3. Circles correspond to each
independent experiment. P values determined by unpaired two-tailed t-test.