Nature | Vol 577 | 2 January 2020 | 111cells (Fig. 3b). Notably, patient PBMCs treated with TNF and SM-164
showed increased levels of not only cleaved caspase-3, but also p-S358-
MLKL, which were both effectively reduced by treatment with Nec-1s
(Fig. 3b). These results suggest that the non-cleavable RIPK1 variant
sensitized the patient PBMCs to both necroptosis and apoptosis in a
RIPK1-dependent manner.
Release of cyclophilin A is a biomarker for necroptosis in cell-based
assays and has also been implicated as a potential biomarker in human
diseases^10 ,^11. We detected the presence of cyclophilin A in a urine sample
from a patient during a fever episode but not in remission, which pro-
vides evidence for enhanced necrotic cell death in the setting of inflam-
mation in vivo (Fig. 3c). These findings indicate that the non-cleavable
RIPK1 variant may promote the activation of RIPK1, which leads to
necrotic cell death in vivo.
Activation of necroptosis promotes a strong inflammatory response
such as the production of pro-inflammatory cytokines^12. Compared to
that of control PBMCs, the patient PBMCs stimulated with TNF plus
SM-164 showed an exacerbated inflammatory response, which was
effectively inhibited by the RIPK1 inhibitor Nec-1s (Fig. 3d). Confirming
the involvement of RIPK1 kinase activity in promoting the inflamma-
tory responses, we found that the increased IL6 expression owing to
cell death induced by TNF plus SM-164 stimulation in patient PBMCs
was suppressed by Nec-1s (Fig. 3e).
The patient data raised the possibility that non-cleavable RIPK1
variants function directly in promoting its own activation, which in
turn mediates apoptosis and necroptosis in a signal-dependent man-
ner. To test this possibility experimentally, we expressed the cleav-
age site D325V and D325H RIPK1 mutants in Ripk1-knockout mouse
embryonic fibroblasts (MEFs). Compared to that of Ripk1-knockout
MEFs and Ripk1-knockout MEFs complemented with wild-type RIPK1,
MEFs expressing the D325V or D325H RIPK1 mutant were consistently
hypersensitive to cell death induced by TNF, which was inhibited by
the addition of Nec-1s. The enhanced cell death could also be blocked
by introducing a RIPK1 kinase inactivation mutation, D138N, in cis with
D325V or D325H, providing direct evidence for the role of RIPK1 kinase
activity in promoting cell death (Fig. 3f, Extended Data Fig. 5a, b). Simi-
lar to patient PBMCs, MEFs expressing D325V or D325H mutant RIPK1
stimulated by TNF alone or TNF plus SM-164 showed increased levels of
p-S166-RIPK1 (Fig. 3g). By contrast, stimulation of Ripk1-knockout MEFs
complemented with wild-type RIPK1 with TNF alone was not sufficient
to promote the activation of RIPK1 (Fig. 3g). These data support the
hypothesis that the non-cleavable variants of RIPK1 directly promote
the activation of RIPK1.
Similar to that of patient PBMCs, RIPK1(D325V)- or RIPK1(D325H)-
complemented Ripk1-knockout MEFs stimulated by TNF or TNF plus
SM-164 showed increased levels of cleaved caspase-3 compared to that
of wild-type-complemented MEFs, which was inhibited by Nec-1s and
by the kinase inactivation mutation D138N in cis with D325V or D325H
construct (Fig. 3g, h, Extended Data Fig. 5c). Also similar to that of
patient PBMCs, the stimulation of Ripk1-knockout MEFs expressing
D325V or D325H RIPK1 mutant with TNF alone or TNF plus SM-164
induced increased levels of p-S345-MLKL (Fig. 3g, Extended Data
Fig. 5c). By contrast and as expected, stimulation of Ripk1-knockout
MEFs or Ripk1-knockout MEFs complemented with wild-type RIPK1
with TNF alone or TNF plus SM-164 was not sufficient to promote
the activation of necroptosis and appearance of p-S345-MLKL. TNF-
induced cell death and the appearance of p-S345-MLKL in D325V- or
D325H-complemented Ripk1-knockout MEFs were both blocked by
Nec-1s and by the inactivation D138N mutation in cis with D325V or
D325H (Fig. 3f–h, Extended Data Fig. 5c). These results suggest that
D325V and D325H are gain-of-function mutations in RIPK1 that pro-
mote the activation of its kinase, which in turn mediates apoptosis
and necroptosis.
Because the expression of non-cleavable RIPK1 promotes both
apoptosis and necroptosis, we next determined whether these two
forms of cell death might be independent of each other by examining
Ripk1D325A/D325ARipk3−/− MEFs from Ripk1D325A/D325A knock-in mice crossed
with necroptosis-deficient Ripk3−/− mice^2. Notably, we found that
Ripk1D325A/D325ARipk3−/− MEFs remained sensitized to apoptosis induced
by TNF alone and TNF plus SM-164 and showed increased levels of
p-S166-RIPK1 and cleaved caspase-3, which are both inhibited by
Nec-1s (Fig. 3i, j, Extended Data Fig. 5d, e). Thus, the activated RIPK1 in
cells expressing non-cleavable RIPK1 is able to drive RIPK1-dependent
apoptosis, independently of necroptosis.UMAP_1UMAP_2–10– 5051015–10–50510–10– 5051015–10–50510UMAP_1UMAP_2C1 P1CXCL2 CXCL3IL8ICAM1SOCS3 IL1BIL6TNF051015200481216020406080024601020304002030400246805152510101020Relative mRNA levelsRelative mRNA levelsC–2 –1 012
C1 C2 C3 P1 C1 C2 C3 P1C1 C2 C3 P1Cell deathNF-κBType I IFNabcd e25/10/175/1/1918/3/1919/3/1925/3/1931/10/177/1/198/4/1905102030050100600
2001,00005100300
200500IL-6TNFIL-10No feverReferenceConcentration (pg ml–1)Concentration (pg ml–1)Concentration (pg ml–1)25/10/175/1/1918/3/1919/3/1925/3/1931/10/17
7/1/198/4/1925/10/17
5/1/1918/3/1919/3/19
25/3/19
31/10/177/1/198/4/19Fever–10–50510–10– 5051015–10– 5051015–10–50510UMAP_1UMAP_1UMAP_2UMAP_2P1–10– 5051015–10–50510–10– 5051015–10–50510UMAP_1UMAP_1UMAP_2UMAP_2C1C1 P1IL8 IL8IL1B IL1BCD4 TB
CD8 TNK
CD14 monoCD16 mono
T memoryγδ T
NK/T doubletsB activated
DendriticNaive B
Eryth
MkPlasma
pDCP1Fig. 2 | Strong activation of inf lammatory signalling in patient P1. a, Serum
levels of cytokines IL-6, TNF and IL-10 from patient P1 (red denotes serum
during fever episodes; blue denotes serum during remission) determined by
cytometric bead array. b, Left, uniform manifold approximation and projection
(UMAP) of 18,928 cells, split between patient P1 and an age- and sex-matched
unaffected control (C1) after alignment. Right, UMAP visualization and marker-
based annotation of 16 cell subtypes, coloured by cluster identity. The patient
P1 displayed higher percentage of monocytes (red frame). Eryth, erythrocytes;
Mk, megakaryocyte; NK, natural killer cell; pDC, plasmacytoid dendritic cell.
c, Visualization of expression of IL8 and IL1B (coloured single cells) on UMAP
plot projecting PBMCs from patient P1 (n = 7,936 cells) and an age- and sex-
matched unaffected control (n = 10,992 cells). d, RNA-sequencing analysis of
cell death, NF-κB and type I IFN pathways in patient PBMCs compared with
three paediatric unaffected controls (C1–C3). Analysis of each sample was
performed in duplicate. For gene names, see Supplementary Fig. 2. e, qPCR
analysis of cytokine and chemokine-related genes in PBMCs from P1 compared
with five paediatric unaffected controls (C). Data are mean ± s.e.m. Circles
correspond to each tested individual. Analysis of each sample was performed
in triplicate. The PBMCs from patient P1 in b–e were obtained during fever
episodes.