Nature | Vol 577 | 2 January 2020 | 113Necroptosis and ferroptosis resistance in fibroblasts
Notably, we observed the opposite response to inducers of cell
death in fibroblasts from patient P1 compared with MEFs and patient
PBMCs. The patient fibroblasts showed resistance to cell death after
stimulation with TNF or LPS plus SM-164 and Z-VAD-FMK (Fig. 4a,
Extended Data Fig. 6a). The cell death resistance was further demon-
strated by reduced phosphorylation of RIPK1 and MLKL in the patient
fibroblasts (Fig. 4b, Extended Data Fig. 6b, c). Patient fibroblasts
also showed diminished gene expression of IL6, IL1B and pro-inflam-
matory chemokines CXCL2 and CXCL3 in response to TNF, SM-164
and Z-VAD-FMK stimulation (Fig. 4c). Levels of RIPK1 protein under
basal and stimulated conditions were lower in patient fibroblasts
than that of controls (Fig. 4d), and the reduction was rescued by
the Nec-1s (Extended Data Fig. 6d). We observed reduction of RIPK1
at the transcriptional level in fibroblasts (Extended Data Fig. 4e,
Extended Data Fig. 6e). Together, these results suggest that decreased
RIPK1 expression may compensate for the presence of the RIPK1-
activating variant in patient fibroblasts. In addition, we found that
patient fibroblasts exhibited reduced expression of TNFR1, which
may provide a further mechanism for the decreased sensitivity to
TNF (Fig. 4d, Extended Data Fig. 6f ). Patient fibroblasts also showed
decreased expression of genes involved in cell death pathways such
as RIPK1 and RIPK3 and a different gene expression pattern (Extended
Data Fig. 6g) compared to PBMCs (Fig. 2d). Together, these findings
provide evidence of compensatory mechanisms to resist cell death
in the patient fibroblasts.
We also characterized the sensitivity of patient fibroblasts to other
cell death stimuli. Notably, we found that the patient fibroblasts were
highly protected against ferroptosis induced by erastin, RSL3 or FIN56
(Fig. 4e)—an effect that was not found in patient PBMCs or MEFs express-
ing the RIPK1 D325V or D325H mutant (Extended Data Fig. 4f, Extended
Data Fig. 5h). Consistent with these findings, erastin-induced degra-
dation of GPX4^14 was blocked in patient fibroblasts (Fig. 4f), but not
in Ripk1−/− MEFs expressing mutant RIPK1 (Extended Data Fig. 5i). To
explore the mechanism of ferroptosis resistance, we analysed gene
expression in patient fibroblasts by RNA sequencing. We found that
the expression of several genes involved in inhibiting ferroptosis—such
as SLC7A11, CISD1 and CD44^15 —were upregulated in patient fibroblasts
(Fig. 4g). This pattern was not observed in the patient PBMCs or MEFs
(Extended Data Figs. 4g, h, 5j). Similarly, the concentration of the anti-
oxidant glutathione (GSH) was much higher in patient fibroblasts than
that of controls (Fig. 4h). By contrast, GSH levels were similar in PBMCs
or MEFs expressing wild-type or mutant RIPK1 (Extended Data Figs. 4i,
5k). Consistent with increased levels of GSH, the amounts of reactive
oxygen species (ROS) (as indicated by the cytosolic ROS sensor carboxy-
H 2 DCFDA) were lower after erastin stimulation in patient fibroblasts
(Fig. 4i), but not in Ripk1-knockout MEFs complemented with mutant
RIPK1 (Extended Data Fig. 5l). These data suggest that restricted release
of ROS by the patient fibroblasts may help to protect against ferropto-
sis, as ROS is known to be crucial for mediating ferroptosis^16. Similarly,
because ROS production can promote RIPK1 activation and necropto-
sis^17 ,^18 , the high levels of antioxidant GSH in the patient fibroblasts may
also contribute to the resistance to necroptosis.C1C2C3P101002003,0004,5006,000Relative intensityErastin (8 h)Ctrl
Erastin
Glutamate0.01.02.03.04.0GSH concentration (ferroptosis/antioxidantGenes involved in μM)–1.501.5C1 C2 C3 P1GPX4 (LE)
GPX4 (SE)
SLC7A11
LAMP2A
COX2
ACSL4
HSC70
p53
Tu bulin- 48– 48– 48– 48
P1 C1 C2 C3
Erastin (h)10050
Cell viability (%) 0
ErastinRSL3FIN56RIPK1
TNFR1
HSP90P1C1C2C3C4P1C1C2C3C4
TNF 0 min 20 minT/S/Z/N
T/NT/ST/S/NT/S/Z
Ctrl N T
T/S/Z/N
T/NT/ST/S/NT/S/Z
Ctrl N T0100200(^300) CXCL3
0
200
400
(^600) CXCL2
0
500
1,000
1,500
2,000
Relative mRNA levels
IL1B
0
100
200
400
800
Relative mRNA levels
IL6
GAPDH
MLKL
p-S358-MLKL
RIPK1
p-S166-RIPK1
Nec-1s ++ ++ ++ ++ ++
VAD ++ ++ ++ ++ ++
SM-164 ++++ ++++ ++++ + +++ ++++
TNF + ++++ +++++ +++++ +++++ +++++
P1 C1 C2 C3 C4
70
70
55
55
37
70
10055
15
15
35
100
70
70
70
55
55
LPS/S/NLPS/S/ZLPS/S/Z/N
T/NT/ST/S/NT/S/ZT/S/Z/NLPSLPS/NLPS/S
N T
100
50
Cell viability (%) 0
ab
c
d
e
f
gh
i
C
P1
C
P1
C
P1
CP1
C1 C2
C3 P1
Fig. 4 | Necroptosis and ferroptosis are repressed in the patient f ibroblasts.
a, Cell viability of fibroblasts from P1 and seven paediatric unaffected controls
after treatment with indicated stimulation for 24 h. N, Nec-1s; S, SM-164; T, TNF;
Z, Z-VAD-FMK. Data are mean ± s.e.m. Circles correspond to each tested
individual. Analysis of each sample was performed in triplicate. b, Western
blots of fibroblasts from patient P1 and four paediatric unaffected controls
after treatment with indicated stimulation for 6 h. For gel source data, see
Supplementary Fig. 1. Results are representative of three independent
experiments. VAD, Z-VAD-FMK. c, Patient and seven paediatric unaffected
control fibroblasts were treated as indicated for 6 h. The mRNA levels of
cytokines were measured by qPCR. Data are mean ± s.e.m. Circles correspond
to each tested individual. Analysis of each sample was performed in triplicate.
d, Western blots of RIPK1 and TNFR1 protein levels at basal state and after TNF
stimulation in fibroblasts from P1 and four paediatric unaffected controls. For
gel source data, see Supplementary Fig. 1. Results are representative of three
independent experiments. e, Cell viability of patient fibroblasts treated with
erastin, RSL3 or FIN56 compared with six paediatric unaffected controls. Data
are mean ± s.e.m. Circles correspond to each tested individual. Analysis of each
sample was performed in triplicate. f, Western blots of fibroblasts from P1 and
three paediatric unaffected controls after treatment with erastin for 4 or 8 h.
For gel source data, see Supplementary Fig. 1. Results are representative of
three independent experiments. g, Expression patterns of genes involved in
ferroptosis and antioxidant by RNA sequencing of fibroblasts from patient and
three paediatric unaffected controls. Analysis of each sample was performed in
duplicate. For gene names, see Supplementary Fig. 2. h, GSH concentrations in
fibroblasts from P1 compared with six paediatric unaffected controls at
baseline and after treatment with erastin or glutamate for 8 h. Data are
mean ± s.e.m. Circles correspond to each tested individual. Analysis of each
sample was performed in triplicate. i, Immunof luorescence (left) and relative
f luorescent intensity (right) of cytosolic ROS (green foci) in patient fibroblasts
after treatment with erastin for 8 h compared with that of three paediatric
unaffected controls. Scale bar, 150 μm. Circles correspond to each tested
individual sample. Analysis of each sample was performed in duplicate.