Induction of ferroptosis-like cell death of eosinophils exerts synergistic effects with glucocorticoids in allergic airway inflammation

Abstract

Introduction
Eosinophils are critical in allergic disorders, and promoting eosinophil death effectively attenuates allergic airway inflammation. Ferroptosis is a recently described novel form of cell death; however, little is known about ferroptosis in eosinophils and related diseases. This study aimed to investigate the effects of ferroptosis-inducing agents (FINs) on eosinophil death and allergic airway inflammation, and to explore their potential synergistic effect with glucocorticoids (GCs).

Methods
Eosinophils isolated from the peripheral blood of humans or mice were incubated with FINs, and eosinophil ferroptosis was assessed. The in vivo effects of FINs alone or in combination with dexamethasone (DXMS) were examined in a mouse model of allergic airway inflammation. Bronchoalveolar lavage fluid and lung tissue were collected to examine airway inflammation.

Results
Treatment with FINs time and dose dependency induced cell death in human and mouse eosinophils. Interestingly, FINs induced non-canonical ferroptosis in eosinophils, which generated morphological characteristics unique to ferroptosis and was iron dependent but was independent of lipid peroxidation. The antioxidants glutathione and N-acetylcysteine significantly attenuated FIN-induced cell death. Treatment with FINs triggered eosinophil death in vivo and eventually relieved eosinophilic airway inflammation in mice. Furthermore, FINs exerted a synergistic effect with DXMS to induce eosinophil death in vitro and to alleviate allergic airway inflammation in vivo.

Conclusions
FINs induced ferroptosis-like cell death of eosinophils, suggesting their use as a promising therapeutic strategy for eosinophilic airway inflammation, especially due to the advantage of their synergy with GCs in the treatment of allergic disorders.

Full text

ORIGINAL RESEARCH
Induction of ferroptosis- like cell death of eosinophils
exerts synergistic effects with glucocorticoids in
allergic airwayinflammation
Yanping Wu,1 Haixia Chen,1 Nanxia Xuan,1 Lingren Zhou,1 Yinfang Wu,1 Chen Zhu,1
Miao Li,1 Qingyu Weng,1 Jiaxin Shen,1 Hao Zhang,1 Bin Zhang,1 Fen Lan,1 Lixia Xia,1
Xuefang Xiong,2 Zhouyang Li ,1 Yun Zhao,1 Mindan Wu,1 Songmin Ying,1 Wen Li,1
Huahao Shen,1,3 Zhihua Chen1
Asthma
To cite: WuY, ChenH,
XuanN, etal. Thorax
2020;75:918–927.
►Additional material is
published online only. To view
please visit the journal online
(http:// dx. doi. org/ 10. 1136/
thoraxjnl- 2020- 214764).
1Key Laboratory of Respiratory
Disease of Zhejiang Province,
Department of Respiratory and
Critical Care Medicine, Zhejiang
University School of Medicine
Second Affiliated Hospital,
Hangzhou, Zhejiang, China
2Department of Respiratory
Medicine, Central Hospital of
Lishui City, Lishui, Zhejiang,
China
3State Key Lab for Respiratory
Diseases, National Clinical
Research Centre for Respiratory
Disease, Guangzhou,
Guangdong, China
Correspondence to
Professor Zhihua Chen;
zhihuachen@ zju. edu. cn
Professor Huahao Shen;
huahaoshen@ zju. edu. cn
Professor Wen Li;
liwen@ zju. edu. cn
YW and HC contributed equally.
Received 5 March 2020
Revised 16 June 2020
Accepted 24 June 2020
Published Online First
5August2020
© Author(s) (or their
employer(s)) 2020. No
commercial re- use. See rights
and permissions. Published
by BMJ.
ABSTRACT
Introduction Eosinophils are critical in allergic
disorders, and promoting eosinophil death effectively
attenuates allergic airway inflammation. Ferroptosis is a
recently described novel form of cell death; however, little
is known about ferroptosis in eosinophils and related
diseases. This study aimed to investigate the effects of
ferroptosis- inducing agents (FINs) on eosinophil death
and allergic airway inflammation, and to explore their
potential synergistic effect with glucocorticoids (GCs).
Methods Eosinophils isolated from the peripheral
blood of humans or mice were incubated with FINs, and
eosinophil ferroptosis was assessed. The in vivo effects
of FINs alone or in combination with dexamethasone
(DXMS) were examined in a mouse model of allergic
airway inflammation. Bronchoalveolar lavage fluid
and lung tissue were collected to examine airway
inflammation.
Results Treatment with FINs time and dose dependency
induced cell death in human and mouse eosinophils.
Interestingly, FINs induced non- canonical ferroptosis
in eosinophils, which generated morphological
characteristics unique to ferroptosis and was iron
dependent but was independent of lipid peroxidation.
The antioxidants glutathione and N- acetylcysteine
significantly attenuated FIN- induced cell death.
Treatment with FINs triggered eosinophil death in vivo
and eventually relieved eosinophilic airway inflammation
in mice. Furthermore, FINs exerted a synergistic effect
with DXMS to induce eosinophil death in vitro and to
alleviate allergic airway inflammation in vivo.
Conclusions FINs induced ferroptosis- like cell death
of eosinophils, suggesting their use as a promising
therapeutic strategy for eosinophilic airway inflammation,
especially due to the advantage of their synergy with
GCs in the treatment of allergic disorders.
INTRODUCTION
Eosinophils, terminally differentiated granulocytic
cells, have been implicated in the pathogenesis of
diverse inflammatory responses.1 They mature from
pluripotent progenitors in the bone marrow and
are released into the circulation in a phenotypically
mature state.2 Eosinophils normally account for less
than 5% of leucocytes in the blood.3 In the absence
of external stimuli, circulating eosinophils usually
end physiologically by spontaneous apoptosis
within 2–5 days.4 In response to diverse stimuli,
eosinophil production is increased. Activated
eosinophils migrate into the bloodstream and are
subsequently recruited to inflammatory foci, where
their lifespan is believed to be prolonged due to the
presence of prosurvival factors in the local micro-
environment.5 Eosinophils are clearly recruited for
defence against invading pathogens at the inflamed
site. On the other hand, eosinophils have also been
shown to serve as major effector cells that induce
tissue injury and dysfunction by secreting toxic
granule proteins and lipid mediators.6
Overwhelming evidence shows that eosino-
phil infiltration in the airways is a key feature of
allergic asthma and is believed to be associated
with the pathogenesis of this disease. The results of
both clinical studies and studies in allergic mouse
models have also demonstrated that eosinophils
contribute to asthma pathogenesis, ongoing inflam-
mation, airway hyper- responsiveness and tissue
remodelling.5 An increasing number of studies
Key messages
What is the key question?
►Here, we questioned whether the induction of
eosinophil ferroptosis could be a new effective
strategy against allergic airway inflammation.
What is the bottom line?
►Treatment with ferroptosis- inducing agents
(FINs) triggered eosinophil death and eventually
relieved eosinophilic airway inflammation;
moreover, FINs exerted a synergistic effect
with dexamethasone to induce eosinophil
death in vitro and to alleviate allergic airway
inflammation in vivo.
Why read on?
►This is the first study to show that FINs induce
ferroptosis- like cell death in eosinophils,
suggesting their use as a promising therapeutic
strategy for eosinophilic airway inflammation,
especially due to the advantage of their synergy
with glucocorticoids in the treatment of allergic
disorders.
918 Wu Y, etal. Thorax 2020;75:918–927. doi:10.1136/thoraxjnl-2020-214764
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Asthma
have proposed that the course of allergic airway inflammation
not only depends on eosinophil recruitment but also partly
depends on the increased lifespan of eosinophils within inflamed
tissue.2 7 The delay of eosinophil apoptosis is a critical mecha-
nism of eosinophil accumulation at an inflammatory site.8 Gluco-
corticoids (GCs) are the most effective therapy for eosinophilic
disorders by both their direct induction of eosinophil apoptosis
and their suppression of prosurvival signals, but the pleiotropic
effects of corticosteroids limit their therapeutic use, especially at
high doses.9 10 Recent studies have focused on the development
of new agents to block eosinophil recruitment and/or to decrease
eosinophil survival and activation.11 12 We recently uncovered
that Bcl-2 inhibitors that promote eosinophil apoptosis eventu-
ally reduced allergic inflammation.13 Thus, selective induction
of eosinophil death is likely to resolve allergic inflammation and
restore tissue homeostasis.
Ferroptosis, a novel form of non- apoptotic cell death, is char-
acterised by the accumulation of reactive oxygen species (ROS)
derived from iron metabolism and lipid peroxidation.14 Multiple
small molecules called ferroptosis- inducing agents (FINs),
including experimental compounds, for example, erastin and
Ras- selective lethal small molecule 3 (RSL3), and clinical drugs,
for example, artesunate (ART), sulfasalazine and sorafenib, have
been discovered. Cell death triggered by erastin was found to
result in glutathione (GSH) depletion and lipid peroxidation
accumulation through its ability to inhibit the import of cystine
by directly inhibiting cystine/glutamate antiporter system xc
-
activity.15 RSL3 was demonstrated to induce ROS production
from lipid peroxidation by inactivating glutathione peroxidase
4 (GPX4).15 In addition, some FINs trigger ferroptosis through
a Fenton- like reaction.16 Generally, ferroptosis should be phar-
macologically inhibited by both an iron chelator and a lipid
peroxidation inhibitor and should involve accumulation of lipid
hydroperoxides. In cases of cell death postulated to be ferroptosis,
other known forms of cell death, such as apoptosis and necrosis,
should be ruled out. Ferroptotic cells lack the morphological and
biochemical characteristics of cells undergoing other forms of cell
death, which exhibit smaller mitochondria with condensed mito-
chondrial membrane densities, outer mitochondrial membrane
rupture and a reduction in mitochondrial crista.17
Recent reports have revealed the connection between ferro-
ptosis and the pathological processes of several diseases and
conditions, including neurodegenerative diseases,18 heart trans-
plantation19 and neoplastic diseases.20 Ferroptosis has been
suggested as a potential contributor for the treatment of cell
death- related diseases. Recently, high levels of lipid peroxides
were found in asthmatic airway epithelial cells, contributing to
epithelial dysfunction, cell death and asthma exacerbation.21
However, the effects of ferroptosis on eosinophils and eosin-
ophilic inflammation have not been studied. A previous study
showed that eosinophils have the highest level of catalytic ferrous
iron (Fe(II)) in normal status, which would further increase in
allergic status.22 Fe(II) is considered as an initiator of the Fenton
reaction and ROS generated through Fenton reaction is known
to contribute to the initiation of ferroptosis.23 Thus, eosinophils
appear to be more likely to occur ferroptosis due to their abun-
dance of iron.
In this study, we show that FIN treatment is associated with
eosinophil death in vitro and exerts therapeutic efficacy in an
allergic airway inflammation model in vivo. Furthermore, FINs
have a synergistic effect with GCs to induce eosinophil death
and alleviate allergic airway inflammation. Accordingly, the
objective of the current study is to identify FINs as a promising
therapeutic strategy for allergic airway inflammation.
METHODS
Additional methods are presented in the online supplementary
file.
Human subjects
Nine hypereosinophilic patients were recruited from the clinical
population at the Department of Respiratory and Critical Care
Medicine of the Second Affiliated Hospital of Zhejiang Univer-
sity School of Medicine. All patients provided written informed
consent and understood that their samples would be used for
research.
Experimental animals and treatments
Male C57BL/6 mice were purchased from Shanghai SLAC Labo-
ratory Animal Co. (Shanghai, China). Mice from each litter were
randomised to different groups. Protocols were approved by the
Ethics Committee for Animal Studies at Zhejiang University,
China.
Erastin (25 mg/kg) and RSL3 (10 mg/kg) were dissolved in
dimethyl sulfoxide (DMSO) and administered intraperitoneally
2 hours after each ovalbumin (OVA) challenge. The controls
were received equal dosage of DMSO for erastin or RSL3 group.
ART (10 or 20 mg/kg) was dissolved in normal saline (NS) and
delivered intraperitoneally once a day for 3 days before the first
challenge, and 2 hours after each challenge. Dexamethasone
(DXMS) (0.25 or 0.5 mg/kg) was diluted in NS and administered
intraperitoneally 2 hours after each challenge. In ART/DXMS
groups, mice were injected with ART (10 mg/kg) alone once a
day for 3 days before the first challenge, and then cotreated with
ART (10 mg/kg) and DXMS (0.25 mg/kg) 2 hours after each chal-
lenge. Control mice were received the same volume of NS for
ART, DXMS or ART/DXMS group. Twenty- four hours after the
last administration, all mice were sacrificed for analysis.
Statistics
All related data are presented as the mean ± SEM. Comparisons
between two groups were calculated by two‐tailed Student’s
t- test, and significant differences between multiple groups were
evaluated by one‐way analysis of variance with Tukey’s honestly
significant difference (HSD) post hoc testing using GraphPad
Prism 8 software (GraphPad Software, La Jolla, California,
USA). The test statistics have been transformed into adjusted p
values following Tukey multiple comparison testing. Differences
were considered statistically significant when the p value was less
than 0.05.
RESULTS
FINs triggered the cell death of eosinophils from human
subjects and mice
To explore whether FIN stimulation could lead to eosinophil
death, we initially harvested peripheral leucocytes from patients
with asthma and non- asthma with increased eosinophils and incu-
bated the leucocytes with FINs. Eosinophils among leucocytes
were distinguished by flow cytometry for Siglec-8 and CCR3
staining (Siglec-8+/CCR3+)24 (online supplementary figure 1A).
Treatment of peripheral eosinophils from both patients with
asthma and non- asthma with a series of FINs (erastin, RSL3,
and ART) resulted in concentration- dependent cell death, as
assayed by flow cytometry (figure 1A, online supplementary
figure 1B,C). However, in this experimental setting, we could
not exclude the potential influence of other types of leucocytes
on eosinophil death. We subsequently isolated eosinophils from
the whole blood of patients with asthma to examine the effects
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Asthma
of FINs on purified eosinophils. A similar effect in response
to FINs was observed in purified eosinophils and eosinophils
among leucocytes, indicating that FIN- treated eosinophil death
was not affected by the presence of other cell types (figure 1B).
To further test whether FINs could exert similar effects on
mouse eosinophils, we isolated eosinophils from the periph-
eral blood of Cd3δ promoter interleukin 5 transgenic (Tg)
mice. Similarly, FINs dose and time dependency induced cell
Figure 1 Ferroptosis- inducing agents (FINs) triggered the cell death of eosinophils from patients with asthma and allergic mice. A total of six
patients with asthma were recruited. (A) Peripheral leucocytes (n=4–6) from patients with asthma were cultured with various concentrations of
FINs for 24 hours prior to viability determination by flow cytometry. Siglec-8+/CCR3+/annexin V−/4',6- diamidino-2- phenylindole (DAPI−) cells were
distinguished as viable peripheral eosinophils. (B) Eosinophils were purified from four of the six patients with asthma and cultured with indicated
concentrations of FINs for 24 hours. Annexin V−/propidium iodide (PI−) cells were defined as viable cells. (C and D) Eosinophils isolated from the
peripheral blood of interleukin 5 transgenic mice were cultured with various concentrations of FINs for 24 hours (C) or FINs (erastin 30 µM, Ras-
selective lethal small molecule 3 (RSL3) 2 µM, artesunate (ART) 100 µM) for the indicated duration before harvest (D). Annexin V−/PI− cells were
defined as viable cells. (E) Bronchoalveolar lavage (BAL) cells from allergic mice were cultured with various concentrations of FINs for 24 hours.
Siglec- F+/CD11c−/annexin V−/DAPI− cells were distinguished as viable BAL eosinophils by flow cytometry. All data are shown as mean±SEM, analysed
by one- way analysis of variance (A, C, D, E) or Student’s t- test (B).
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Asthma
death in mouse eosinophils (figure 1C,D, online supplemen-
tary figure 2A). To confirm that FINs can induce cell death
in local inflammatory eosinophils, we next collected bron-
choalveolar lavage (BAL) cells from allergic mice and then
stimulated the BAL cells with FINs. Mouse BAL eosinophils
were distinguished by Siglec- F and CD11c staining (Siglec- F+/
CD11c−)25 (online supplementary figure 2B). Again, FIN
treatment efficiently triggered concentration- dependent cell
death in BAL eosinophils, though eosinophils in the broncho-
alveolar lavage fluid (BALF) were slightly more resistant to
erastin and ART stimulation than those in blood (figure 1E,
online supplementary figure 2C). Taken together, these results
indicate that FINs could remarkably trigger the cell death of
eosinophils from both human subjects and mice, regardless of
their allergic status.
FINs induced non-canonical ferroptosis in eosinophils
We further examined the mode of cell death caused by FINs in
eosinophils. We first investigated whether the observed cytotox-
icity was related to apoptotic or necrotic death or autophagy. As
expected, Z- VAD- FMK (a pan- caspase inhibitor), necrostatin-1
(a potent necroptosis inhibitor targeting the death domain kinase
RIP), or spautin-1 (a specific autophagy inhibitor targeting the
activity of ubiquitin- specific peptidases) failed to reverse erastin-
induced or RSL3- induced cell death (figure 2A,B). In ART- treated
Figure 2 Ferroptosis- inducing agents (FINs) induced non- canonical ferroptosis in eosinophils. Eosinophils were isolated from the peripheral blood
of interleukin 5 transgenic mice. Annexin V−/PI− cells were defined as viable eosinophils. (A–C) Eosinophils were cultured with FINs (erastin 30 µM,
Ras- selective lethal small molecule 3 (RSL3) 2 µM, artesunate (ART) 100 µM) with or without Z- VAD- FMK (100 µM), necrostatin-1 (100 µM) and
spautin-1 (5 µM) for 24 hours prior to viability determination by flow cytometry. (D) Transmission electron microscopy of eosinophils treated with
dimethylsulfoxide (DMSO) or FINs for 12 hours. Single red arrowheads point to normal mitochondria; paired red arrowheads point to damaged
mitochondria. (E and F) Effects of ferrostatin-1 (fer-1; 2 µM) and liproxstatin-1 (lip-1; 2 µM) on the cell viability of eosinophils treated with FINs for
24 hours. (G and H) Effects of deferoxamine (DFO; 100 µM) and ciclopirox olamine (CPX; 500 nM) on the cell viability of eosinophils treated with
FINs for 24 hours. (I) Effects of ferrous iron (Fe(II); 200 µM) and FIN cotreatment on the cell viability of eosinophils for 24 hours. All data are shown as
mean±SEM, analysed by one- way analysis of variance. CTL, control; PI, propidium iodide.
921Wu Y, etal. Thorax 2020;75:918–927. doi:10.1136/thoraxjnl-2020-214764
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Asthma
eosinophils, cell death was partly suppressed by spautin-1, but
not by Z- VAD- FMK or necrostatin-1 (figure 2C). The bioactivity
of these two compounds was confirmed in other cases (online
supplementary figure 3A,B). These data suggest that FINs induce
predominantly non- apoptotic cell death in eosinophils and that
autophagy is likely involved in ART- induced cell death.
We next sought to test whether ferroptosis was genuinely
activated in the eosinophils treated with FINs. As ferro-
ptosis has its unique morphological characteristics,14 we used
transmission electron microscopy to monitor morphological
changes caused by FINs. FIN- treated eosinophils exhibited
shrunken and damaged mitochondria, features unique to
ferroptosis, with no other obvious changes prior to the occur-
rence of cell death (figure 2D). For direct comparison, eosin-
ophils were treated with staurosporine or hydrogen peroxide
solution to trigger apoptosis or necroptosis, respectively, and
the representative morphologies induced by these types of cell
death were clearly different from that induced by FINs (online
supplementary figure 3C).
Lipid peroxidation is a defining event in ferroptosis. We
stained FIN- treated eosinophils with C11- BODIPY, a membrane-
targeted lipid ROS sensor, to detect changes in lipid ROS by
flow cytometry. Not surprisingly, the levels of lipid ROS were
significantly increased on FIN treatment (online supplementary
figure 3D). We then pretreated eosinophils with ferrostatin-1
(fer-1) or liproxstatin-1 (lip-1) to eliminate the accumulation of
lipid peroxides. Surprisingly, these lipid ROS inhibitors failed
to exert any protective effect against FIN- induced cell death in
eosinophils (figure 2E,F), although lipid ROS were effectively
abolished (online supplementary figure 3E). These results indi-
cate that lipid ROS accumulation by FINs may not be necessary
for FIN- induced cytotoxicity in eosinophils.
We confirmed the effects of these ferroptosis inhibitors in
classical ferroptosis- sensitive cells. Mouse embryonic fibroblasts
(MEFs), in which ferroptosis had been verified, were stimulated
with FINs at lethal concentrations. Cell death induced by FINs in
MEFs was almost completely suppressed by fer-1 or lip-1 (online
supplementary figure 3F), and lipid ROS were eliminated from
the cells (online supplementary figure 3G).
Ferroptotic cell death has been characterised by its depen-
dency on iron; thus, we investigated the effects of the iron
chelators deferoxamine (DFO) and ciclopirox olamine (CPX) on
FIN- induced cell death in eosinophils. Interestingly, both iron
chelators markedly reversed FIN- induced cell death in eosino-
phils (figure 2G,H). On the other hand, the addition of Fe(II)
to the eosinophil culture medium further augmented cell death
induced by FINs (figure 2I).
Altogether, these data indicate that FINs induce non- classical
cell death in eosinophils, which display the cardinal morpholog-
ical features of ferroptosis, and that this cell death is iron depen-
dent but independent of lipid ROS. We therefore suggest that
FIN- induced cell death of eosinophils is non- canonical ferro-
ptosis or ferroptosis- like cell death.
FINs induced ferroptosis-like cell death through cytosolic ROS
Since lipid ROS were not critical for FIN- induced cell death in
eosinophils, we next attempted to identify the other types of ROS
that might be involved. All eosinophils treated with FINs exhibited
increased CM- H2DCFDA fluorescence, indicating the production
of cytosolic ROS (figure 3A,B). Moreover, we examined the func-
tional requirement of cytosolic ROS during ferroptosis by treating
eosinophils with FINs in the presence of the ROS scavengers N- ace-
tylcysteine (NAC) or GSH. Notably, the addition of NAC or GSH
completely reversed RSL3- induced cell death and partly blocked
erastin- induced or ART- induced cell death (figure 3C,D). Indeed,
NAC and GSH could suppress cytosolic ROS accumulation induced
by all FINs, but fer-1 failed to decrease the generation of cytosolic
Figure 3 Ferroptosis- inducing agents (FINs) induced ferroptosis- like cell death through cytosolic reactive oxygen species (ROS). Eosinophils were
isolated from the peripheral blood of interleukin 5 transgenic mice. Annexin V−/PI− cells were defined as viable eosinophils. (A and B) Eosinophils
were cultured with FINs (erastin 30 µM, Ras- selective lethal small molecule 3 (RSL3) 2 µM, artesunate (ART) 100 µM). Cytosolic ROS production at
indicated times (6, 12 and 24 hours) was assessed by flow cytometry using CM- H2DCFDA. Representative histograms are shown in (A), and cumulative
data expressed relative to the control are represented in (B). (C and D) Effects of N- acetylcysteine (NAC; 5 mM) and glutathione (GSH; 5 mM) on the
lethality of FINs in eosinophils. All drug treatments were administered for 24 hours. (E) Ability of ferrostatin-1 (fer-1; 2 µM), NAC (5 mM) and GSH
(5 mM) to prevent accumulation of cytosolic ROS when used to cotreat FINs for 24 hours. All data are shown as mean±SEM, analysed by one- way
analysis of variance. CTL, control; PI, propidium iodide.
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ROS (figure 3E). In ART- treated eosinophils, we observed a time-
dependent increase in mitochondrial ROS production, whereas
no change was observed in erastin- treated or RSL3- treated cells
(online supplementary figure 4A,B). However, MitoTEMPO, a
mitochondria- targeted antioxidant, exerted no appreciable effects
on the ART- induced cell death of eosinophils (online supplementary
figure 4C).
Altogether, these data suggest that FINs induce ferroptosis-
like cell death in eosinophils most likely through cytosolic ROS,
but not lipid or mitochondrial ROS, and that ferroptosis path-
ways in eosinophils differ from the canonical pathways previ-
ously identified in tumour cells.14 17
FINs induced ferroptosis-like cell death in eosinophils via
differential mechanisms and exerted a synergistic effect
Ferroptosis induced by erastin or RSL3 is due to GSH depletion
or reduced GPX4 activity, respectively15; however, little is known
about the mechanisms of ART in regulating of ferroptosis. Consis-
tent with previous reports, erastin time dependency decreased the
cellular levels of GSH in eosinophils, while the other two FINs
tested only slightly decreased GSH levels (figure 4A). Interestingly,
RSL3 time dependency attenuated the expression of GPX4 in eosin-
ophils, whereas neither erastin nor ART affected GPX4 expression
(figure 4B). As ART- triggered cell death was totally dependent on
iron, we hypothesised that ART generates a Fenton- type reaction
to initiate oxidative damage in eosinophils. We then measured the
oxidation state of iron and observed that cellular Fe(II) levels were
modestly reduced with ART treatment, while erastin- treated and
RSL3- treated cells showed no change in the abundance of Fe(II)
(figure 4C).
As erastin, RSL3 and ART likely induce eosinophil death
through different intracellular signals, we stimulated eosinophils
with these three compounds in pairs. Every pair of compounds
exerted a synergistic effect (figure 4D), laterally suggesting that
these three compounds induce ferroptosis- like cell death in
eosinophils via distinct mechanisms.
FINs attenuated allergic airway inflammation and induced
eosinophil death in vivo
We next questioned whether facilitating the ferroptosis- like cell
death of eosinophils would attenuate allergic airway inflammation
in vivo. In the classical OVA- induced model of allergy, FINs were
administered 2 hours after each OVA challenge, except ART was
also treated for 3 days before the first challenge. BALF and lung
tissues were collected 24 hours after the last administration of FINs
(online supplementary figure 5A). Significantly decreased infiltra-
tion of total BAL cells and eosinophils was observed in the FIN-
treated group (figure 5A–C). H&E staining further revealed that the
accumulation of airway inflammatory cells in the peribronchiolar
and perivascular regions was clearly reduced in FIN- treated mice
(figure 5D,E). The mRNA levels of Il13 or Il25 were also dramat-
ically decreased by FIN treatment (figure 5F). Moreover, mucus
hyperproduction induced by OVA was dramatically attenuated in
response to FIN treatment, as evidenced by periodic acid- schiff
staining (online supplementary figure5B,C). Each compound alone
exhibited no appreciable toxicity in mouse lungs (figure 5A–F), and
mouse weight and BAL protein levels were not affected by FINs
(online supplementary figure 5D,E), indicating the inappreciable
pulmonary toxicity of these FINs in vivo.
To further examine possible eosinophil cell death in vivo,
we acquired BAL cells from allergic mice treated with FINs or
vehicle control. BAL cells were collected and subsequently stained
with anti- CD11c and anti- Siglec- F (online supplementary figure
5F). As expected, FIN treatment led to reduced cell viability
(Annexin V−/4',6- diamidino-2- phenylindole−) of BAL eosinophils
(figure 5G,H). In addition, BAL eosinophils from allergic mice
treated with FINs exhibited morphological changes indicative of
ferroptosis (shrunken and damaged mitochondria) (figure 5I).
Synergistic effect of FINs with DXMS in vitro and in vivo
GCs such as betamethasone or DXMS are conventionally used as
an effective anti- inflammatory therapy for asthma, and the partial
anti- inflammatory effects of GCs have been ascribed to their ability
to facilitate eosinophil apoptosis.26 As expected, DXMS induced
eosinophil death in a concentration- dependent manner, which
could be effectively prevented by Z- VAD- FMK (figure 6A,B).
However, DXMS- induced eosinophil death was not responsive to
iron chelation or supplementation. In addition, fer-1 also failed to
protect eosinophils from DXMS- induced cell death (figure 6C).
These results suggest that DXMS- induced death of eosinophils
is likely apoptosis. A recent study found that the combination of
ferroptosis and apoptosis serves as a promising modality to improve
anticancer treatment efficacy.27 As long- term use of GCs in high
Figure 4 Ferroptosis- inducing agents (FINs) induced ferroptosis-
like cell death in eosinophils via differential mechanisms and exerted
synergistic effects. Eosinophils were isolated from the peripheral blood
of interleukin 5 transgenic mice and cultured with FINs (erastin 30 µM,
Ras- selective lethal small molecule 3 (RSL3) 2 µM, artesunate (ART)
100 µM). (A) Total glutathione (GSH) levels were assessed over the
indicated duration. (B) Glutathione peroxidase 4 (GPX4) protein at
indicated times (3, 6, 9 and 12 hours) was assessed by western blot
analysis. Actin beta (ACTB) was used as a loading control. (C) Cellular
Fe(II) levels at indicated times (6 and 12 hours) were assessed. (D)
Synergistic effect of FINs (erastin 20 µM, RSL3 1.5 µM, ART 50 µM)
on the cell viability of eosinophils administered in pairs for 24 hours.
Annexin V−/PI− cells were defined as viable eosinophils. All data are
shown as mean±SEM, analysed by one- way analysis of variance. CTL,
control; Fe(II), ferrous iron; PI, propidium iodide.
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Asthma
doses is associated with considerable side effects, we were therefore
interested in whether FINs could synergize with GCs to induce cell
death in eosinophils. To investigate the efficacy of FINs and DXMS
combined, we administrated both FINs and low dose of DXMS
(0.4 mM) to eosinophils in vitro. Intriguingly, eosinophil death was
markedly enhanced when the eosinophils were cotreated with FINs
and DXMS, indicating the apparent synergistic effect of FINs and
DXMS (figure 6D–F).
As ART is now widely used for clinical treatment and is well
tolerated, we studied the possible synergistic effect of ART with
Figure 5 Ferroptosis- inducing agents (FINs) attenuated allergic airway inflammation and induced eosinophil death in vivo. (A) Total bronchoalveolar
lavage (BAL) cell counts. (B) BAL eosinophil percentage. (C) BAL eosinophil counts. (D) Representative lung tissue sections stained with H&E. (E)
Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. (F) Cytokine levels in lung tissues
were determined by qPCR. (G) The percentage of viable cells of total BAL eosinophils was determined by flow cytometry. Siglec- F+/CD11c−/annexin
V−/4',6- diamidino-2- phenylindole (DAPI−) cells were distinguished as viable BAL eosinophils. (H) Viable BAL eosinophil counts. (I) Transmission
electron microscopy of BAL eosinophils from allergic mice treated with control or FINs. Single red arrowheads point to normal mitochondria; paired
red arrowheads point to damaged mitochondria. All data are shown as the mean±SEM of six to eight mice per group, analysed by one- way analysis
of variance (A, B, C, E, F) or Student’s t- test (G, H). ART, artesunate; BALF, bronchoalveolar lavage fluid; CTL, control; DMSO, dimethyl sulfoxide; Il,
interleukin; NS, normal saline; OVA, ovalbumin; RSL3, Ras- selective lethal small molecule 3.
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Asthma
DXMS in vivo. ART and DXMS at doses of 10 and 0.25 mg/
kg/day, respectively, were injected intraperitoneally alone or in
combination. Interestingly, the infiltration of total BAL cells
and eosinophils in the combination group was lower than that
in the groups treated with either ART or DXMS (figure 6G–I).
Consistently, levels of the TH2- related cytokines Il4 and Il13,
as measured by real- time quantitative PCR, were more signifi-
cantly decreased in the combined treatment group (figure 6J).
Figure 6K summarises the potential synergistic effects of FINs
with GCs in attenuating allergic airway inflammation.
DISCUSSION
In this study, we demonstrated that FINs induced the
ferroptosis- like cell death of eosinophils in a time- dependent
and concentration- dependent manner. However, unlike the
Figure 6 Ferroptosis- inducing agents (FINs) showed a synergistic effect with dexamethasone (DXMS) in vitro and in vivo. Eosinophils were isolated
from the peripheral blood of interleukin (Il) 5 transgenic mice. Annexin V−/PI− cells were defined as viable eosinophils. (A) Eosinophils were cultured
with various concentrations of DXMS for 24 hours. (B and C) Effects of Z- VAD- FMK (100 µM), deferoxamine (DFO; 100 µM), ferrous iron (Fe(II);
200 µM) and ferrostatin-1 (fer-1; 2 µM) on the cell viability of eosinophils treated with DXMS (0.4 mM) for 24 hours. (D–F) Viability of eosinophils
coadministrated FINs (erastin 30 µM, Ras- selective lethal small molecule 3 (RSL3) 2 µM, artesunate (ART) 100 µM) and DXMS (0.4 mM) at indicated
times (12 and 18 hours) was assessed by flow cytometry. (G–J) ART (10 mg/kg) and DXMS (0.25 mg/kg or 0.5 mg/kg) were administered alone or in
combination (ART 10 mg/kg and DXMS 0.25 mg/kg), with acquisition and analysis 24 hours after the last administration. Total bronchoalveolar lavage
(BAL) cell counts (G), BAL eosinophil percentage (H), BAL eosinophil counts (I) and cytokine levels in lung tissues determined by qPCR (J) are shown.
(K) Schematic summarising the synergistic effects of FINs with glucocorticoids in allergic airway disease. All data are shown as mean±SEM, analysed
by one- way analysis of variance. CTL, control; PI, propidium iodide.
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Asthma
ferroptosis of engineered tumour cells, the ferroptosis of eosin-
ophils occurred via a non- canonical pathway that was iron
dependent and likely required cytosolic ROS, but not lipid or
mitochondrial ROS, to trigger cell death. We further demon-
strated that promoting the ferroptosis- like cell death of eosin-
ophils relieved allergic airway inflammation in mice. Moreover,
FINs exerted a synergistic effect when combined with DXMS
in vitro, and the combined administration of ART and DXMS
improved the therapeutic effect and reduced the required dosage
of DXMS in vivo.
Eosinophils are thought to play important roles in both the
maintenance of tissue homeostasis and exacerbation of disease
in allergic diseases.28 Timely resolution of inflammation is essen-
tial for the host to prevent severe tissue damage and regain
homeostasis. Otherwise, non- resolving inflammation can lead
to host tissue injury and organ failure.2 Of note, it is increas-
ingly recognised that eosinophils at an inflammatory site have
a prolonged life span, which may impede the timely clearance
of inflammatory cells and delay the resolution of airway inflam-
mation.8 Recently, pharmacological agents targeting eosinophil
apoptosis have been studied, and consequent benefits were
observed in preclinical models of inflammation.29 30 Thus, treat-
ments that specifically promote eosinophil resolution are likely
to be effective in controlling allergic inflammatory diseases.
Ferroptosis is a non- apoptotic form of cell death that was
recently identified during exposure to FINs, including erastin
and RSL3, and other clinical drugs, including ART.31 Emerging
studies suggest that FINs exhibit strong antitumour activity in
different cancer cells.32 We first showed here that FINs could
effectively induce ferroptosis- like cell death in eosinophils,
which eventually protected against allergic airway inflamma-
tion in vivo. This study may provide a novel strategy for airway
inflammation therapy similar to other antieosinophil therapies,
such as GCs or anti- IL5 treatment. More importantly, we found
that FINs showed a synergistic effect with DXMS in triggering
eosinophil death in vitro. The drug combination of ART and
DXMS also obviously improved the therapeutic effect of steroids
in vivo. Therefore, treatments that specifically target eosinophil
ferroptosis seem to be effective in controlling allergic airway
inflammation and may reduce the dose and adverse effects of
GCs. ART, a drug for falciparum malaria, shows remarkable
safety and is widely used in the clinic. Thus, our results suggest
that combined ferroptosis- apoptosis therapy might serve as a
safe and effective treatment for allergic airway inflammation.
Although erastin, RSL3 and ART serve as canonical ferro-
ptosis inducers, accumulating evidence indicates that the mode
of cell death may vary in different cell types.33 Ferroptosis is
distinct from known forms of cell death and characterised by the
iron- dependent accumulation of ROS, especially lipid ROS, to
lethal levels.14 17 We found that erastin- induced or RSL3- induced
eosinophil death could not be prevented by small- molecule inhib-
itors of apoptosis, necroptosis or autophagy. However, we found
that eosinophil death triggered by FINs was iron dependent, as
it was inhibited by the presence of the iron chelators DFO and
CPX. Mitochondrial changes typical of cell ferroptosis were
observed in FIN- treated eosinophils, but few morphological
characteristics of other types of cell death were found. There-
fore, we assumed that the form of cell death induced by FINs
in eosinophils was non- canonical ferroptosis or ferroptosis- like
cell death. Nonetheless, we verified that these FINs could trigger
typical ferroptosis in MEFs, which was dependent on lipid ROS.
Thus, our results suggest that the mechanisms of FIN- triggered
ferroptosis are cell type specific and provide a paradigm showing
that ferroptosis is not always dependent on lipid ROS.
Ferroptosis is related to metabolic dysfunction that leads to
the accumulation of both cytosolic and lipid ROS, independent
of mitochondria.14 It is possible that different types of ROS are
involved in mediating cell death in response to lethal stimula-
tion with different FINs. We found that cytosolic ROS were
induced by FINs in eosinophils and that FIN- stimulated cyto-
solic ROS production and cell death could be reversed by NAC
or GSH. However, although mitochondrial ROS accumulated in
only ART- treated eosinophils, they were not essential for ART
lethality, as MitoTEMPO failed to protect against ART- induced
cell death. Thus, the initiation of ferroptosis in eosinophils seems
to be solely dependent on cytosolic ROS.
Previous studies have reported that the antimalarial mech-
anism of ART is Fe(II) dependent, as Fe(II) can cause the
cleavage of an endoperoxide bridge in ART and lead to the
generation of ROS.34 In addition, ART derivatives have been
shown to induce iron- dependent programmed ferroptosis in
tumour cells.35 36 Consistently, in our study, we observed that
ART- induced ferroptosis- like cell death of eosinophils was iron
dependent. Moreover, it is worth noting that autophagy is asso-
ciated with ferroptosis in eosinophils following ART treatment
but does not contribute to erastin- induced or RSL3- induced
ferroptosis. Autophagy has been shown to contribute to ferro-
ptosis by degrading ferritin in fibroblasts and cancer cells.37
Increased ferritin expression is thought to limit ferroptosis.38
Thus, increased autophagy might increase iron levels leading to
oxidative injury by the Fenton reaction.
In conclusion, our study suggests that induction of the
ferroptosis- like cell death of eosinophils might be a promising
therapeutic strategy for allergic airway inflammation, especially
due to the advantage of its synergy with GCs in the treatment of
eosinophil- related disorders.
Acknowledgements The authors thank James J Lee (Department of Biochemistry
and Molecular Biology, Mayo Clinic, USA) for the generous gift of Il5 Tg mice. They
thank for the technical support by the Core Facilities, Zhejiang University School
of Medicine. They thank Chenyu Yang and Beibei Wang in the Centre of Cryo-
Electron Microscopy (CCEM), Zhejiang University for their technical assistance on
Transmission Electron Microscopy.
Contributors ZC, HS and WL designed and supervised the study. YW, HC, NX, LZ,
YW, CZ, ML, QW, JS, ZL, YZ and MW performed experiments. XX, HZ, BZ, FL and LX
assisted in the collection of human samples. YW, HC and ZC prepared figures and
drafted manuscript. SY, WL, HS and ZC analysed data and revised manuscript. All
authors approved the final manuscript.
Funding This work was supported by the State Key Program (2016YFA0501802 to
ZC) from Ministry of Science and Technology of the People’s Republic of China, and
the Key Project (81930003 to HS), the Major Research plan (91642202 to WL) and
the General Program (81873403 to WL) from National Natural Science Foundation
of China.
Competing interests None declared.
Patient consent for publication Not required.
Ethics approval This study was approved by the Ethics Committee of the Second
Affiliated Hospital of Zhejiang University School of Medicine.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement All data relevant to the study are included in the
article or uploaded as supplementary information.
ORCID iD
ZhouyangLi http:// orcid. org/ 0000- 0002- 7721- 0175
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