Caspase-4: A Therapeutic Target for Peptic Ulcer Disease
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Caspase-4: A Therapeutic Target for Peptic Ulcer Disease
Zbigniew Zaslona, Ewelina Flis, Ciara Nulty, Jay Kearney, Rebecca Fitzgerald, Atiyekeogbebe R.
Douglas, Deirdre McNamara, Sinead Smith, Luke A. J. O'Neill and Emma M. Creagh
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ImmunoHorizons 2020, 4 (10) 627-633
doi: https://doi.org/10.4049/immunohorizons.2000080
http://www.immunohorizons.org/content/4/10/627
This information is current as of August 19, 2021.
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ISSN 2573-7732.RESEARCH ARTICLE
Innate Immunity
Caspase-4: A Therapeutic Target for Peptic Ulcer Disease
Zbigniew Zaslona,*,1 Ewelina Flis,*,1 Ciara Nulty,* Jay Kearney,* Rebecca Fitzgerald,† Atiyekeogbebe R. Douglas,†
Deirdre McNamara,†,‡ Sinead Smith,† Luke A. J. O’Neill,* and Emma M. Creagh*
*School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland; †Department of Clinical
Medicine, School of Medicine, Trinity College Dublin, Dublin 2, Ireland; and ‡Department of Gastroenterology, Tallaght University Hospital, Dublin
D24 NR04, Ireland
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ABSTRACT
Peptic ulcers are caused by the interaction between bacterial and host factors. This study demonstrates enhanced expression of
caspase-4 in peptic ulcer patient biopsies, indicating that pyroptosis and noncanonical inflammasome activity may be processes
involved in peptic ulcer disease. We show that primary murine macrophages infected with Helicobacter pylori upregulate caspase-11
(the ortholog of human caspase-4), activate caspase-1, and secrete IL-1b. We demonstrate that misoprostol (a stable PGE1 analogue)
decreased IL-1b secretion and delayed lethality in vivo in a murine peritonitis model. PGE2 was shown to inhibit caspase-11–driven
pyroptosis and IL-1b secretion in macrophages. Overall, we provide evidence for a pathological role of caspase-4/11 in peptic ulcer
disease and propose that targeting caspase-4 or inhibiting pyroptosis may have therapeutic potential in the management of peptic
ulcers. ImmunoHorizons, 2020, 4: 627–633.
INTRODUCTION plays an essential role (12, 13). As caspase-4 expression is induced
in asthmatic patients and correlates with disease severity, this
Several cell death mechanisms are capable of fueling inflamma- suggests that its inhibition may have therapeutic potential (13, 14).
tory responses, causing tissue damage and contributing to the In this study, we report that caspase-4 is induced in peptic ulcer
pathologic condition of inflammatory disease (1, 2). Pyroptosis is tissue, suggesting that noncanonical inflammasome activation and
a highly inflammatory type of cell death that is triggered by pyroptosis are processes participating in ulcer formation. Caspase-4
the pore-forming N-terminal cleavage fragment of gasdermin D has previously been linked to decreased adenosine deaminase activity
(GSDMD) (3). Inflammatory caspases (caspase-1, -4, and -5 in and implicated as a factor inducing gastric ulcers (15). Immune cells,
humans; caspase-1 and -11 in mice), are responsible for GSDMD such as macrophages, contribute to peptic ulcer formation through
cleavage in mice and humans (3–6). Caspase-11, and its human bacterial-induced inflammation (16). Helicobacter pylori infection
orthologs caspase-4 and -5, can directly bind cytosolic LPS from alters the gastric microbiome (17) and induces inflammatory
Gram-negative bacteria, leading to GSDMD processing, responses by recruiting immune cells, including macrophages,
pyroptosis, and NLRP3-dependent activation of capase-1, known dendritic cells, neutrophils, and lymphocytes (18, 19). The main
as the noncanonical inflammasome (6–8). Murine caspase-11 is an cytokine involved in modulating the inflammatory response
essential mediator of sepsis (8). Although originally limited to bacterial during ulcer disease is IL-1b (20).
infection, pyroptosis has recently been implicated in the pathogenesis Perforated peptic ulcers can lead to peritonitis and, subsequently,
of disorders, including asthma and psoriasis (9–12), in which caspase-11 sepsis—a common emergency condition worldwide with associated
Received for publication September 16, 2020. Accepted for publication September 18, 2020.
Address correspondence and reprint requests to: Dr. Zbigniew Zaslona, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Room
4.32, Dublin 2, Ireland. E-mail address: zaslonaz@tcd.ie
ORCIDs: 0000-0003-1889-0171 (C.N.); 0000-0002-0684-0436 (J.K.); 0000-0002-7056-0682 (R.F.); 0000-0001-7631-4370 (E.M.C.).
1
Z.Z. and E.F. contributed equally to the study.
This work was supported by the European Union Horizon 2020 Research and Innovation Programme (Marie Skłodowska-Curie Actions Grant Agreement 721906).
Abbreviations used in this article: BMDM, bone marrow–derived macrophage; Casp-112/2, caspase-11–deficient; DAMP, damage-associated molecular pattern;
GSDMD, gasdermin D; LDH, lactate dehydrogenase; NSAID, nonsteroidal anti-inflammatory drug; WT, wild-type.
This article is distributed under the terms of the CC BY 4.0 Unported license.
Copyright © 2020 The Authors
https://doi.org/10.4049/immunohorizons.2000080 627
ImmunoHorizons is published by The American Association of Immunologists, Inc.628 CASPASE-4 EXPRESSION IN GASTRIC ULCERS ImmunoHorizons
mortality rates of up to 30% (21). This study demonstrates that Patient samples
PGE2 inhibits IL-1b production and delays mortality in a murine The study was conducted after ethical approval was granted from
model of LPS-induced peritonitis. We show that PGE2 inhibits the St. James’ Hospital/Adelaide and Meath hospital, incorporat-
caspase-11–driven pyroptosis in macrophages, which can also ing the National Children’s Hospital Research Ethics Committee
subsequently limit canonical and noncanonical inflammasome in accordance with European Communities (Clinical Trials on
activation to effectively block the inflammatory cascade. Our in Medicinal Products for Human Use) regulations 2004 and
vivo data provide a possible explanation for the beneficial effects International Conference on Harmonisation – Good Clinical
of misoprostol (PGE1 analogue) observed in the management of Practice guidelines. Patients participating in the study were
peptic ulcers. Because misoprostol has side effects and is poorly between 18 and 80 y of age, referred by their treating clinician
tolerated (22), we suggest that caspase-4 inhibition represents for a gastroscopy based on standard clinical assessment. Peptic
a novel therapeutic strategy for the management of peptic ulcers and biopsies were taken at the side of the antral ulcer, and antral
the prevention of ulcer perforation and its associated complications. biopsies from unaffected patients served as controls. Patients
with a known history of peptic malignancy (carcinoma or
lymphoma), stromal tumor, or previous upper gastrointestinal
MATERIALS AND METHODS
surgery, as well as those with the endoscopic description of a
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mass, polyp, nodule, or lump anywhere in the esophagus, stomach,
Mice
or small intestine, were excluded from the study. Biopsies were
Casp112/2 and wild-type (WT) mice were bred under specific
anonymized and stored at 280°C until analysis. Procedures had no
pathogen-free conditions and under license and the approval of
effect on patient care, and primary consultant interactions were
the local animal research ethics committee and the Irish Health
carried out as normal.
Protection Regulatory Agency.
Cell culture List of Abs for Western blot
WT and Casp112/2 bone marrow–derived macrophages (BMDM) b-Actin (1:15,000, AC-74; Sigma-Aldrich), IL-1b (pro–IL-1b and
were isolated and cultured in 20% L929 media until day 6, after cleaved; 1:1000, AF-401; R&D Systems), caspase-11 (1:1000, 17D9;
which they were plated for experimentation. THP-1 monocytes Sigma-Aldrich), caspase-1 p20 (Asp297; 1:1000, D57A2; Cell Signaling
were differentiated into macrophages with 10 ng/ml PMA for 72 h Technology), pro–caspase-1 p45 (A-19, 1:2000, sc-622; Santa
prior to H. pylori infection. Cruz Biotechnology), caspase-4 (1:1000, no. 4450; Cell Signaling
FIGURE 1. Caspase-4 expression is increased in patient ulcers and following H. pylori infection in macrophages.
(A) Caspase-4 and b-actin levels in biopsy tissue from control and antrum ulcer patients were assessed by Western blot, and (B) subsequent
densitometric analysis of caspase-4 is presented relative to actin as a housekeeping gene, n = 5. *p , 0.05, two-tailed Student t test. (C) Relative
mRNA expression levels of caspase-4 in gastric epithelium (GE) biopsies taken from the antrum region of H. pylori (HP)-infected patients with
normal, mild, and severe gastritis were analyzed using the published dataset GSE60427. Mann–Whitney U test for nonparametric data were used to
evaluate significant differences between the groups. *p , 0.05.
https://doi.org/10.4049/immunohorizons.2000080ImmunoHorizons CASPASE-4 EXPRESSION IN GASTRIC ULCERS 629
Technology), and HRP-conjugated secondary Abs were from inflammation observed in the gastric corpus (23). We observed
Jackson ImmunoResearch Laboratories. increased caspase-4 expression in gastric antrum biopsies from
ulcer patients, compared with healthy patients (Fig. 1A, 1B).
ELISA Moreover, dataset analysis reveals that CASP4 mRNA expres-
IL-1b concentrations in supernatants were measured by ELISA sion in antrum biopsies correlates with disease severity in
(R&D Systems) or by MesoScale Diagnostics according to the H. pylori–infected patients (Fig. 1C). These findings confirm
manufacturer’s instructions. previously reported findings, suggesting that caspase-4 con-
tributes to the inflammation and mucosal damage that occur
Lactate dehydrogenase assay during ulcer formation (15).
Lactate dehydrogenase (LDH) release into the cell culture
supernatants was determined using CytoTox 96 Non-Radioactive Caspase-11 regulates caspase-1 activity and IL-1b
Cytotoxicity Assay Technical Bulletin (Promega) according to the production in H. pylori–infected macrophages
manufacture’s protocol. H. pylori is one of the most common risk factors for peptic ulcer
disease, with colonization occurring predominantly in the gastric
Statistical analysis antrum (24). To determine whether H. pylori can induce caspase-4
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Data are presented as mean 6 SEM. Statistical significance was expression in macrophages, mRNA levels of caspase-4 and
analyzed using the GraphPad Prism 5.0 statistical program pro–IL-1b were determined in THP-1 cells infected over a 24-h
(GraphPad Software, La Jolla, CA). Datasets were analyzed using time course. Results revealed that H. pylori infection significantly
the parametric Student t test or ANOVA. Data were considered increased caspase-4 and pro–IL-1b expression (Fig. 2A, 2B).
significant when *p , 0.05, **p , 0.01, and ***p , 0.001. Caspase-11 is the murine ortholog of caspase-4 (25); therefore, to
assess the importance of caspase-4 during H. pylori infection, we
compared the effects of infection in primary macrophages from
RESULTS WT and caspase-11–deficient (Casp-112/2) mice. H. pylori–infected
BMDM from Casp-112/2 mice had an impaired ability to upregulate
Increased caspase-4 expression in patient ulcer biopsies pro–IL-1b and expressed lower levels of cleaved caspase-1 p10 when
The majority of gastric ulcers are situated in the gastric antrum, as compared with WT BMDM (Fig. 2C). Importantly, mature IL-1b,
gastritis is usually more severe in the antrum, with little or no which contributes to gastric ulcer pathogenesis (26), was also
FIGURE 2. Caspase-4 expression is increased following H. pylori infection in macrophages.
(A and B) PMA-differentiated THP-1 macrophages were H. pylori NCTC11638 (multiplicity of infection [MOI] 100) infected for 0–24 h, as indicated,
prior to RNA extraction and RT qPCR determination of (A) caspase-4 and (B) IL-1b mRNA levels. Changes in expression were evaluated using the
comparative cycle threshold method. Results were normalized to GAPDH levels and expressed relative to uninfected cells. Paired Student t test was used to
compare gene expression in cell culture samples. *p , 0.05, **p , 0.01. (C and D) H. pylori strain NCTC11638 infection (MOI 100) of WT and Caspase-112/2
(C112/2) BMDM for 0–48 h, as indicated. (C) Inflammatory caspase-1 and -11, pro–IL-1b, and b-actin levels were determined by Western blot. (D) Secreted
levels of IL-1b were determined by ELISA (MesoScale Discovery). Values represent the mean 6 SEM; two-way ANOVA was used to compare mean values WT
and C112/2 BMDM. Blots shown are representative of three independent experiments. **p , 0.01. HP, H. pylori strain NCTC11638.
https://doi.org/10.4049/immunohorizons.2000080630 CASPASE-4 EXPRESSION IN GASTRIC ULCERS ImmunoHorizons
significantly decreased in Casp-112/2 BMDM after H. pylori infection Caspase-11 regulates caspase-1 activity and
(Fig. 2D). We have previously shown that the PGE1 analogue, IL-1b production
misoprostol, decreases caspase-11 expression in vivo (13). Caspase-11–mediated pyroptosis causes the release of damage-
associated molecular patterns (DAMPs) and ATP, which can
PGE2 inhibits IL-1b production and pyroptosis in a murine subsequently activate NLRP3 and caspase-1, which will serve to
model of peritonitis further boost pyroptosis levels. PGE2 would therefore have
We have initially verified that H. pylori is capable of inducing potential to limit this process based on its well-established tissue
caspase-11 expression, and more importantly, this induction is protective characteristics (28). We designed an experiment to
completely inhibited by PGE2 treatment (27) (Fig. 3A). Peritonitis determine whether PGE2 could limit DAMP production and
is a complication that occurs following ulcer perforation. Non- thereby indirectly inhibit NLRP3 and caspase-1 activation (Fig.
canonical inflammasome activation is driven by caspase-11, rather 4A). WT BMDM were pretreated with PGE2 or vehicle before a
than canonical caspase-1, and is responsible for LPS-induced 4-h LPS prime and subsequent LPS transfection to activate the
mortality in a murine model of peritonitis. PGE2 is an inhibitor of noncanonical, caspase-11–dependent pathway. To support the
caspase-11–driven pyroptosis (13). Given the clinically proven hypothesis that protective effects of PGE2-driven caspase-11
protective role of the stable PGE1 analogue, misoprostol, against inhibition can indirectly attenuate canonical inflammasome
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ulcers, we tested its effects in a peritonitis model. We observed that (caspase-1) activation, Casp-112/2 BMDM were similarly treated
mice treated with misoprostol before LPS challenge had significantly to compare their responses against PGE2-treated WT BMDM.
decreased IL-1b serum levels and delayed LPS-induced lethality In vitro, canonical inflammasome activation requires LPS
(Fig. 3B, 3C). To confirm that the protective effects of PGE2 are priming, followed by addition of ATP or nigericin to imitate
mediated by its ability to inhibit pyroptosis, LPS-primed DAMPs. Western blotting revealed that supernatants from PGE2-
BMDM (to upregulate caspase-11 expression) were pretreated pretreated and LPS-primed and -transfected WT BMDM (donors),
with PGE2 and subsequently transfected with LPS (to activate incubated with LPS-primed WT BMDM (recipients), caused
caspase-11). Consistent with previous findings (10), data revealed significantly lower secretion levels of mature IL-1b and processed
that PGE2 inhibited caspase-11–induced pyroptosis (Fig. 3D). caspase-1 (p10) when compared with donor supernatants from
FIGURE 3. PGs inhibit caspase-11 upregulation, IL-1b production, and pyroptosis in mice.
(A) BMDM were pretreated with 1 mM PGE2 for 30 min and then treated with 10 ng/ml LPS from H. pylori NCTC11637 for 4 h. Caspase-11 expression
was assessed in protein lysates by Western blot. (B) Mice (n = 6 per group) were i.p. injected with 100 ml saline containing 1% DMSO or 50 mg misoprostol
in 1% DMSO (misoprostol). Following 2 h, half of the mice from each group were administered LPS (2.5 mg/ml, i.p. injection) or PBS. All mice were
sacrificed 90 min later, and blood serum was analyzed for IL-1b by ELISA. Data are representative from three independent experiments. *p , 0.05. (C) Mice
(n = 15 per group) were i.p. injected with 100 ml saline containing 1% DMSO or 50 mg misoprostol in 1% DMSO. Following 2 h, mice were administered LPS
(i.p., 25 mg/ml), and survival was monitored over 72 h. (D) Murine BMDM were primed with LPS (100 ng/ml, 4 h) prior to 30 min pretreatment with PGE2
(1 mM) or DMSO. Cells were transfected with LPS (2 mg) using FuGENE 0.25% v/v liposomes o/n. Supernatants were collected and analyzed for cell death
using LDH assay. Data shown as percentage of total cell death from three independent experiments, each using three mice per group. Data are represented
as means 6 SEM. Statistical analysis was performed using unpaired two-tail Student t test to compare mean values. ***p , 0.001.
https://doi.org/10.4049/immunohorizons.2000080ImmunoHorizons CASPASE-4 EXPRESSION IN GASTRIC ULCERS 631
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FIGURE 4. PGE2 inhibits IL-1b production and pyroptosis by inhibiting the noncanonical inflammasome in murine macrophages.
(A) Schematic representation of the experiment designed to explore paracrine caspase-11 signaling to mediate pyroptosis. WT and Casp-112/2
BMDM were pretreated with PGE2 or vehicle (DMSO), primed with 100 ng/ml LPS for 4 h, and subsequently transfected with 2 mg LPS to generate
supernatants (donors, supernatant [Sup.]A1–Sup.D1). A separate plate of LPS-primed (4 h) WT BMDM (recipients) were treated with Sup.A1–Sup.D1,
and the resulting lysates and supernatants (Sup.A2–Sup.D2) were assayed for inflammatory and cell death readouts (n = 3). (B) Western blot analysis
of recipient BMDM was performed to determine the levels of procaspase-1, pro–IL-1b, and actin (loading control) in lysates from LPS-primed WT
BMDM treated with donor Sup.A1 or Sup.B1 alongside relevant control treatments (PGE2, LPS prime [pLPS], or tLPS treatments alone or combined).
Levels of mature IL-1b (p17) and caspase-1 (p10) in supernatants from the same experiment. (C) LDH assay was performed on Sup.A2–Sup.D2 to
determine cell death levels. Statistical analysis was performed using two-way ANOVA test to compare mean values. *p , 0.05, **p , 0.01. (D) IL-1b
levels in supernatants (Sup.A2 and Sup.B2) were determined using ELISA. Data represent mean 6 SEM of three independent experiments. Statistical
analysis was performed using unpaired two-tail Student t test to compare mean values. ***p , 0.001.
similarly stimulated WT BMDM that received no PGE2 pre- with H. pylori and associated Gram-negative microbiota activates
treatment (Fig. 4B; compare LPS prime [pLPS] 1 supernatant pyroptosis in ulcers of damaged and infected gastric mucosa. A
[Sup.]A1 with pLPS + Sup.B1). Importantly, PGE2 alone did not hypothesis that combination of H. pylori and other bacteria,
affect IL-1b secretion (Fig. 4C). Fig. 4C revealed that recipient possibly Escherichia coli infection, activates caspase-4 and
LPS-primed WT BMDM exhibited significantly less cell death pyroptosis was suggested by our in vitro study that demonstrated
when incubated with donor supernatants from PGE2-treated WT that LPS from H. pylori alone was not able to induce caspase-4
(compare A2 with B2) or Casp-112/2 BMDM (compare A2 with expression or activation; however, in vivo biopsies from patients
C2). The inhibitory effect of PGE2 on IL-1b secretion was also with gastric ulcers exhibited a clear induction of caspase-4
confirmed by ELISA (Fig. 4D). These data confirm that PGE2 can expression. Therefore, a model is likely in which H. pylori may
indirectly limit NLRP3 and caspase-1–dependent IL-1b pro- sensitize macrophages for caspase-4 activation by other infectious
duction. We therefore propose that in vivo inhibition of caspase-11 bacteria or even commensals capable of effectively activating
will effectively attenuate the inflammatory and pyroptotic cas- caspase-4–driven noncanonical pyroptosis. Inhibiting inflamma-
cades that occur during ulcer development or perforation. tory cascades that lead to abdominal and systemic inflammation in
this context is critically important, as peptic ulcer complications
include perforations that lead to peritonitis and often sepsis.
DISCUSSION Although an anti-inflammatory role for PGE2 in tissue homeostasis
has been known for years (32), the mechanism underlying its tissue
Caspase-11/4 expression and functions are studied in the context protective activity has not been fully elucidated. We have shown
of bacterial infection and LPS recognition (29). Infection with that H. pylori induces caspase-11, mediating caspase-1 activation
H. pylori is associated with peptic ulcers (24) and is known to cause and IL-1b production. As LPS extracted from H. pylori is a weak
cell death (30), induce NLRP3 activation, and produce IL-1b (31). inducer of inflammatory responses (33), additional virulence factors
Although not yet reported, it is therefore likely that colonization are probably needed to induce inflammasome activation (18, 19).
https://doi.org/10.4049/immunohorizons.2000080632 CASPASE-4 EXPRESSION IN GASTRIC ULCERS ImmunoHorizons
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