Second-Generation H₁-Receptor Antagonists, Mequitazine, Azelastine and Desloratadine Activate Caspase-8, Caspase-3, and Gasdermin E and Induce Cell Death Showing Pyroptotic-Like Features in Macrophages at Excessively High Concentrations

Abstract

Histamine H₁ receptor (H₁R) is expressed in various cells, including neurons, smooth muscle cells, hepatocytes, T and B cells, neutrophils, dendritic cells, monocytes, and macrophages. Antagonists that target the H₁R are used to relieve the symptoms of allergy and inflammation. Here, we examined the inflammation-modulating and cell death-inducing effects of 10 second-generation H₁R antagonists on mouse intraperitoneal macrophages, which include ketotifen, mequitazine, azelastine, oxatomide, epinastine, bepotastine, fexofenadine, loratadine, levocetirizine, and desloratadine, to assess an anticipated adverse reaction. Three of these antagonists, namely, mequitazine, azelastine, and desloratadine, induced the secretion of interleukin-1α (IL-1α), a marker of pyroptosis and an inflammatory cytokine, from the macrophages at excessively high concentrations, while reducing the secretion of another inflammatory cytokine, IL-6. We found that the macrophages treated with the 3 antagonists showed pyroptotic-like, but not apoptotic-like, morphology and died. Western blotting analysis revealed that caspase-8, caspase-3, and gasdermin E (GSDME), but not gasdermin D, were cleaved and activated in the macrophages. These results suggest that second-generation H₁R antagonists such as mequitazine, azelastine, and desloratadine induce pyroptotic-like cell death accompanied by caspase-8, caspase-3, and GSDME activation in macrophages.

INTRODUCTION

Histamine is a major mediator of acute and chronic inflammatory responses and plays an important role in various pathophysiological conditions. Four types of histamine receptors, such as the H₁ receptor (H₁R), H₂R, H₃R, and H₄R, have been reported. Antagonists that target the H₁R are used to relieve the symptoms of allergy and inflammation. H₁R is expressed in various cells, including neurons, smooth muscle cells, hepatocytes, T and B cells, neutrophils, dendritic cells, monocytes, and macrophages.^1,2)

A few decades ago, cell death was mainly classified into 2 types: apoptosis and necrosis. Apoptosis is a regulated cell death (RCD) and is described as an active and autonomous cellular process without eliciting inflammation. Necrosis is characterized as a passive and accidental cell death with uncontrolled release of inflammatory cellular contents. At the end of the last century, researchers discovered other types of RCDs, such as necroptosis and pyroptosis, which show necrotic-like morphology with the release of inflammatory cellular contents. Both necroptosis and pyroptosis induce inflammation by releasing various damage/danger-associated molecular patterns (DAMPs), such as ATP, double-stranded DNA, single-stranded RNA, and high-mobility group box 1 (HMGB1), from dying cells.^3–5) A part of the mechanisms of these 2 RCDs has been revealed.^6–12) Necroptosis occurs in a variety of pathological conditions and is known to be initiated by RIP1/RIP3.^13,14) Pyroptosis is mainly induced by activation of the canonical inflammatory caspase-1 and noncanonical caspase-11 (caspase-4/5 in human), both of which are followed by cleavage of gasdermin D (GSDMD). The cleaved N-terminal fragment of GSDMD forms nonselective pores¹⁵⁾ and induces plasma membrane rupture and cell death. Recently, another type of pyroptosis has been reported. This newly reported pyroptosis involves the activation of caspase-8/caspase-3 and nonselective pore formation, which is constructed with the cleaved N-terminal fragment of gasdermin E (GSDME), a member of the gasdermin superfamily.^16–18) In contrast to the release of interleukin-1β (IL-1β) and IL-1α that accompanies GSDMD-mediated pyroptosis, GSDME-mediated pyroptosis is accompanied by the release of only IL-1α, but not IL-1β.¹⁹⁾

Macrophages exist in various tissues and have roles in development, homeostasis, tissue repair, and immunity, including cell death, in mammals. Accordingly, macrophages are involved in the pathophysiology of several diseases.^20–22) In the present study, we examined the inflammation-modulating and cell death-inducing effects of the second-generation H₁R antagonists on macrophages to assess an anticipated adverse reaction and found that some of the antagonists modulate the release of inflammatory cytokines and induce cell death showing pyroptotic-like features in macrophages.

MATERIALS AND METHODS

Reagents

Ketotifen Fumarate (Ket, K0048), mequitazine (Meq, M2807), azelastine hydrochloride (Aze, A2340), epinastine hydrochloride (Epi, E0799), bepotastine besilate (Bep, B5943), fexofenadine hydrochloride (Fex, F0698), loratadine (Lor, L0223), levocetirizine dihydrochloride (Lev, L0264), and desloratadine (Des, D3787) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Oxatomide (Oxa, O9387) and lipopolysaccharide (LPS, 0111 Escherichia coli B4, L2630) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). All H₁R antagonists were dissolved at a concentration of 100 mM in dimethyl sulfoxide (DMSO). When antagonists were used, the final concentration of DMSO was 0.1% in all cultures, including the controls.

Animal Experiments

Male C57BL/6J mice were purchased from SLC (Shizuoka, Japan) at 6–7 weeks of age, maintained under controlled temperature and light (23°C and 12-h light/dark, respectively), and used at 7–10 weeks of age; all of the animals received food and water ad libitum. All experimental procedures were in accordance with the guidelines for animal experimentation of the Animal Care and Use Committee of Matsuyama University (Matsuyama, Japan). The certification numbers verifying study approval were as follows: 24-005 on 2024.3.19. and 23-008 on 2023.3.30.

Mouse Primary Peritoneal Macrophage Culture

Mouse primary peritoneal macrophage culture was performed as described.²³⁾ Thioglycolate medium (3%) was prepared with BD BBL^TM Brewer’s modified thioglycolate medium (#211716, BD, Franklin Lakes, NJ, U.S.A.) and aged for over 1 month. To obtain peritoneal macrophages, 7–10-week-old male C57BL/6 mice were intraperitoneally injected with 1.3 mL of the 3% thioglycolate medium. Four days later, peritoneal macrophages were harvested as exudative macrophages and cultured at a density of 1.2 × 10⁵ cells/cm².

Morphological Analysis

Peritoneal macrophages were treated with test drugs for 2 h and observed with an All-in-One fluorescence microscope BZ-8000 (Keyence, Osaka, Japan). Hoechst 33342 (H33342) and propidium iodide (PI) were added to the cultures at a concentration of 15 μg/mL, 30 min before fluorescent observation.

Assays for Assessing Cell Viability and Cell Death

Peritoneal macrophages were treated with test drugs at the indicated concentrations and cultured for the indicated time. We used the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation kit (Roche Diagnostics, Basel, Switzerland) to assess the degree of cell viability, and the lactate dehydrogenase (LDH) assay kit (Cytotoxicity LDH Assay Kit-WST, DOJINDO, Kumamoto, Japan) to assess the degree of cell death, following the manufacturer’s instructions.

Western Blotting Analysis

Western blotting analysis was performed as described previously.²⁴⁾ The following antibodies were used: anti-GSDMD antibody (#39754, Cell Signaling, Danvers, MA, U.S.A.), anti-GSDME antibody (#88874, Cell Signaling), anti-caspase-3 antibody (#14220, Cell Signaling), anti-caspase-8 antibody (#4790, Cell Signaling), anti-HMGB1 antibody (#6893, Cell Signaling), anti-actin antibody (#4970, Cell Signaling), and anti-rabbit IgG antibody–horseradish peroxidase conjugate (#7074, Cell Signaling).

Enzyme-Linked Immunosorbent Assay (ELISA)

Peritoneal macrophages were treated with test drugs at the indicated concentrations and cultured for the indicated time. The culture media were harvested for ELISA. The concentrations of IL-6, IL-1α, and IL-1β in the culture media were measured with the Mouse IL-6 ELISA MAX Deluxe Set (#431304, BioLegend, San Diego, CA, U.S.A.), Mouse IL-1α ELISA MAX Deluxe Set (#433404), and Mouse IL-1β ELISA MAX Deluxe Set (#432604), respectively.

Statistical Analysis

Data were analyzed using one-factor ANOVA followed by Tukey’s multiple comparison test and are expressed as mean ± standard error of the mean. p < 0.05 was considered significant.

RESULTS

H₁R Antagonists Modulate the Release of Inflammatory Cytokines and Induce Pyroptotic-Like Cell Death

We 1st examined the inflammation-modulating effect of 10 second-generation H₁R antagonists on mouse intraperitoneal macrophages treated with LPS, which include (#1) Ket, (#2) Meq, (#3) Aze, (#4) Oxa, (#5) Epi, (#6) Bep, (#7) Fex, (#8) Lor, (#9) Lev, and (#10) Des (Fig. 1). Seven of the 10 antagonists, except for (#6) Bep, (#7) Fex, and (#9) Lev, reduced the release of an inflammatory cytokine, IL-6, while 3 of these 7 antagonists, such as (#2) Meq, (#3) Aze, and (#10) Des, at 20 μM induced the release of another inflammatory cytokine, IL-1α, from the macrophages. Interestingly, the 3 antagonists most intensively reduced the viability of macrophages.

Fig. 1. Effects of H₁R Antagonists on IL-6 and IL-1α Release from Macrophages Pretreated with LPS and Their Viability

(A) Chemical structures of H₁R antagonists. (#1) Ket, (#2) Meq, (#3) Aze, (#4) Oxa, (#5) Epi, (#6) Bep, (#7) Fex, (#8) Lor, (#9) Lev, and (#10) Des. (B and C) Macrophages were treated with the indicated concentrations of H₁R antagonists. One hour later, LPS was added. After 24 h of incubation, the supernatant was collected for measuring IL-6 (B) and IL-1α (C) levels by ELISA. (D) Macrophages were treated with the indicated concentrations of H₁R antagonists. After 2 h of incubation, the cultures were used for the assessment of cell viability with MTT reduction. The scale of the ordinate axis is repeated in the right panels of (B–D). Data are shown as mean ± S.E.M. (number of samples = 4). *p < 0.05.

Fig. 1. Effects of H₁R Antagonists on IL-6 and IL-1α Release from Macrophages Pretreated with LPS and Their Viability

(A) Chemical structures of H₁R antagonists. (#1) Ket, (#2) Meq, (#3) Aze, (#4) Oxa, (#5) Epi, (#6) Bep, (#7) Fex, (#8) Lor, (#9) Lev, and (#10) Des. (B and C) Macrophages were treated with the indicated concentrations of H₁R antagonists. One hour later, LPS was added. After 24 h of incubation, the supernatant was collected for measuring IL-6 (B) and IL-1α (C) levels by ELISA. (D) Macrophages were treated with the indicated concentrations of H₁R antagonists. After 2 h of incubation, the cultures were used for the assessment of cell viability with MTT reduction. The scale of the ordinate axis is repeated in the right panels of (B–D). Data are shown as mean ± S.E.M. (number of samples = 4). *p < 0.05.

We next examined the release of IL-1β in addition to IL-1α (Fig. 2). In the absence of LPS, Meq, Aze, and Des did not induce the release of IL-1β but induced the release of IL-1α in a dose-dependent manner. Meq, Aze, and Des also induced LDH release, which is a marker of cell death, from macrophages. These contrasting modes of the IL-1α and IL-1β release suggest that cell death induced by Meq, Aze, and Des shows pyroptotic-like features.¹⁹⁾

Fig. 2. Meq, Aze, and Des Induce the Release of LDH and IL-1α, but Not IL-1β

Macrophages were treated with the indicated concentrations of H₁R antagonists. After 1.5 h of incubation, the supernatant was collected for measuring IL-1α (A) and IL-1β (B) levels by ELISA. The supernatants were also used for the assessment of the LDH release with LDH assay (C). The scale of the ordinate axis is repeated in the right panels. Data are shown as mean ± S.E.M. (number of samples = 4). *p < 0.05.

Figure 3 shows the macrophage images under phase-contrast microscopy and fluorescence projected with PI and H33342. Phase-contrast microscopic observation demonstrates that macrophages treated with Meq, Aze, and Des remain tightly attached to culture plates with flattened cytoplasm. Observation of PI and H33342 staining indicates plasma membrane rupture and retention of nuclear morphology in macrophages treated with the 3 antagonists. Nuclear condensation or DNA fragmentation, which is characteristic of apoptosis, was not observed. These morphological changes induced by Meq, Aze, and Des resemble those observed in pyroptosis.^25,26)

Fig. 3. Meq, Aze, and Des Induce Cell Death Showing Necrotic-Like Morphology in Macrophages

Macrophages were treated with DMSO (Con) (A), Meq (B), Aze (C), or Des (D) for 2 h. Phase-contrast microscopy and fluorescence images using H33342 and PI of macrophages are presented. Bar = 0.1 mm.

Meq, Aze, and Des Induce Cell Death Accompanied by Caspase-8, Caspase-3, and GSDME Activation

We next examined the possible activation of pyroptosis-related key molecules such as GSDMD and GSDME. Western blotting analysis revealed that all of Meq, Aze, and Des cleaved and activated GSDME, but not GSDMD, in macrophages. HMGB1, one of the DAMPs, was released into the supernatant of the macrophage cultures treated with the antagonists (Fig. 4).

Fig. 4. Meq, Aze, and Des Induce the Cleavage of GSDME, but Not GSDMD, in Macrophages

Macrophages were treated with the indicated concentrations of H₁R antagonists. After 0, 1, or 4 h of incubation, the supernatant and the lysate of the macrophage cultures were collected for Western blotting analysis of GSDME, GSDMD, and HMGB1. Lys: lysate; Sup: supernatant; FL: full length.

Activation of caspase-8 and caspase-3, which are prerequisites for the cleavage of GSDME,¹⁹⁾ was also examined by Western blotting analysis. All of Meq, Aze, and Des cleaved and activated caspase-8 and caspase-3 (Fig. 5), which is consistent with the activation of GSDME as shown in Fig. 4.

Fig. 5. Meq, Aze, and Des Induce the Cleavage of Caspase-3 and Caspase-8 in Macrophages

Macrophages were treated with the indicated concentrations of H₁R antagonists. After 0, 1, or 4 h of incubation, the supernatant and the lysate of the macrophage cultures were collected for Western blotting analysis of caspase-3 and caspase-8. Lys: lysate; Sup: supernatant; FL: full length.

Cell Death Induced by Meq, Aze, and Des Is Reduced by Pretreatment with Ket

To probe the targets of the cell death-inducing effect of Meq, Aze, and Des, we examined the impact of pretreatment with Ket. Because Meq, Aze, and Des are all H₁R antagonists, the most likely target for cell death induction would be H₁R itself. To confirm this hypothesis, macrophages were preincubated with Ket, which has low Ki values for H₁R^27,28) and a lesser cell death-inducing effect on macrophages (Fig. 1D), and then treated with Meq, Aze, and Des. MTT reduction in macrophages challenged with Aze or Des at 20 μM was significantly upregulated by pretreatment with Ket (Figs. 6B, 6C). MTT reduction in macrophages challenged with Meq showed a tendency to be upregulated by Ket pretreatment (Fig. 6A).

Fig. 6. Ketotifen Rescues Macrophages from the Cell Death Induced by Meq, Aze, and Des

Macrophages were treated with Ket (20 μM). One hour later, Meq, Aze, or Des was added. After 2 h of incubation, the cultures were used for the assessment of cell viability with MTT reduction. The scale of the ordinate axis is repeated even in the right panels. Data are shown as mean ± S.E.M. (number of samples = 4). *p < 0.05. FL: full length.

DISCUSSION

In the present study, we demonstrated that 3 second-generation H₁-receptor antagonists, Meq, Aze, and Des, induce an RCD in macrophages, which is accompanied by the release of IL-1α and one of the DAMPs, HMGB1. This inflammation-inducing characteristic should be paid attention to, because the second-generation H₁-receptor antagonists are expected to relieve the symptoms of allergy and inflammation. The concentrations of H₁R antagonists used in this study are excessively high; thus, clinically prescribed doses would not have such effects. We must examine the inflammation-inducing effects of these antagonists on H₁R-expressing cells other than macrophages, as well as their in vivo effects.

We analyzed the cleavage of GSDME and GSDMD, which trigger pyroptosis. Meq, Aze, and Des clearly induced the p37 cleaved GSDME fragment but did not induce the p30 cleaved GSDMD²⁹⁾ (Fig. 4). Concerning the release of IL-1 cytokines, Meq, Aze, and Des induced the release of IL-1α but did not induce that of IL-1β. It is reported that GSDME cleavage accompanies the release of IL-1α, but not IL-1β, while GSDMD cleavage accompanies the release of both IL-1α and IL-1β¹⁹⁾; thus, cell death induced by Meq, Aze, and Des shows pyroptotic-like features.

We performed caspase inhibitor experiments to confirm the necessity of the activation of caspase-3 and caspase-8 to execute GSDME-mediated cell death, because many groups have reported the involvement of caspase-3 and/or caspase-8 in GSDME-mediated pyroptosis.^30,31) However, we failed to demonstrate that caspase inhibitors such as Z-DEVD-FMK, Z-IETD-FMK, or Z-VAD-FMK, after 1 or 3 h of preincubation at 20 or 40 μM, rescue macrophages from the cell death caused by Meq, Aze, and Des (data not shown). Inhibitors of caspase-8 and/or caspase-3 might promote alternative cell death pathways, as reported by others.^32–36) We recognize that experiments using GSDME-deficient macrophages are lacking to examine the GSDME dependency of the cell death-inducing activity of Meq, Aze, and Des. At present, we cannot conclude that the cell death induced by Meq, Aze, and Des is GSDME-mediated pyroptosis. Thus, Meq, Aze, and Des are suggested to induce pyroptotic-like cell death accompanied by caspase-8, caspase-3, and GSDME activation in macrophages.

The mode of cell death induced by the 3 H₁R antagonists, Meq, Aze, and Des, is unique and resembles that of “incomplete pyroptosis” defined by Aizawa et al.¹⁹⁾ They reported that nigericin induces caspase-1-independent necrotic-like cell death via apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). This cell death is accompanied by ASC-mediated caspase-8 activation and subsequent GSDME processing, followed by IL-1α release but not IL-1β release. As the macrophages we used in the present study are not caspase-1-deficient cells, the 3 antagonists could be considered to affect the dual effects of caspase-1 inhibition and NLRP3 inflammasome activation. The precise mechanisms of the cell death-inducing effect of Meq, Aze, and Des must be clarified.

We attempted to probe the possible targets of Meq, Aze, and Des in their cell death-inducing effect. Because second-generation H₁R antihistamines are highly selective for the histamine H₁R, do not cross the blood–brain barrier, and have minimal adverse events,³⁷⁾ we examined whether H₁R itself is a target of the 3 drugs. For this purpose, we used Ket as an inhibitor to block the binding of the 3 antihistamines to H₁R, as its Ki value is at very low levels compared to those of Meq, Aze, and Des against H₁R.^27,28) As we expected, the reduced viability caused by Meq, Aze, and Des was improved and upregulated by pretreatment with Ket, implying the possibility that the cell death induced by Meq, Aze, and Des is mediated by the binding of these antihistamines to H₁R. Experiments using H₁R-deficient cells must be performed to conclude the involvement of H₁R in the Meq-, Aze-, and Des-induced cell death.

In the present study, we found that 3 of 10 second-generation H₁R antagonists, such as Meq, Aze, and Des, induced pyroptotic-like cell death with IL-1α secretion in macrophages, while the other 7 antagonists did not. The reason for the difference between the 2 groups in cell death induction is unclear. We could not identify points of similarity in the chemical structures among Meq, Aze, and Des. The slight structural difference between Des and Lor might provide a hint. The former induces pyroptotic-like cell death with IL-1α secretion in macrophages, while the latter does not. A study using cryo-electron microscopy reports detailed interactions of these 2 antagonists in the H₁R ligand-binding pocket.¹⁾ Further interaction analysis concerning the other H₁R antagonists might provide a hint for revealing the mechanisms of cell death induction.

Funding

This study was supported by JSPS KAKENHI (Grant No. JP22K11819).

Author Contributions

AI: Investigation. AS: Investigation and Methodology. SO: Methodology and writing—original draft. MN: Conceptualization and writing—original draft, writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1)  Wang D, Guo Q, Wu Z, Li M, He B, Du Y, Zhang K, Tao Y. Molecular mechanism of antihistamines recognition and regulation of the histamine H₁ receptor. Nat. Commun., 15, 84 (2024).
• 2)  Jutel M, Akdis M, Akdis CA. Histamine, histamine receptors and their role in immune pathology. Clin. Exp. Allergy, 39, 1786–1800 (2009).
• 3)  Fink SL, Cookson BT. Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect. Immun., 73, 1907–1916 (2005).
• 4)  Shi J, Gao W, Shao F. Pyroptosis: Gasdermin-mediated programmed necrotic cell death. Trends Biochem. Sci., 42, 245–254 (2017).
• 5)  Martin SJ, Henry CM, Cullen SP. A perspective on mammalian caspases as positive and negative regulators of inflammation. Mol. Cell, 46, 387–397 (2012).
• 6)  Bertheloot D, Latz E, Franklin BS. Necroptosis, pyroptosis and apoptosis: an intricate game of cell death. Cell. Mol. Immunol., 18, 1106–1121 (2021).
• 7)  Tsuchiya K, Hosojima S, Hara H, Kushiyama H, Mahib MR, Kinoshita T, Suda T. Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes. Cell Rep., 34, 108887 (2021).
• 8)  Chao YY, Puhach A, Frieser D, Arunkumar M, Lehner L, Seeholzer T, Garcia-Lopez A, van der Wal M, Fibi-Smetana S, Dietschmann A, Sommermann T, Ćiković T, Taher L, Gresnigt MS, Vastert SJ, van Wijk F, Panagiotou G, Krappmann D, Groß O, Zielinski CE. Human T_H17 cells engage gasdermin E pores to release IL-1apha on NLRP3 inflammasome activation. Nat. Immunol., 24, 295–308 (2023).
• 9)  Tsuchiya K, Nakajima S, Hosojima S, Thi Nguyen D, Hattori T, Manh Le T, Hori O, Mahib MR, Yamaguchi Y, Miura M, Kinoshita T, Kushiyama H, Sakurai M, Shiroishi T, Suda T. Caspase-1 initiates apoptosis in the absence of gasdermin D. Nat. Commun., 10, 2091 (2019).
• 10)  Liu S, Perez P, Sun X, Chen K, Fatirkhorani R, Mammadova J, Wang Z. MLKL polymerization-induced lysosomal membrane permeabilization promotes necroptosis. Cell Death Differ., 31, 40–52 (2024).
• 11)  Zhan C, Huang M, Yang X, Hou J. MLKL: Functions beyond serving as the Executioner of Necroptosis. Theranostics, 11, 4759–4769 (2021).
• 12)  Ueda H, Fujita R. Cell death mode switch from necrosis to apoptosis in brain. Biol. Pharm. Bull., 27, 950–955 (2004).
• 13)  Han J, Zhong CQ, Zhang DW. Programmed necrosis: backup to and competitor with apoptosis in the immune system. Nat. Immunol., 12, 1143–1149 (2011).
• 14)  Chan FK, Luz NF, Moriwaki K. Programmed necrosis in the cross talk of cell death and inflammation. Annu. Rev. Immunol., 33, 79–106 (2015).
• 15)  Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat. Rev. Immunol., 13, 397–411 (2013).
• 16)  Fritsch M, Günther SD, Schwarzer R, Albert MC, Schorn F, Werthenbach JP, Schiffmann LM, Stair N, Stocks H, Seeger JM, Lamkanfi M, Krönke M, Pasparakis M, Kashkar H. Caspase-8 is the molecular switch for apoptosis, necroptosis and pyroptosis. Nature, 575, 683–687 (2019).
• 17)  Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES. Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat. Commun., 8, 14128 (2017).
• 18)  Wang Y, Gao W, Shi X, Ding J, Liu W, He H, Wang K, Shao F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature, 547, 99–103 (2017).
• 19)  Aizawa E, Karasawa T, Watanabe S, Komada T, Kimura H, Kamata R, Ito H, Hishida E, Yamada N, Kasahara T, Mori Y, Takahashi M. GSDME-dependent incomplete pyroptosis permits selective IL-1alpha release under caspase-1 inhibition. iScience, 23, 101070 (2020).
• 20)  Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature, 496, 445–455 (2013).
• 21)  Satoh T, Kidoya H, Naito H, Yamamoto M, Takemura N, Nakagawa K, Yoshioka Y, Morii E, Takakura N, Takeuchi O, Akira S. Critical role of Trib1 in differentiation of tissue-resident M2-like macrophages. Nature, 495, 524–528 (2013).
• 22)  Li Z, Liu W, Fu J, Cheng S, Xu Y, Wang Z, Liu X, Shi X, Liu Y, Qi X, Liu X, Ding J, Shao F. Shigella evades pyroptosis by arginine ADP-riboxanation of caspase-11. Nature, 599, 290–295 (2021).
• 23)  Okabe Y, Medzhitov R. Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell, 157, 832–844 (2014).
• 24)  Ishikawa S, Sawamoto A, Okuyama S, Nakajima M. T-cell activation-inhibitory assay to screen caloric restriction mimetics drugs for drug repositioning. Biol. Pharm. Bull., 44, 550–556 (2021).
• 25)  Jorgensen I, Miao EA. Pyroptotic cell death defends against intracellular pathogens. Immunol. Rev., 265, 130–142 (2015).
• 26)  Chen X, He WT, Hu L, Li J, Fang Y, Wang X, Xu X, Wang Z, Huang K, Han J. Pyroptosis is driven by non-selective gasdermin-D pore and its morphology is different from MLKL channel-mediated necroptosis. Cell Res., 26, 1007–1020 (2016).
• 27)  Anthes JC, Gilchrest H, Richard C, Eckel S, Hesk D, West RE Jr, Williams SM, Greenfeder S, Billah M, Kreutner W, Egan RE. Biochemical characterization of desloratadine, a potent antagonist of the human histamine H₁ receptor. Eur. J. Pharmacol., 449, 229–237 (2002).
• 28)  Kubo N, Shirakawa O, Kuno T, Tanaka C. Antimuscarinic effects of antihistamines: quantitative evaluation by receptor-binding assay. Jpn. J. Pharmacol., 43, 277–282 (1987).
• 29)  Miyawaki S, Sawamoto A, Okuyama S, Nakajima M. Sulconazole induces pyroptosis promoted by interferon-γ in monocyte/macrophage lineage cells. J. Pharmacol. Sci., 154, 166–174 (2024).
• 30)  Yu J, Li S, Qi J, Chen Z, Wu Y, Guo J, Wang K, Sun X, Zheng J. Cleavage of GSDME by caspase-3 determines lobaplatin-induced pyroptosis in colon cancer cells. Cell Death Dis., 10, 193 (2019).
• 31)  Wei Y, Lan B, Zheng T, Yang L, Zhang X, Cheng L, Tuerhongjiang G, Yuan Z, Wu Y. GSDME-mediated pyroptosis promotes the progression and associated inflammation of atherosclerosis. Nat. Commun., 14, 929 (2023).
• 32)  Ma F, Ghimire L, Ren Q, Fan Y, Chen T, Balasubramanian A, Hsu A, Liu F, Yu H, Xie X, Xu R, Luo HR. Gasdermin E dictates inflammatory responses by controlling the mode of neutrophil death. Nat. Commun., 15, 386 (2024).
• 33)  Vandenabeele P, Vanden Berghe T, Festjens N. Caspase inhibitors promote alternative cell death pathways. Sci. STKE, 2006, pe44 (2006).
• 34)  Los M, Mozoluk M, Ferrari D, Stepczynska A, Stroh C, Renz A, Herceg Z, Wang ZQ, Schulze-Osthoff K. Activation and caspase-mediated inhibition of PARP: a molecular switch between fibroblast necrosis and apoptosis in death receptor signaling. Mol. Biol. Cell, 13, 978–988 (2002).
• 35)  Prabhakaran K, Li L, Borowitz JL, Isom GE. Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation. Toxicol. Appl. Pharmacol., 195, 194–202 (2004).
• 36)  Lafont E, Milhas D, Teissié J, Therville N, Andrieu-Abadie N, Levade T, Benoist H, Ségui B. Caspase-10-dependent cell death in Fas/CD95 signalling is not abrogated by caspase inhibitor zVAD-fmk. PLoS One, 5, e13638 (2010).
• 37)  Kalpaklioglu F, Baccioglu A. Efficacy and safety of H₁-antihistamines: an update. Antiinflamm. Antiallergy Agents Med. Chem., 11, 230–237 (2012).