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Induction of Mast Cell Apoptosis via Granule Permeabilization: A Novel Approach to Target Mast Cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Mast cells are densely granulated tissue-resident immune cells that play an important role in orchestrating inflammatory responses. Dysregulated increases in the numbers and activation status of mast cells can have deleterious consequences for the body in various inflammatory diseases. Mast cells are best-known for their detrimental roles in allergic diseases, e.g., asthma. Thus, strategies that target mast cells and their harmful activities in such pathological conditions are potentially attractive therapeutic options. An efficient strategy to accomplish a full blockade of the harmful events mediated by various mast cell mediators is to locally eliminate mast cell populations altogether by inducing their apoptosis.

Using in vitro-cultured mast cells, we identified that mefloquine, an antimalarial drug with lysosomotropic activity, causes permeabilization of secretory granules, increased production of reactive oxygen species (ROS), release of granule-localized proteases into the cytosol and apoptotic cell death (Paper I). Moreover, intraperitoneal injections of mefloquine in mice resulted in a reduced peritoneal mast cell population in vivo.

To evaluate the possibility of using lysosomotropic agents for selectively depleting human lung mast cells by induction of apoptosis, human lung specimens were used. Exposure of either intact human lung tissue, purified lung mast cells or mixed populations of lung cells to mefloquine revealed that human lung mast cells are highly susceptible to ROS-induced apoptosis in this setting. In contrast, other cell populations of the lung were largely refractory (Paper II).

Lastly, in an attempt to gain a deeper insight into the mechanism underlying ROS production and the downstream events in response to lysosomotropic challenge, we identified that the mast cell secretory granules comprise major subcellular compartments for ROS production in response to mefloquine (Paper III). Lysosomal iron, granzyme B and the ERK1/2 MAP kinase signaling pathway were found to contribute to production of ROS in response to mefloquine. Furthermore, granule acidification was shown to be essential for mefloquine-mediated effects in mast cells, i.e., granule permeabilization, ROS production and cell death. Collectively, the present thesis introduces the possibility of inducing selective mast cell apoptosis via granule permeabilization as a novel strategy to target mast cells. Thus, this strategy has a potential to be used therapeutically to ameliorate mast cell-mediated detrimental effects in inflammatory diseases, such as asthma.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 68
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1628
Keywords [en]
Mast cells, Granules, Apoptosis, Reactive oxygen species, ROS, Lysosomotropic agents, Mefloquine
National Category
Immunology Biochemistry and Molecular Biology
Research subject
Immunology; Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-402203ISBN: 978-91-513-0848-7 (print)OAI: oai:DiVA.org:uu-402203DiVA, id: diva2:1384913
Public defence
2020-02-27, Room C8:301, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2020-02-06 Created: 2020-01-13 Last updated: 2020-02-06
List of papers
1. Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway.
Open this publication in new window or tab >>Mefloquine, an anti-malaria agent, causes reactive oxygen species-dependent cell death in mast cells via a secretory granule-mediated pathway.
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2014 (English)In: Pharmacology research & perspectives, ISSN 2052-1707, Vol. 2, no 6, article id e00066Article in journal (Refereed) Published
Abstract [en]

Mast cells are known to have a detrimental impact on a variety of pathological conditions. There is therefore an urgent need of developing strategies that limit their harmful effects. The aim of this study was to accomplish this by developing a means of inducing mast cell apoptosis. The strategy was to identify novel compounds that induce mast cell apoptosis by permeabilization of their secretory lysosomes (granules). As a candidate, we assessed mefloquine, an anti-malarial drug that has been proposed to have lysosome-permeabilizing activity. Mefloquine was added to mast cells and administered in vivo, followed by assessment of the extent and mechanisms of mast cell death. Mefloquine was cytotoxic to murine and human mast cells. Mefloquine induced apoptotic cell death of wild-type mast cells whereas cells lacking the granule compounds serglycin proteoglycan or tryptase were shown to undergo necrotic cell death, the latter finding indicating a role of the mast cell granules in mefloquine-induced cell death. In support of this, mefloquine was shown to cause compromised granule integrity and to induce leakage of granule components into the cytosol. Mefloquine-induced cell death was refractory to caspase inhibitors but was completely abrogated by reactive oxygen species inhibition. These findings identify mefloquine as a novel anti-mast cell agent, which induces mast cell death through a granule-mediated pathway. Mefloquine may thus become useful in therapy aiming at limiting harmful effects of mast cells.

Keywords
Apoptosis, granules, mast cells, mefloquine, mMCP-6, reactive oxygen species, serglycin, tryptase
National Category
Natural Sciences
Research subject
Immunology
Identifiers
urn:nbn:se:uu:diva-401708 (URN)10.1002/prp2.66 (DOI)25505612 (PubMedID)
Available from: 2020-01-08 Created: 2020-01-08 Last updated: 2020-02-05Bibliographically approved
2. Induction of Human Lung Mast Cell Apoptosis by Granule Permeabilization: A Novel Approach for Targeting Mast Cells
Open this publication in new window or tab >>Induction of Human Lung Mast Cell Apoptosis by Granule Permeabilization: A Novel Approach for Targeting Mast Cells
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2017 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 8, article id 1645Article in journal (Refereed) Published
Abstract [en]

Mast cells are implicated as detrimental players in inflammatory lung diseases, particularly asthma. Mast cells respond to activating stimuli by releasing a wide panel of pro-inflammatory compounds that can contribute profoundly to the pathology, and there is currently an unmet need for strategies that efficiently ameliorate harmful effects of mast cells under such conditions. Here, we sought to evaluate a novel concept for targeting human lung mast cells, by assessing the possibility of selectively depleting the lung mast cells by induction of apoptosis. For this purpose, we used lysosomotropic agents, i.e., compounds that are known to permeabilize the secretory granules of mast cells, thereby releasing the contents of the granules into the cytosol. Either intact human lung tissue, purified human lung mast cells or mixed populations of human lung cells were incubated with the lysosomotropic agents mefloquine or siramesine, followed by measurement of apoptosis, reactive oxygen species (ROS) production, and release of cytokines. We show that human lung mast cells were highly susceptible to apoptosis induced by this strategy, whereas other cell populations of the lung were largely refractory. Moreover, we demonstrate that apoptosis induced by this mode is dependent on the production of ROS and that the treatment of lung tissue with lysosomotropic agents causes a decrease in the release of pathogenic cytokines. We conclude that selective apoptosis of human lung mast cells can be accomplished by administration of lysosomotropic agents, thus introducing the possibility of using such drugs as novel therapeutics in the treatment of inflammatory lung disorders such as asthma.

Keywords
apoptosis, granules, lysosomotropic agents, mast cells, mefloquine
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-339681 (URN)10.3389/fimmu.2017.01645 (DOI)000416221900001 ()29230220 (PubMedID)
Available from: 2018-01-22 Created: 2018-01-22 Last updated: 2020-01-13Bibliographically approved
3. Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production
Open this publication in new window or tab >>Lysosomotropic challenge of mast cells causes intra-granular reactive oxygen species production
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2019 (English)In: CELL DEATH DISCOVERY, ISSN 2058-7716, Vol. 5, article id 95Article in journal (Refereed) Published
Abstract [en]

Mast cells contribute to the pathology of allergic and other disorders. Strategies to interfere with harmful mast cell-related activities are therefore warranted. Previously we established a principle for inducing selective apoptosis of mast cells, by the use of lysosomotropic agents that cause secretory granule permeabilization, leading to production of reactive oxygen species (ROS). However, the mechanism of ROS production has not been known. Here we addressed this issue. Live microscopy analysis showed that the secretory granules comprise major subcellular compartments for ROS production in response to mefloquine. As further signs for the primary involvement of secretory granules, both ROS production and cell death was blunted in mast cells lacking serglycin, a secretory granule-restricted proteoglycan. Inhibition of granule acidification caused an essentially complete blockade of granule permeabilization, ROS production and cell death in response to mefloquine. ROS production was also attenuated in the presence of an iron chelator, and after inhibition of either granzyme B or the ERK1/2 MAP kinase signaling pathway. Together, our findings reveal that the mast cell secretory granules constitute major sites for ROS production in mast cells subjected to lysosomotropic challenge. Moreover, this study reveals a central role for granule acidification in ROS generation and the pro-apoptotic response triggered downstream of secretory granule permeabilization.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-387281 (URN)10.1038/s41420-019-0177-3 (DOI)000468005600001 ()31123601 (PubMedID)
Funder
Swedish Research CouncilKnut and Alice Wallenberg Foundation
Available from: 2019-06-24 Created: 2019-06-24 Last updated: 2020-01-13Bibliographically approved

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