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Mastocytosis is a KIT-related myeloproliferative disease characterised by abnormal expansion of neoplastic mast cells (MC) in the skin or virtually any other organ system. The cutaneous form of adult-onset mastocytosis is almost invariably combined with indolent systemic involvement for which curative therapy is yet not available. Here we evaluated a concept of depleting cutaneous MCs in mastocytosis lesions ex vivo by targeting their secretory granules. Skin biopsies from mastocytosis patients were incubated with or without mefloquine, an antimalarial drug known to penetrate into acidic organelles such as MC secretory granules. Mefloquine reduced the number of dermal MCs without affecting keratinocyte proliferation or epidermal gross morphology at drug concentrations up to 40 mu M. Flow cytometric analysis of purified dermal MCs showed that mefloquine-induced cell death was mainly due to apoptosis and accompanied by caspase-3 activation. However, caspase inhibition provided only partial protection against mefloquine-induced cell death, indicating predominantly caspase-independent apoptosis. Further assessments revealed that mefloquine caused an elevation of granule pH and a corresponding decrease in cytosolic pH, suggesting drug-induced granule permeabilisation. Extensive damage to the MC secretory granules was confirmed by transmission electron microscopy analysis. Further, blockade of granule acidification or serine protease activity prior to mefloquine treatment protected MCs from apoptosis, indicating that granule acidity and granule-localised serine proteases play major roles in the execution of mefloquine-induced cell death. Altogether, these findings reveal that mefloquine induces selective apoptosis of MCs by targeting their secretory granules and suggest that the drug may potentially extend its range of medical applications.

Chronic local inflammation of adipose tissue is an important feature of obesity. Serglycin is a proteoglycan highly expressed by various immune cell types known to infiltrate adipose tissue under obese conditions. To investigate if serglycin expression has an impact on diet-induced adipose tissue inflammation, we subjected Srgn+/+ and Srgn−/− mice (C57BL/6J genetic background) to an 8-wk high-fat and high-sucrose diet. The total body weight was the same in Srgn+/+ and Srgn−/− mice after diet treatment. Expression of white adipose tissue genes linked to inflammatory pathways were lower in Srgn−/− mice. We also noted reduced total macrophage abundance, a reduced proportion of proinflammatory M1 macrophages, and reduced formation of crown-like structures in adipose tissue of Srgn−/− compared with Srgn+/+ mice. Further, Srgn−/− mice had more medium-sized adipocytes and fewer large adipocytes. Differentiation of preadipocytes into adipocytes (3T3-L1) was accompanied by reduced Srgn mRNA expression. In line with this, analysis of single-cell RNA sequencing data from mouse and human adipose tissue supports that Srgn mRNA is predominantly expressed by various immune cells, with low expression in adipocytes. Srgn mRNA expression was higher in obese compared with lean humans and mice, accompanied by an increased expression of immune cell gene markers. SRGN and inflammatory marker mRNA expression was reduced upon substantial weight loss in patients after bariatric surgery. Taken together, this study introduces a role for serglycin in the regulation of obesity-induced adipose inflammation.

Background/Aim: Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. Materials and Methods: Here, we adopted an established hanging-drop spheroid system to investigate how mast cells influence melanoma growth and phenotype in a 3-D context. To address the underlying mechanism, we conducted transcriptomic and pathway analyses. Results: In the presence of mast cells or mast cell-conditioned medium, growth of melanoma spheroids was profoundly reduced. Transcriptomic analysis revealed that mast cell-conditioned medium had extensive effects on the gene-expression patterns of melanoma. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced up-regulation of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced growth and adhesive properties. However, when assessing the possible impact of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on growth of melanoma spheroids could potentially be explained by such a scenario. Conclusion: Our findings suggest that the phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Our findings may provide insight into the effects on gene-expression events that occur in melanoma tumors under nutrient stress.

Mast cells are abundant in melanoma tumors, and studies suggest that they can be either detrimental or protective for melanoma growth. However, the underlying mechanisms are not fully understood. Here, we adopted a hanging drop-established spheroid system to investigate how mast cells can influence melanoma growth and phenotype in a 3-D context. In the presence of mast cells or mast cell-conditioned medium, melanoma spheroid growth was profoundly reduced. To address the underlying mechanism, we conducted a transcriptomic analysis, which revealed that mast cell-conditioned medium had extensive effects on the melanoma gene expression patterns. Pathway analyses revealed profound effects on the expression of genes related to amino acid and protein metabolism. The conditioned medium also induced an upregulated expression of cancer-related genes, including adhesion molecules implicated in metastatic spreading. In line with this, after transfer to a Matrigel extracellular matrix milieu, spheroids that had been developed in the presence of mast cell-conditioned medium displayed enhanced elevated growth and adhesive properties. However, when assessing for possible effects of nutrient starvation, i.e., reduced nutrient content in mast cell-conditioned medium, we found that the observed effects on melanoma spheroid growth potentially could be explained by such effects. Hence, it cannot be excluded that the observed phenotypic alterations of melanoma spheroids grown in the presence of mast cells or mast cell-conditioned media are, at least partly, due to nutrient starvation rather than to the action of factors secreted by mast cells. Instead, our findings may provide insight into the effects on gene expression events that occur in melanoma tumors under nutrient stress.

Background Mast cells (MCs) have a profound impact on allergic asthma. Under such conditions, MCs undergo degranulation, resulting in the release of exceptionally large amounts of MC-restricted proteases. However, the role of these proteases in asthma is only partially understood. Objectives We sought to test our hypothesis that MC proteases can influence the functionality of human lung fibroblasts (HLFs). Methods Primary HLFs were treated with MC chymase or tryptase, followed by assessment of parameters related to fibroblast function. Results HLFs underwent major morphologic changes in response to chymase, showing signs of cellular contraction, but were refractory to tryptase. However, no effects of chymase on HLF viability or proliferation were seen. Chymase, but not tryptase, had a major impact on the output of extracellular matrix–associated compounds from the HLFs, including degradation of fibronectin and collagen-1, and activation of pro–matrix metalloprotease 2. Further, chymase induced the release of various chemotactic factors from HLFs. In line with this, conditioned medium from chymase-treated HLFs showed chemotactic activity on neutrophils. Transcriptome analysis revealed that chymase induced a proinflammatory gene transcription profile in HLFs, whereas tryptase had minimal effects. Conclusions Chymase, but not tryptase, has a major impact on the phenotype of primary airway fibroblasts by modifying their output of extracellular matrix components and by inducing a proinflammatory phenotype.

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-β1 and TGF-β2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

Mast cells (MCs) are versatile effector cells of the immune system, characterized by a large content of secretory granules containing a variety of inflammatory mediators. They are implicated in the host protection toward various external insults, but are mostly well known for their detrimental impact on a variety of pathological conditions, including allergic disorders such as asthma and a range of additional disease settings. Based on this, there is currently a large demand for therapeutic regimens that can dampen the detrimental impact of MCs in these respective pathological conditions. This can be accomplished by several strategies, including targeting of individual mediators released by MCs, blockade of receptors for MC-released compounds, inhibition of MC activation, limiting mast cell growth or by inducing mast cell apoptosis. Here, we review the currently available and emerging regimens to interfere with harmful mast cell activities in asthma and other pathological settings and discuss the advantages and limitations of such strategies.

Letter to the Editor.

It has been recognized for a long time that the secretory granules of mast cells are acidic, but the functional importance of maintaining an acidic pH in the mast cell granules is not fully understood. Here we addressed this issue by examining the effects of raising the pH of the mast cell secretory granules. Mast cells were incubated with bafilomycin A1, an inhibitor of the vacuolar-type ATPase proton pump. Supporting a role of vacuolar-type ATPase in mast cell granule acidification, bafilomycin A1 treatment caused a robust increase in granule pH. This was accompanied by marked effects on mast cell granules, including swelling and acquisition of vacuole-like morphology. Moreover, bafilomycin A1 caused extensive, yet selective effects on the granule content. These included aberrant processing of pro-carboxypeptidase A3 and a reduction in the level of intracellular histamine, the latter being accompanied by an increase in extracellular histamine. In contrast, the storage of β-hexosaminidase, a prototype lysosomal hydrolase known to be stored in mast cell granules, was not affected by abrogation of granule acidification. Moreover, bafilomycin A1 caused a reduction of tryptase enzymatic activity and appearance of tryptase degradation products. Tryptase inhibition prevented the formation of such degradation products, suggesting that the pH elevation causes tryptase to undergo autoproteolysis. Taken together, our findings reveal that mast cell secretory granule homeostasis is critically dependent on an acidic milieu.

SummaryBackground Skin mast cells are implicated as detrimental effector cells in various inflammatory skin diseases such as contact eczema, atopic dermatitis and psoriasis. Selective reduction of cutaneous mast cells, e.g. by inducing targeted apoptosis, might prove a rational and efficient therapeutic strategy in dermatoses negatively influenced by mast cells. Objectives The objective of the present study was to evaluate whether a lysosomotropic agent such as siramesine can cause apoptosis of mast cells present in psoriatic lesions. Methods Punch biopsies were obtained from lesional and uninvolved skin in 25 patients with chronic plaque psoriasis. After incubation with siramesine, the number of tryptase-positive mast cells, and their expression of IL-6 and IL-17 was analyzed. Skin biopsies were digested to allow flow cytometry analysis of the drug's effect on cutaneous fibroblasts and keratinocytes. Results Siramesine caused a profound reduction in the total number of mast cells in both lesional and uninvolved psoriatic skin biopsies without affecting the gross morphology of the tissue. The drug reduced the density of IL-6- and IL-17-positive mast cells, and showed antiproliferative effects on epidermal keratinocytes but had no apparent cytotoxic effect on keratinocytes or dermal fibroblasts. Conclusions Considering the pathophysiology of psoriasis, the effects of siramesine on cutaneous mast cells may prove favourable from the therapeutic aspect. The results encourage further studies to assess the usefulness of siramesine and other lysosomotropic agents in the treatment of cutaneous mastocytoses and inflammatory skin diseases aggravated by dermal mast cells. This article is protected by copyright. All rights reserved.

Mast cells are key effector cells in allergic reactions. When activated to degranulate, they release a plethora of bioactive compounds from their secretory granules, including mast cell-restricted proteases such as tryptase. In a previous study, we showed that tryptase, in addition to its intragranular location, can be found within the nuclei of mast cells where it truncates core histones at their N-terminal ends.Considering that the N-terminal portions of the core histones constitute sites for posttranslational modifications of major epigenetic impact, we evaluated whether histone truncation by tryptase could have an impact on epigenetic events in mast cells.Mast cells were cultured from wild-type and tryptase null mice, followed by an assessment of their profile of epigenetic histone modifications and their phenotypic characteristics.We show that tryptase truncates nucleosomal histone 3 and histone 2B (H2B) and that its absence results in accumulation of the epigenetic mark, lysine 5-acetylated H2B. Intriguingly, the accumulation of lysine 5-acetylated H2B was cell age-dependent and was associated with a profound upregulation of markers of non-mast cell lineages, loss of proliferative control, chromatin remodeling as well as extensive morphological alterations.These findings introduce tryptase-catalyzed histone clipping as a novel epigenetic regulatory mechanism, which in the mast cell context may be crucial for maintaining cellular identity.

Mast cells are known to have a detrimental impact on numerous types of inflammatory skin diseases such as contact dermatitis, atopic eczema and cutaneous mastocytosis. Regimens that dampen skin mast cell-mediated activities can thus offer an attractive therapeutic option under such circumstances. As mast cells are known to secrete a large array of potentially pathogenic compounds, both from preformed stores in secretory lysosomes (granules) and after de novo synthesis, mere inhibition of degranulation or interference with individual mast cell mediators may not be sufficient to provide an effective blockade of harmful mast cell activities. An alternative strategy may therefore be to locally reduce skin mast cell numbers. Here, we explored the possibility of using lysosomotropic agents for this purpose, appreciating the fact that mast cell granules contain bioactive compounds prone to trigger apoptosis if released into the cytosolic compartment. Based on this principle, we show that incubation of human skin punch biopsies with the lysosomotropic agents siramesine or Leu-Leu methyl ester preferably ablated the mast cell population, without causing any gross adverse effects on the skin morphology. Subsequent analysis revealed that mast cells treated with lysosomotropic agents predominantly underwent apoptotic rather than necrotic cell death. In summary, this study raises the possibility of using lysosomotropic agents as a novel approach to targeting deleterious mast cell populations in cutaneous mastocytosis and other skin disorders negatively influenced by mast cells.

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.