Early development and functional properties of tryptase/chymase double-positive mast cells from human pluripotent stem cells.
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ABSTRACT:
SUBMITTER: Bian G
PROVIDER: S-EPMC8104937 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chronic respiratory diseases are often characterized by impaired epithelial function and remodeling. Mast cells (MCs) are known to home into the epithelium in respiratory diseases, but the MC-epithelial interactions remain less understood. Therefore, this study aimed to investigate the effect of MC proteases on bronchial epithelial morphology and function. Bronchial epithelial cells were stimulated with MC tryptase and/or chymase. Morphology and epithelial function were performed using cell tracking analysis and holographic live-cell imaging. Samples were also analyzed for motility-associated gene expression. Immunocytochemistry was performed to compare cytoskeletal arrangement. Stimulated cells showed strong alterations on gene, protein and functional levels in several parameters important for maintaining epithelial function. The most significant increases were found in cell motility, cellular speed and cell elongation compared to non-stimulated cells. Also, cell morphology was significantly altered in chymase treated compared to non-stimulated cells. In the current study, we show that MC proteases can induce cell migration and morphological and proliferative alterations in epithelial cells. Thus, our data imply that MC release of proteases may play a critical role in airway epithelial remodeling and disruption of epithelial function.
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Project description:Mast cell secretory granules are densely packed with various bioactive mediators including proteases of chymase, tryptase and CPA3 type. Previous studies have indicated that mast cells can affect the outcome of melanoma but the contribution of the mast cell granule proteases to such effects has not been clear. Here we addressed this issue by assessing mice lacking either the chymase Mcpt4, the tryptase Mcpt6 or carboxypeptidase A3 (Cpa3), as well as mice simultaneously lacking all three proteases, in a model of melanoma dissemination from blood to the lung. Although mice with individual deficiency in the respective proteases did not differ significantly from wildtype mice in the extent of melanoma colonization, mice with multiple protease deficiency (Mcpt4/Mcpt6/Cpa3-deficient) exhibited a higher extent of melanoma colonization in lungs as compared to wildtype animals. This was supported by higher expression of melanoma-specific genes in lungs of Mcpt4/Mcpt6/CPA3-deficient vs. wildtype mice. Cytokine profiling showed that the levels of CXCL16, a chemokine with effects on T cell populations and NKT cells, were significantly lower in lungs of Mcpt4/Mcpt6/Cpa3-deficient animals vs. controls, suggesting that multiple mast cell protease deficiency might affect T cell or NKT cell populations. In line with this, we found that the Mcpt4/Mcpt6/Cpa3-deficiency was associated with a reduction in cells expressing CD1d, a MHC class 1-like molecule that is crucial for presenting antigen to invariant NKT (iNKT) cells. Together, these findings indicate a protective role of mast cell-specific proteases in melanoma dissemination, and suggest that this effect involves a CXCL16/CD1d/NKT cell axis.
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Project description:BackgroundMast cells (MCs) are known to contribute to both acute and chronic inflammation. Bronchial epithelial cells are the first line of defence against pathogens and a deficient anti-viral response has been suggested to play a role in the pathogenesis of asthma exacerbations. However, effects of MC mediators on bronchial epithelial immune response have been less studied. The aim of this study is to investigate the direct effects of stimulation with MC proteases, tryptase and chymase, on inflammatory and anti-viral responses in human bronchial epithelial cells (HBECs).MethodCultured BEAS-2b cells and primary HBECs from 3 asthmatic patients were stimulated with tryptase or chymase (0.1 to 0.5 μg/ml) for 1, 3, 6 and 24 h. To study the effects of MC mediators on the anti-viral response, cells were stimulated with 10 μg/ml of viral mimic Poly (I:C) for 3 and 24 h following pre-treatment with 0.5 μg/ml tryptase or chymase for 3 h. Samples were analysed for changes in pro-inflammatory and anti-viral mediators and receptors using RT-qPCR, western blot and Luminex.ResultsTryptase and chymase induced release of the alarmin ATP and pro-inflammatory mediators IL-8, IL-6, IL-22 and MCP-1 from HBECs. Moreover, tryptase and chymase decreased the expression of E-cadherin and zonula occludens-1 expression from HBECs. Pre-treatment of HBECs with tryptase and chymase further increased Poly (I:C) induced IL-8 release at 3 h. Furthermore, tryptase significantly reduced type-I and III interferons (IFNs) and pattern recognition receptor (PRR) expression in HBECs. Tryptase impaired Poly (I:C) induced IFN and PRR expression which was restored by treatment of a serine protease inhibitor. Similar effects of tryptase on inflammation and anti-viral responses were also confirmed in primary HBECs from asthmatic patients.ConclusionMC localization within the epithelium and the release of their proteases may play a critical role in asthma pathology by provoking pro-inflammatory and alarmin responses and downregulating IFNs. Furthermore, MC proteases induce downregulation of epithelial junction proteins which may lead to barrier dysfunction. In summary, our data suggests that mast cells may contribute towards impaired anti-viral epithelial responses during asthma exacerbations mediated by the protease activity of tryptase.
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Project description:Mast cells are tissue-resident immune cells. Their overgrowth/overactivation results in a range of common distressing, sometimes life-threatening disorders, including asthma, psoriasis, anaphylaxis, and mastocytosis. Currently, drug discovery is hampered by use of cancer-derived mast cell lines or primary cells. Cell lines provide low numbers of mature mast cells and are not representative of in vivo mast cells. Mast cell generation from blood/bone marrow gives poor reproducibility, requiring 8-12 weeks of culture. Here we report a method for the rapid/robust production of mast cells from pluripotent stem cells (PSCs). An advantageous Gata2Venus reporter enriches mast cells and progenitors as they differentiate from PSCs. Highly proliferative mouse mast cells and progenitors emerge after 2 weeks. This method is applicable for rapid human mast cell generation, and could enable the production of sufficient numbers of physiologically relevant human mast cells from patient induced PSCs for the study of mast cell-associated disorders and drug discovery.
| S-EPMC6178197 | biostudies-literature
Project description:Incomplete excision of pleomorphic adenoma (PA) may result in recurrent pleomorphic adenoma (RPA). Furthermore, long-term neglected PA may become carcinoma ex pleomorphic adenoma (CXPA). In the present study, the relationships between mast cell-derived chymase and these tumors were examined. The tumor tissues of PA consisted of either or both glandular and fibrotic structures. Histological features of RPA were almost similar to those of PA, except that they showed multinodular structures. CXPA is composed of a mixture of PA and carcinoma. The main stromal cells in PA were myofibroblasts, whereas fibroblasts constituted the main cellular portion in the stromal tissue of RPA. Cancer-associated fibroblasts (CAFs) were present abundantly in CXPA. With increased VEGF expression, neovascularization tended to increase in RPA or CXPA. Compared with PA, chymase-positive mast cells, as well as chymase gene expression, were increased in the tumor tissues from patients with RPA or CXPA. SCF, TGFβ1, and PCNA-positive staining was widely observed in these tumor tissues. The above results suggest that mast cell-derived chymase through its direct or cooperative effects with other mediators may participate in the pathophysiology of RPA and CXPA.
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Project description:Our previous study demonstrated significantly more degranulating mast cells (MCs) in choroids from subjects with age-related macular degeneration compared to aged controls. This study examined the immunolocalization of tryptase, the most abundant MC secretory granule-derived serine protease, in aged control eyes and eyes with geographic atrophy (GA).Postmortem human eyes with and without GA were obtained from the National Disease Research Interchange. Tissue was fixed, cryopreserved, sectioned, and immunostained with a monoclonal antibody against tryptase. Sections were imaged on a Zeiss 710 Confocal Microscope.In the posterior pole of all aged control eyes, tryptase was confined to choroidal MCs, which were located primarily in Sattler's layer. In eyes with GA, many MCs were located in the inner choroid near choriocapillaris and Bruch's membrane (BM). Tryptase was found not only in MCs but also diffusely around them in stroma, suggesting they had degranulated. In contrast with aged control eyes, eyes with GA also had strong tryptase staining in BM. Tryptase was observed within BM in regions of RPE atrophy, at the border of atrophy, and extending well into the nonatrophic region.Our results demonstrate that tryptase, released during choroidal MC degranulation, binds to BM in GA in advance of RPE atrophy. Tryptase activates MMPs that can degrade extracellular matrix (ECM) and basement membrane components found in BM. ECM modifications are likely to have a profound effect on the function and health of RPE and choroidal thinning in GA.
| S-EPMC5699534 | biostudies-literature
Project description:Although mast cells (MCs) often are abundant in the synovial tissues of patients with rheumatoid arthritis, the contribution of MCs to joint inflammation and cartilage loss remains poorly understood. MC-restricted tryptase/heparin complexes have proinflammatory activity, and significant amounts of human tryptase beta (hTryptase-beta) are present in rheumatoid arthritis synovial fluid. Mouse MC protease-6 (mMCP-6) is the ortholog of hTryptase-beta, and this serine protease is abundant in the synovium of arthritic mice. We now report that C57BL/6 (B6) mice lacking their tryptase/heparin complexes have attenuated arthritic responses, with mMCP-6 as the dominant tryptase responsible for augmenting neutrophil infiltration in the K/BxN mouse serum-transfer arthritis model. While inflammation in this experimental arthritis model was not dependent on protease-activated receptor-2, it was dependent on the chemokine receptor CXCR2. In support of the latter data, exposure of synovial fibroblasts to hTryptase-beta/heparin or mMCP-6/heparin complexes resulted in expression of the neutrophil chemotactic factors CXCL1/KC, CXCL5/LIX, and CXCL8/IL-8. Our proteomics, histochemistry, and immunohistochemistry data also revealed substantial loss of cartilage-derived aggrecan proteoglycans in the arthritic joints of wild-type B6 mice but not mMCP-6-null B6 mice. These observations demonstrate the functional contribution of MC-restricted tryptase/heparin complexes in the K/BxN mouse arthritis model and connect our mouse findings with rheumatoid arthritis pathophysiology.
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Project description:BackgroundMast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation.MethodsHuman lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells.ResultsThe migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration.ConclusionsMast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases.
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Project description:Human embryonic stem cells (hESCs) can self-renew while maintaining their pluripotency. Direct reprogramming of adult somatic cells to induced pluripotent stem cells (iPSCs) has been reported. Although hESCs and human iPSCs have been shown to share a number of similarities, such basic properties as the electrophysiology of iPSCs have not been explored. Previously, we reported that several specialized ion channels are functionally expressed in hESCs. Using transcriptomic analyses as a guide, we observed tetraethylammonium (TEA)-sensitive (IC(50) = 3.3 +/- 2.7 mM) delayed rectifier K(+) currents (I(KDR)) in 105 of 110 single iPSCs (15.4 +/- 0.9 pF). I(KDR) in iPSCs displayed a current density of 7.6 +/- 3.8 pA/pF at +40 mV. The voltage for 50% activation (V(1/2)) was -7.9 +/- 2.0 mV, slope factor k = 9.1 +/- 1.5. However, Ca(2+)-activated K(+) current (I(KCa)), hyperpolarization-activated pacemaker current (I(f)), and voltage-gated sodium channel (Na(V)) and voltage-gated calcium channel (Ca(V)) currents could not be measured. TEA inhibited iPSC proliferation (EC(50) = 7.8 +/- 1.2 mM) and viability (EC(50) = 5.5 +/- 1.0 mM). By contrast, 4-aminopyridine (4-AP) inhibited viability (EC(50) = 4.5 +/- 0.5 mM) but had less effect on proliferation (EC(50) = 0.9 +/- 0.5 mM). Cell cycle analysis further revealed that K(+) channel blockers inhibited proliferation primarily by arresting the mitotic phase. TEA and 4-AP had no effect on iPSC differentiation as gauged by ability to form embryoid bodies and expression of germ layer markers after induction of differentiation. Neither iberiotoxin nor apamin had any function effects, consistent with the lack of I(KCa) in iPSCs. Our results reveal further differences and similarities between human iPSCs and hESCs. A better understanding of the basic biology of iPSCs may facilitate their ultimate clinical application.
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Project description:Mast cells (MCs) represent a population of hematopoietic cells with a key role in innate and adaptive immunity and are well known for their detrimental role in allergic responses. Yet, MCs occur in low abundance, which hampers their detailed molecular analysis. Here, we capitalized on the potential of induced pluripotent stem (iPS) cells to give rise to all cells in the body and established a novel and robust protocol for human iPS cell differentiation toward MCs. Relying on a panel of systemic mastocytosis (SM) patient-specific iPS cell lines carrying the KIT D816V mutation, we generated functional MCs that recapitulate SM disease features: increased number of MCs, abnormal maturation kinetics and activated phenotype, CD25 and CD30 surface expression and a transcriptional signature characterized by upregulated expression of innate and inflammatory response genes. Therefore, human iPS cell-derived MCs are a reliable, inexhaustible, and close-to-human tool for disease modeling and pharmacological screening to explore novel MC therapeutics.
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