Mast Cell Chymase Research Articles

Thymic Stromal Lymphopoietin (TSLP) Is Cleaved by Human Mast Cell Tryptase and Chymase.

Thymic stromal lymphopoietin (TSLP), mainly expressed by epithelial cells, plays a central role in asthma. In humans, TSLP exists in two variants: the long form TSLP (lfTSLP) and a shorter TSLP isoform (sfTSLP). Macrophages (HLMs) and mast cells (HLMCs) are in close proximity in the human lung and play key roles in asthma. We evaluated the early proteolytic effects of tryptase and chymase released by HLMCs on TSLP by mass spectrometry. We also investigated whether TSLP and its fragments generated by these enzymes induce angiogenic factor release from HLMs. Mass spectrometry (MS) allowed the identification of TSLP cleavage sites caused by tryptase and chymase. Recombinant human TSLP treated with recombinant tryptase showed the production of 1-97 and 98-132 fragments. Recombinant chymase treatment of TSLP generated two peptides, 1-36 and 37-132. lfTSLP induced the release of VEGF-A, the most potent angiogenic factor, from HLMs. By contrast, the four TSLP fragments generated by tryptase and chymase failed to activate HLMs. Long-term TSLP incubation with furin generated two peptides devoid of activating property on HLMs. These results unveil an intricate interplay between mast cell-derived proteases and TSLP. These findings have potential relevance in understanding novel aspects of asthma pathobiology.

EFFECT OF POST MORTEM INTERVAL TOWARD Β-TRYPTASE AND CHYMASE EXPRESSION OF ANAPHYLACTIC HEART MAST CELLS

Background: The effects of post mortem interval on tryptase levels are unclear and have been challenging to quantify due to limited and at times contradictory research. Purpose: Analyze the effects of Post Mortem Interval (PMI) due to anaphylactic shock on the expression of β-tryptase and mast cell chymase in the heart organ. Method: This type of research was an experimental study with a randomized block design (RBD) with the same subjects and a time series for 24 hours, using 5 (five) rabbits. The measurement of β-tryptase and chymase expression was based on immunohistochemical IRS (immunoreactive score) in the heart organs of rabbits experiencing anaphylactic shock sensitization and induction of anaphylactic shock using ovalbumin. Result: Based on the results of statistical tests using univariate analysis, there was a relationship between the length of death (post mortem interval) and the expression of mast cells β-Tryptase and cardiac chymase (p-value = 0.006) and chymase (p-value = 0.002) with (p-value <0.05). The effective test time with β-tryptase markers using cardiac organs was prone to long death time of 0 - 15 hours. The effective check-up time with chymase markers using the heart organ was at a long time of death of 9 hours. Conclusion: The results of the study showed that tryptase and chymase could be used as markers of anaphylactic shock.

Abstract 14288: α1-Antichymotrypsin Complex (SERPINA3) is an Independent Predictor of Long-Term All-Cause Mortality, but Did Not Predict CVD Events

Introduction: SERPINA3 is an acute phase protein triggered by inflammation. It inhibits neutrophil cathepsin which cleaves protease-activated receptor 4 (PAR-4) via a thrombin-independent pathway, and mast cell chymase which mediates the production of angiotensin-II via an ACE-independent pathway. SERPINA3 is upregulated after an acute myocardial infarction (MI), but data on its long-term prognostic value in MI patients are scarce. Aim: To assess the utility of SERPINA3 as a prognostic marker in patients hospitalized for chest pain of suspected coronary origin. Methods: A total of 871 consecutive patients were included (ClinicalTrials.gov Identifier: NCT00521976). Stepwise Cox regression models, applying continuous log e -transformed values, were fitted for each of the biomarkers with all-cause mortality and cardiac death within median 24-months (mo) and all-cause mortality within median 7 years (y) as the dependent variables, adding an analysis of 1) all-cause mortality or MI, and 2) all-cause mortality or MI or stroke at 7 y follow up (FU). The hazard ratio (HR) (95% CI) was assessed in a univariate and multivariable model, adjusting for traditional clinical cardiovascular risk factors, adding BNP, hsCRP, TnT. Results: Plasma samples from 847 patients were available. At 24 mo FU, 138 (15.8%) patients had died, of which 86 were cardiac deaths. The univariate analysis showed a positive, significant association between SERPINA3 and total death [HR 1.42 (95% 1.19-1.68), p =0.000], but was not significant for cardiac death. Associations after multivariable adjustment were statistically non-significant. At 7 y FU, 332 (38.1%) patients had died. After adjustment, SERPINA3 was independently associated with all-cause mortality [HR 1.14 (95% CI, 1.02-1.28), p=0.022], but not for MI (n=203), and stroke (n=55), respectively, and remained essentially unaffected for all-cause mortality or MI combined (n=404) [HR 1.12 (95% CI, 1.01-1.24), p=0.031], slightly weakened when stroke also was added (n=422) [HR 1.10 (95% CI, 1.00-1.22), p=0.060]. Conclusions: SERPINA3 predicts long-term all-cause mortality, but had no prognostic bearing on short-term cardiac death. Introducing non-fatal CVD events during long-term FU did not improve its prognostic value.

Febrile Phase Soluble Urokinase Plasminogen Activator Receptor and Olfactomedin 4 as Prognostic Biomarkers for Severe Dengue in Adults.

Dengue cases continue to rise and can overwhelm healthcare systems during outbreaks. In dengue, neutrophil mediators, soluble urokinase plasminogen activator receptor (suPAR) and olfactomedin 4, and mast cell mediators, chymase and tryptase, have not been measured longitudinally across the dengue phases. The utility of these proteins as prognostic biomarkers for severe dengue has also not been assessed in an older adult population. We prospectively enrolled 99 adults with dengue-40 dengue fever, 46 dengue with warning signs and 13 severe dengue, along with 30 controls. Plasma levels of suPAR, olfactomedin 4, chymase and tryptase were measured at the febrile, critical and recovery phases in dengue patients. The suPAR levels were significantly elevated in severe dengue compared to the other dengue severities and controls in the febrile (P < .001), critical (P < .001), and recovery (P = .005) phases. In the febrile phase, suPAR was a prognostic biomarker of severe dengue, with an AUROC of 0.82. Using a cutoff derived from Youden's index (5.4 ng/mL) and an estimated prevalence of severe dengue (16.5%) in our healthcare institution, the sensitivity was 71.4% with a specificity of 87.9% in the febrile phase, and the positive and negative predictive values were 54.7% and 95.8%, respectively. Olfactomedin 4 was elevated in dengue patients but not in proportion to disease severity in the febrile phase (P = .04) There were no significant differences in chymase and tryptase levels between dengue patients and controls. In adult dengue, suPAR may be a reliable prognostic biomarker for severe dengue in the febrile phase.

Aedes albopictus salivary adenosine deaminase is an immunomodulatory factor facilitating dengue virus replication

The Asian tiger mosquito, Aedes albopictus, is an important vector for the transmission of arboviruses such as dengue virus (DENV). Adenosine deaminase (ADA) is a well-characterized metabolic enzyme involved in facilitating blood feeding and (or) arbovirus transmission in some hematophagous insect species. We previously reported the immunologic function of ADA by investigating its effect on mast cell activation and the interaction with mast cell tryptase and chymase. The 2-D gel electrophoresis and mass spectrometry analysis in the current study revealed that ADA is present and upregulated following mosquito blood feeding, as confirmed by qRT-PCR and western blot. In addition, the recombinant ADA efficiently converted adenosine to inosine. Challenging the Raw264.7 and THP-1 cells with recombinant ADA resulted in the upregulation of IL-1β, IL-6, TNF-α, CCL2, IFN-β, and ISG15. The current study further identified recombinant ADA as a positive regulator in NF-κB signaling targeting TAK1. It was also found that recombinant Ae. albopictus ADA facilitates the replication of DENV-2. Compared with cells infected by DENV-2 alone, the co-incubation of recombinant ADA with DENV-2 substantially increased IL-1β, IL-6, TNF-α, and CCL2 gene transcripts in Raw264.7 and THP-1 cells. However, the expression of IFN-β and ISG15 were markedly downregulated in Raw264.7 cells but upregulated in THP-1 cells. These findings suggest that the immunomodulatory protein, Ae. albopictus ADA is involved in mosquito blood feeding and may modulate DENV transmission via macrophage or monocyte-driven immune response.

Space-Flight- and Microgravity-Dependent Alteration of Mast Cell Population and Protease Expression in Digestive Organs of Mongolian Gerbils.

Mast cell (MC)-specific proteases are of particular interest for space biology and medicine due to their biological activity in regulating targets of a specific tissue microenvironment. MC tryptase and chymase obtain the ability to remodel connective tissue through direct and indirect mechanisms. Yet, MC-specific protease expression under space flight conditions has not been adequately investigated. Using immunohistochemical stainings, we analyzed in this study the protease profile of the jejunal, gastric, and hepatic MC populations in three groups of Mongolian gerbils-vivarium control, synchronous experiment, and 12-day orbital flight on the Foton-M3 spacecraft-and in two groups-vivarium control and anti-orthostatic suspension-included in the experiment simulating effects of weightlessness in the ground-based conditions. After a space flight, there was a decreased number of MCs in the studied organs combined with an increased proportion of chymase-positive MCs and MCs with a simultaneous content of tryptase and chymase; the secretion of specific proteases into the extracellular matrix increased. These changes in the expression of proteases were observed both in the mucosal and connective tissue MC subpopulations of the stomach and jejunum. Notably, the relative content of tryptase-positive MCs in the studied organs of the digestive system decreased. Space flight conditions simulated in the synchronous experiment caused no similar significant changes in the protease profile of MC populations. The space flight conditions resulted in an increased chymase expression combined with a decreased total number of protease-positive MCs, apparently due to participating in the processes of extracellular matrix remodeling and regulating the state of the cardiovascular system.

Two granzyme A/K homologs in Zebra mbuna have different specificities, one classical tryptase and one with chymase activity

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTLs) and natural killer (NK) cells. The locus encoding these two proteases is the first of the hematopoietic serine protease loci to appear during vertebrate evolution. This locus is found in all jawed vertebrates including the cartilaginous fishes. Granzyme A is the most abundant of the different granzymes expressed by CTLs and NK cells and its potential function has been studied extensively for many years. However, no clear conclusions concerning its primary role in the immune defense has been obtained. In all mammals, there are only one copy each of granzyme A and K, whereas additional copies are found in both cartilaginous and ray finned fishes. In cichlids two of these copies seem to encode new members of the granzyme A/K family. These two new members appear to have changed primary specificity and to be pure chymases based on the amino acids in their active site substrate binding pockets. Interestingly, one of these gene copies is located in the middle of the granzyme A/K locus, while the other copy is present in another locus, the met-ase locus. We here present a detailed characterization of the extended cleavage specificity of one of these non-classical granzymes, a Zebra mbuna granzyme positioned in the granzyme A/K locus. This enzyme, named granzyme A2, showed a high preference for tyrosine in the P1 position of substrates, thereby being a strict chymase. We have also characterized one of the classical granzyme A/Ks of the Zebra mbuna, granzyme A1, which is a tryptase with preference for arginine in the P1 position of substrates. Based on their extended specificities, the two granzymes showed major similarities, but also some differences in preferred amino acids in positions surrounding the cleavable amino acid. Fish lack one of the hematopoietic serine protease loci of mammals, the chymase locus, where one of the major mast cell enzymes is located. An interesting question is now if cichlids have by compensatory mechanisms generated a mast cell chymase from another locus, and if similar chymotryptic enzymes have appeared also in other fish species.

Amyloid Beta Peptides Lead to Mast Cell Activation in a Novel 3D Hydrogel Model.

Alzheimer's disease (AD) is a prevalent neurodegenerative disease and the world's primary cause of dementia among the elderly population. The aggregation of toxic amyloid-beta (Aβ) is one of the main pathological hallmarks of the AD brain. Recently, neuroinflammation has been recognized as one of the major features of AD, which involves a network of interactions between immune cells. The mast cell (MC) is an innate immune cell type known to serve as a first responder to pathological changes and crosstalk with microglia and neurons. Although an increased number of mast cells were found near the sites of Aβ deposition, how mast cells are activated in AD is not clear. We developed a 3D culture system to culture MCs and investigated the activation of MCs by Aβ peptides. Because collagen I is the major component of extracellular matrix (ECM) in the brain, we encapsulated human LADR MCs in gels formed by collagen I. We found that 3D-cultured MCs survived and proliferated at the same level as MCs in suspension. Additionally, they can be induced to secrete inflammatory cytokines as well as MC proteases tryptase and chymase by typical MC activators interleukin 33 (IL-33) and IgE/anti-IgE. Culturing with peptides Aβ1-42, Aβ1-40, and Aβ25-35 caused MCs to secrete inflammatory mediators, with Aβ1-42 inducing the maximum level of activation. These data indicate that MCs respond to amyloid deposition to elicit inflammatory responses and demonstrate the validity of collagen gel as a model system to investigate MCs in a 3D environment to understand neuroinflammation in AD.

Massive platelet-rich thrombus formation in small pulmonary vessels in amniotic fluid embolism: an autopsy study.

To identify pulmonary/uterine thrombus formation in amniotic fluid embolism (AFE). Retrospective, observational. Nationwide. Eleven autopsy cases of AFE and control cases. We assessed pulmonary and uterine thrombus formation and thrombus area in AFE and pulmonary thromboembolism (PTE) as a control. The area of platelet glycoprotein IIb/IIIa, fibrin, neutrophil elastase, citrullinated histone H3 (a neutrophil extracellular trap marker) and mast cell chymase immunopositivity was measured in 90 pulmonary emboli, 15 uterine thrombi and 14 PTE. Pathological evidence of thrombus formation and its components in AFE. Amniotic fluid embolism lung showed massive thrombus formation, with or without amniotic emboli in small pulmonary arteries and capillaries. The median pulmonary thrombus size in AFE (median, 0.012 mm2 ; P < 0.0001) was significantly smaller than that of uterine thrombus in AFE (0.61 mm2 ) or PTE (29 mm2 ). The median area of glycoprotein IIb/IIIa immunopositivity in pulmonary thrombi in AFE (39%; P < 0.01) was significantly larger than that of uterine thrombi in AFE (23%) and PTE (15%). The median area of fibrin (0%; P < 0.001) and citrullinated histone H3 (0%; P < 0.01) immunopositivity in pulmonary thrombi in AFE was significantly smaller than in uterine thrombi (fibrin: 26%; citrullinated histone H3: 1.1%) and PTE (fibrin: 42%; citrullinated histone H3: 0.4%). No mast cells were identified in pulmonary thrombi. Amniotic fluid may induce distinct thrombus formation in the uterus and lung. Pulmonary and uterine thrombi formation may contribute to cardiorespiratory collapse and/or consumptive coagulopathy in AFE.

Identification of potential human chymase inhibitors using molecular docking and molecular dynamics simulation

Chymase is a hydrolase class of enzymes that involves  hydrolysis of peptide bonds. It is abundant in secretory granules of mast cells. Mast cell chymase is involved in the synthesis of angiotensin-II from its precursor protein. In addition, chymase is involved in converting TGF-β and matrix metalloproteinase to their active form. Chymase involved in heart failure has been proven, and its inhibition may reduce the progression. Hence, to identify the potential inhibitors against the chymase, the present study employed structure-based virtual screening, molecular docking, and molecular dynamics simulation to identify potential chymase inhibitors. Initially, compounds were selected based on their physicochemical and pharmacokinetic properties. Further, the binding affinities using molecular docking and interaction analyses were performed to find potential chymase inhibitors. The study identified chymase inhibitor ZINC000008382327, bearing significant binding affinity, specificity, and efficacy towards the chymase. Next, the stability and binding mode of chymase with ZINC000008382327 were assessed using molecular dynamics simulations. The simulation analysis using root mean square deviations and fluctuations revealed that inhibitor ZINC000008382327 affected the structure and dynamics of the chymase protein. It was also shown that the chymase forms a stable complex with ZINC000008382327 during the simulation. Thus, the present computational study put forward that the identified compound can be further exploited as a potential chemical scaffold to design and develop new human chymase inhibitors.

Expressions of β-Tryptase and Chymase in Lung Mast Cells due to Anaphylactic Shock through Histopathological Appearance at Different Post-Mortem Intervals

Highlights: The post-mortem interval is related to tryptase and chymase expressions in anaphylactic shock incidence Forensic experts can utilize tryptase and chymase as markers of anaphylactic (non-anaphylactoid) shock that occurs in the lungs. Abstract: Anaphylactic shock is a hypersensitivity response, a commonly type I hypersensitivity involving immunoglobulin E (IgE). It is caused by an antigen-antibody reaction that occurs immediately after a sensitive antigen enters the circulation. Anaphylactic shock is a clinical manifestation of anaphylaxis that is distributive shock, characterized by hypotension due to sudden blood vessel vasodilation and accompanied by a collapse in blood circulation that can result in death. β-tryptase and mast cell chymase expressions in the lungs of histopathological specimens that had experienced anaphylactic shock were examined at different post-mortem intervals in this study. A completely randomized design (CRD) method was employed by collecting lung samples every three hours within 24 hours of death, and then preparing histopathological and immunohistochemical preparations. The mast cell tryptase and chymase expressions were counted and summed up in each field of view, and the average was calculated to represent each field of view. The univariate analysis yielded p-values of 0.008 at the 15-hour post-mortem interval, and 0.002 at the 12-hour post-mortem interval. It was concluded that tryptase and chymase can be utilized as markers of anaphylactic (non-anaphylactoid) shock in the lungs.  

A Role for Mast Cell-Mediated Antibodies in the Formation of Cholesteatoma and Cholesteatoma-Induced Bone Erosion.

The study aimed to evaluate the effects and relationships between mast cells in the matrix, mast cell enzymes tryptase and chymase, epithelial proliferation, microvascular density, and bone destruction in cholesteatoma. Thirty-five biopsies diagnosed with cholesteatoma and seven healthy skin tissues taken from the retro-auricular region for control were evaluated. Immunohistochemical studies were performed with CD117, CD34, Ki-67, chymase, and tryptase antibodies, in a single session for all cases and the control group. The relationship between erosion size and antibody load was determined. The mean cholesteatoma epithelium Ki-67 was higher than the control group (p < 0.001). CD117-positive mast cells, chymase-positive mast cells, tryptase-positive mast cells, and microvessel density were significantly higher in the cholesteatoma matrix compared to the control group (p < 0.002, p < 0.001, p < 0.005). In the group with bone erosion scores of two and above, immunohistochemical markers tended to be higher. A positive correlation was found between CD117 and chymase, tryptase, and microvessel density; between tryptase, chymase, and microvessel density; and between chymase and microvessel density. CD117-positive mast cells and chymase-positive mast cells stimulate angiogenesis, increase the epithelium's proliferative capacity in the cholesteatoma matrix, and form cholesteatoma. The increased proliferation of cholesteatoma epithelium and increased vascular density in the matrix exacerbate bone erosion.

Mast cell chymase is in contact with melanoma cells in vivo and it detaches melanoma cells from the substratum in vitro.

Mast cell chymase, a chymotryptic serine proteinase, is a powerful enzyme that may liberate adherent tumor cells thereby promoting tumor spread. This study investigated interactions between cells containing immunoreactive chymase and melanoma cells in 101 melanoma and 50 nevus specimens as well as used recombinant human (rh)-chymase and WM115 and G361 primary melanoma cell lines in culture. Rh-chymase at low concentration (0.1 µg/mL) detached both melanoma cell lines from noncoated or collagen-coated plastic surface, but this effect was regulated by heparin and heparinase. After prolonged treatment at high rh-chymase (1-5 µg/mL) the growth pattern of detached and re-seeded cells was abnormal. Decreased migration and proliferation at up to 0.005-0.01 µg/mL rh-chymase was noticed in WM115 cells, while rh-chymase at 1-5 µg/mL partially reduced the viability of G361 cells. Of importance is the finding that melanoma cell lines are not uniform as there was variation between cell lines with regard to the concentration of rh-chymase needed for these effects. Sonicates prepared from the cell lysates did not inhibit the enzymatic activity of rh-chymase. The interactions were confirmed morphologically by detecting apparent contacts between chymase+ cells and melanoma cells in melanoma specimens. These results suggest that chymase can detach melanoma cells from the tumor, but this effect is regulated by heparin, and their proliferation, migration or viability can be reduced by chymase. Thus, the effect of chymase may be antitumorigenic, rather than protumorigenic.

Immune-related SERPINA3 as a biomarker involved in diabetic nephropathy renal tubular injury.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and has become a serious medical issue globally. Although it is known to be associated with glomerular injury, tubular injury has been found to participate in DN in recent years. However, mechanisms of diabetic renal tubular injury remain unclear. Here, we investigated the differentially expressed genes in the renal tubules of patients with DN by analyzing three RNA-seq datasets downloaded from the Gene Expression Omnibus database. Gene set enrichment analysis and weighted gene co-expression network analysis showed that DN is highly correlated with the immune system. The immune-related gene SERPINA3 was screened out with lasso regression and Kaplan–Meier survival analyses. Considering that SERPINA3 is an inhibitor of mast cell chymase, we examined the expression level of SERPINA3 and chymase in human renal tubular biopsies and found that SERPINA3 was upregulated in DN tubules, which is consistent with the results of the differential expression analysis. Besides, the infiltration and degranulation rates of mast cells are augmented in DN. By summarizing the biological function of SERPINA3, chymase, and mast cells in DN based on our results and those of previous studies, we speculated that SERPINA3 is a protective immune-related molecule that prevents renal tubular injury by inhibiting the proliferation and activation of mast cells and downregulating the activity of chymase.

Mast Cell Proteases Promote Diverse Effects on the Plasminogen Activation System and Wound Healing in A549 Alveolar Epithelial Cells.

Tissue damage, epithelial alterations, and intraepithelial presence of mast cells (MCs) are characteristics of asthma pathogenesis. Increased alveolar infiltration of MC populations has also been identified as a feature of asthma and other chronic respiratory diseases. The asthma associated receptor, urokinase plasminogen activator receptor (uPAR), has been shown to regulate bronchial epithelial repair responses. However, the impact of MC tryptase and chymase on functional properties and expression of uPAR in alveolar epithelial cells have not been fully investigated. Alveolar epithelial cell migration and wound healing were investigated using holographic live cell imaging of A549 cells in a wound scratch model post stimulation with tryptase or chymase. The expression of uPAR was investigated on the protein and gene level from cellular supernatants and in bronchoalveolar lavage fluid fractions from allergic asthmatics. We found that tryptase improved wound healing capacity, cellular migration and membrane bound uPAR expression. Chymase reduced gap closure capacity, cellular migration and membrane bound uPAR expression but increased soluble uPAR release. Our data suggest a dual regulatory response from the MC proteases in events related to uPAR expression and wound healing which could be important features in asthmatic disease.

Aerosol-Administered Adelmidrol Attenuates Lung Inflammation in a Murine Model of Acute Lung Injury.

Acute lung injury (ALI) is a common and devastating clinical disorder with a high mortality rate and no specific therapy. The pathophysiology of ALI is characterized by increased alveolar/capillary permeability, lung inflammation, oxidative stress and structural damage to lung tissues, which can progress to acute respiratory distress syndrome (ARDS). Adelmidrol (ADM), an analogue of palmitoylethanolamide (PEA), is known for its anti-inflammatory and antioxidant functions, which are mainly due to down-modulating mast cells (MCs) and promoting endogenous antioxidant defense. The aim of this study is to evaluate the protective effects of ADM in a mice model of ALI, induced by intratracheal administration of lipopolysaccharide (LPS) at the dose of 5 mg/kg. ADM 2% was administered by aerosol 1 and 6 h after LPS instillation. In this study, we clearly demonstrated that ADM reduced lung damage and airway infiltration induced by LPS instillation. At the same time, ADM counteracted the increase in MC number and the expression of specific markers of MC activation, i.e., chymase and tryptase. Moreover, ADM reduced oxidative stress by upregulating antioxidant enzymes as well as modulating the Nf-kB pathway and the resulting pro-inflammatory cytokine release. These results suggest that ADM could be a potential candidate in the management of ALI.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5′ and 3′ region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of β-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex

The pulmonary pathological findings associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result from the release of multiple proinflammatory cytokines, which causes the subsequential damage of the lungs. The current study was undertaken to investigate the responses of mast cells to viral inoculation and their contribution to host defenses from the point of view of viral entry. Pseudovirions, in which the spike glycoprotein of SARS-CoV-2 was incorporated, triggered activation of mast cells, and a mast cell-derived chymase, MCP2, formed a complex with spike protein, which promoted protease-dependent viral entry. According to the quantification results of viral entry, 10 μM quercetin, a mast cell stabilizer, potentially potently inhibited 41.3% of viral entry, while 100 μM chymostatin, which served as a chymase inhibitor, suppressed 52.1% of viral entry, compared to non-treated cells. Study using mast cell-deficient mice showed that the absence of mast cells may influence early viral loading in the upper respiratory tract, which consequently increases the risk of viral invasion into the lower respiratory system. Furthermore, mast cell-deficient mice exhibited ongoing infection in the late phase post-viral inoculation, while clearance of virus-positive cells was observed in wild-type mice. In conclusion, mast cells act as a multifaceted immune modulator that is equipped with both protective effects and pathogenic influences on viral entry of SARS-CoV-2. The utility of mast cell stabilizers and chymase inhibitors in the treatment of SARS-CoV-2-induced acute respiratory syndrome should be optimized regarding the infection stage and the risk of cytokine storm.

Mast cell chymase has a negative impact on human osteoblasts

Mast cells have been linked to osteoporosis and bone fractures, and in a previous study we found that mice lacking a major mast cell protease, chymase, develop increased diaphyseal bone mass. These findings introduce the possibility that mast cell chymase can regulate bone formation, but the underlying mechanism(s) has not previously been investigated. Here we hypothesized that chymase might exert such effects through a direct negative impact on osteoblasts, i.e., the main bone-building cells. Indeed, we show that chymase has a distinct impact on human primary osteoblasts. Firstly, chymase was shown to have pronounced effects on the morphological features of osteoblasts, including extensive cell contraction and actin reorganization. Chymase also caused a profound reduction in the output of collagen from the osteoblasts, and was shown to degrade osteoblast-secreted fibronectin and to activate pro-matrix metallopeptidase-2 released by the osteoblasts. Further, chymase was shown to have a preferential impact on the gene expression, protein output and phosphorylation status of TGFβ-associated signaling molecules. A transcriptomic analysis was conducted and revealed a significant effect of chymase on several genes of importance for bone metabolism, including a reduction in the expression of osteoprotegerin, which was confirmed at the protein level. Finally, we show that chymase interacts with human osteoblasts and is taken up by the cells. Altogether, the present findings provide a functional link between mast cell chymase and osteoblast function, and can form the basis for a further evaluation of chymase as a potential target for intervention in metabolic bone diseases.

Mast cell chymase regulates extracellular matrix remodeling-related events in primary human small airway epithelial cells

Mast cells are implicated in the pathogenesis of asthma, but the underlying mechanisms are not fully elucidated. Under asthmatic conditions, mast cells can relocalize to the epithelial layer and may thereby affect the functional properties of the airway epithelial cells. Activated mast cells release large quantities of proteases from their secretory granules, including chymase and tryptase. Here we investigated whether these proteases may affect airway epithelial cells. Primary small airway epithelial cells were treated with tryptase or chymase, and the effects on epithelial cell viability, proliferation, migration, cytokine output, and transcriptome were evaluated. Airway epithelial cells were relatively refractory to tryptase. In contrast, chymase had extensive effects on multiple features of the epithelial cells, with a particular emphasis on processes related to extracellular matrix (ECM) remodeling. These included suppressed expression of ECM-related genes such as matrix metalloproteinases, which was confirmed at the protein level. Further, chymase suppressed the expression of the fibronectin gene and also caused degradation of fibronectin released by the epithelial cells. Chymase was also shown to suppress the migratory capacity of the airway epithelial cells and to degrade the cell-cell contact protein E-cadherin on the epithelial cell surface. Our findings suggest that chymase may affect the regulation of ECM remodeling events mediated by airway epithelial cells, with implications for the impact of mast cells in inflammatory lung diseases such as asthma.