The Chymase, Mouse Mast Cell Protease 4, Constitutes the Major Chymotrypsin-like Activity in Peritoneum and Ear Tissue. A Role for Mouse Mast Cell Protease 4 in Thrombin Regulation and Fibronectin Turnover

To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 null cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 null peritoneal cells to process endogenous fibronectin

Serglycin-Deficiency Causes Reduced Weight Gain and Changed Intestinal Cytokine Responses in Mice Infected With Giardia intestinalis

The proteoglycan serglycin (SG) is expressed by different innate and adaptive immune cells, e.g. mast cells, macrophages, neutrophils, and cytotoxic T lymphocytes, where SG contributes to correct granule storage and extracellular activity of inflammatory mediators. Here the serglycin-deficient (SG(-/-)) mouse strain was used to investigate the impact of SG on intestinal immune responses during infection with the non-invasive protozoan parasite Giardia intestinalis. Young (asymptotic to 11 weeks old) oral gavage-infected congenic SG(-/-) mice showed reduced weight gain as compared with the infected SG(+/+) littermate mice and the PBS-challenged SG(-/-) and SG(+/+) littermate mice. The infection caused no major morphological changes in the small intestine. However, a SG-independent increased goblet cell and granulocyte cell count was observed, which did not correlate with an increased myeloperoxidase or neutrophil elastase activity. Furthermore, infected mice showed increased serum IL-6 levels, with significantly reduced serum IL-6 levels in infected SG-deficient mice and decreased intestinal expression levels of IL-6 in the infected SG-deficient mice. In infected mice the qPCR analysis of alarmins, chemokines, cytokines, and nitric oxide synthases (NOS), showed that the SG-deficiency caused reduced intestinal expression levels of TNF-alpha and CXCL2, and increased IFN-gamma, CXCL1, and NOS1 levels as compared with SG-competent mice. This study shows that SG plays a regulatory role in intestinal immune responses, reflected by changes in chemokine and cytokine expression levels and a delayed weight gain in young SG(-/-) mice infected with G. intestinalis

Giardia excretory-secretory proteins modulate the enzymatic activities of mast cell chymase and tryptase

Background: Mast cells are involved in the host immune response controlling infection with the non-invasive intestinal protozoan parasite Giardia intestinalis. Experimental infections in rodents with G. intestinalis showed increased intestinal expression of mucosal and connective mast cell specific proteases suggesting that both mucosal and connective tissue mast cells are recruited and activated during infection. During infection Giardia excretory-secretory proteins (ESPs) with immunomodulatory capacity are released. However, studies investigating potential interactions between Giardia ESPs and the connective tissue mast cell specific serine proteases, ie. human chymase and mouse mast cell protease (mMCP)-4 and, human and mouse tryptase (mMCP-6) remain scarce.Results: We first investigated if soluble Giardia proteins (sGPs), which over-lap extensively in protein content with ESP fractions, from the isolates GS, WB and H3, could induce mast cell activation. sGPs induced a minor activation of bone marrow derived mucosal-like mast cells, as indicated by increased IL-6 secretion and no degranulation. Furthermore, sGPs were highly resistant to degradation by human tryptase while human chymase degraded a 65 kDa sGP and, wild-type mouse ear tissue extracts degraded several protein bands in the 10 to 75 kDa range. In striking contrast, sGPs and ESPs were found to increase the enzymatic activity of human and mouse tryptase and to reduce the activity of human and mouse chymase.Conclusion: Our finding suggests that Giardia ssp. via enhancement or reduction of mast cell protease activity may modulate mast cell-driven intestinal immune responses. ESP-mediated modulation of the mast cell specific proteases may also increase degradation of tight junctions, which may be beneficial for Giardia ssp. during infection

The Chymase Mouse Mast Cell Protease-4 Regulates Intestinal Cytokine Expression in Mature Adult Mice Infected with Giardia intestinalis

Mast cells have been shown to affect the control of infections with the protozoan parasite Giardia intestinalis. Recently, we demonstrated that Giardia excretory-secretory proteins inhibited the activity of the connective tissue mast cell-specific protease chymase. To study the potential role of the chymase mouse mast cell protease (mMCP)-4 during infections with Giardia, mMCP-4(+/+) and mMCP-4(-/-) littermate mice were gavage-infected with G. intestinalis trophozoites of the human assemblage B isolate GS. No significant changes in weight gain was observed in infected young (approximate to 10 weeks old) mMCP-4(-/-) and mMCP-4(+/+) littermate mice. In contrast, infections of mature adult mice (>18 weeks old) caused significant weight loss as compared to uninfected control mice. We detected a more rapid weight loss in mMCP-4(-/-) mice as compared to littermate mMCP-4(+/+) mice. Submucosal mast cell and granulocyte counts in jejunum increased in the infected adult mMCP-4(-/-) and mMCP-4(+/+) mice. This increase was correlated with an augmented intestinal trypsin-like and chymotrypsin-like activity, but the myeloperoxidase activity was constant. Infected mice showed a significantly lower intestinal neutrophil elastase (NE) activity, and in vitro, soluble Giardia proteins inhibited human recombinant NE. Serum levels of IL-6 were significantly increased eight and 13 days post infection (dpi), while intestinal IL-6 levels showed a trend to significant increase 8 dpi. Strikingly, the lack of mMCP-4 resulted in significantly less intestinal transcriptional upregulation of IL-6, TNF-alpha, IL-25, CXCL2, IL-2, IL-4, IL-5, and IL-10 in the Giardia-infected mature adult mice, suggesting that chymase may play a regulatory role in intestinal cytokine responses

Transcriptomics of ivermectin response in Caenorhabditis elegans: Integrating abamectin quantitative trait loci and comparison to the Ivermectin-exposed DA1316 strain

Parasitic nematodes pose a significant threat to human and animal health, as well as cause economic losses in the agricultural sector. The use of anthelmintic drugs, such as Ivermectin (IVM), to control these parasites has led to widespread drug resistance. Identifying genetic markers of resistance in parasitic nematodes can be challenging, but the free-living nematode Caenorhabditis elegans provides a suitable model. In this study, we aimed to analyze the transcriptomes of adult C. elegans worms of the N2 strain exposed to the anthelmintic drug Ivermectin (IVM), and compare them to those of the resistant strain DA1316 and the recently identified Abamectin Quantitative Trait Loci (QTL) on chromosome V. We exposed pools of 300 adult N2 worms to IVM (10(-7) and 10(-8) M) for 4 hours at 20 degrees C, extracted total RNA and sequenced it on the Illumina NovaSeq6000 platform. Differentially expressed genes (DEGs) were determined using an in-house pipeline. The DEGs were compared to genes from a previous microarray study on IVM-resistant C. elegans and Abamectin-QTL. Our results revealed 615 DEGs (183 up-regulated and 432 down-regulated genes) from diverse gene families in the N2 C. elegans strain. Of these DEGs, 31 overlapped with genes from IVM-exposed adult worms of the DA1316 strain. We identified 19 genes, including the folate transporter (folt-2) and the transmembrane transporter (T22F3.11), which exhibited an opposite expression in N2 and the DA1316 strain and were deemed potential candidates. Additionally, we compiled a list of potential candidates for further research including T-type calcium channel (cca-1), potassium chloride cotransporter (kcc-2), as well as other genes such as glutamate-gated channel (glc-1) that mapped to the Abamectin-QTL

Quantitative Transcriptome Analysis of Purified Equine Mast Cells Identifies a Dominant Mucosal Mast Cell Population with Possible Inflammatory Functions in Airways of Asthmatic Horses

Asthma is a chronic inflammatory airway disease and a serious health problem in horses as well as in humans. In humans and mice, mast cells (MCs) are known to be directly involved in asthma pathology and subtypes of MCs accumulate in different lung and airway compartments. The role and phenotype of MCs in equine asthma has not been well documented, although an accumulation of MCs in bronchoalveolar lavage fluid (BALF) is frequently seen. To characterize the phenotype of airway MCs in equine asthma we here developed a protocol, based on MACS Tyto sorting, resulting in the isolation of 92.9% pure MCs from horse BALF. We then used quantitative transcriptome analyses to determine the gene expression profile of the purified MCs compared with total BALF cells. We found that the MCs exhibited a protease profile typical for the classical mucosal MC subtype, as demonstrated by the expression of tryptase (TPSB2) alone, with no expression of chymase (CMA1) or carboxypeptidase A3 (CPA3). Moreover, the expression of genes involved in antigen presentation and complement activation strongly implicates an inflammatory role for these MCs. This study provides a first insight into the phenotype of equine MCs in BALF and their potential role in the airways of asthmatic horses

Aedes albopictus salivary proteins adenosine deaminase and 34k2 interact with human mast cell specific proteases tryptase and chymase

When mosquitoes probe to feed blood, they inoculate a mixture of salivary molecules into vertebrate hosts' skin causing acute inflammatory reactions where mast cell-derived mediators are involved. Mosquito saliva contains many proteins with largely unknown biological functions. Here, two Aedes albopictus salivary proteins - adenosine deaminase (alADA) and al34k2 - were investigated for their immunological impact on mast cells and two mast cell-specific proteases, the tryptase and the chymase. Mouse bone marrow-derived mast cells were challenged with increased concentrations of recombinant alADA or al34k2 for 1, 3, and 6 h, and to measure mast cell activation, the activity levels of beta-hexosaminidase and tryptase and secretion of IL-6 were evaluated. In addition, a direct interaction between alADA or al34k2 with tryptase or chymase was investigated. Results show that bone marrow-derived mast cells challenged with 10 mu g/ml of alADA secreted significant levels of beta-hexosaminidase, tryptase, and IL-6. Furthermore, both al34k2 and alADA are cut by human tryptase and chymase. Interestingly, al34k2 dose-dependently enhance enzymatic activity of both tryptase and chymase. In contrast, while alADA enhances the enzymatic activity of tryptase, chymase activity was inhibited. Our finding suggests that alADA and al34k2 via interaction with mast cell-specific proteases tryptase and chymase modulate mast cell-driven immune response in the local skin microenvironment. alADA- and al34k2-mediated modulation of tryptase and chymase may also recruit more inflammatory cells and induce vascular leakage, which may contribute to the inflammatory responses at the mosquito bite site

Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis

Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin, a proteoglycan with heparin side chains. Hence, serglycin-protease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation, whereas serglycin(-/-) MCs completely lacked this ability. Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist, which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex. Moreover, IL-13 degradation was abrogated in MC-CPA(-/-) MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein. Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4(-/-)) mice and Mcpt4(+/+) C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4(-/-) mice compared with Mcpt4(+/+) controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4(-/-) mice. Relative to infected Mcpt4(+/+) mice, ileal MC accumulation in Mcpt4(-/-) mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4(+/+) but not Mcpt4(-/-) mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-alpha, IL-12p40 and IL-3 were observed in Mcpt4(-/-) mice, while levels of IL-2, IL-10 and MIP1 beta (CCL4) were increased over the same period in Mcpt4(+/+) mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4(-/-) mice and type-2 response in Mcpt4(+/+) mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4(-/-) parasitemia with TNF-alpha and IFN-gamma, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4(+/+) mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4(-/-) mice compared with infected Mcpt4(+/+) mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-alpha and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility

Mast Cell Degranulation Exacerbates Skin Rejection by Enhancing Neutrophil Recruitment

Recent evidences indicate an important role of tissue inflammatory responses by innate immune cells in allograft acceptance and survival. Here we investigated the role of mast cells (MC) in an acute male to female skin allograft rejection model using red MC and basophil (RMB) mice enabling conditional MC depletion. Kinetic analysis showed that MCs markedly accelerate skin rejection. They induced an early inflammatory response through degranulation and boosted local synthesis of KC, MIP-2, and TNF. This enhanced early neutrophil infiltration compared to a female-female graft-associated repair response. The uncontrolled neutrophil influx accelerated rejection as antibody-mediated depletion of neutrophils delayed skin rejection. Administration of cromolyn, a MC stabilizer and to a lesser extent ketotifen, a histamine type I receptor antagonist, and absence of MCPT4 chymase also delayed graft rejection. Together our data indicate that mediators contained in secretory granules of MC promote an inflammatory response with enhanced neutrophil infiltration that accelerate graft rejection