Biosynthesis of prostaglandin 15dPGJ2 -glutathione and 15dPGJ2-cysteine conjugates in macrophages and mast cells via MGST3.

Inhibition of microsomal prostaglandin E synthase-1 (mPGES-1) results in decreased production of proinflammatory PGE2 and can lead to shunting of PGH2 into the prostaglandin D2 (PGD2)/15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) pathway. 15dPGJ2 forms Michael adducts with thiol-containing biomolecules such as GSH or cysteine residues on target proteins and is thought to promote resolution of inflammation. We aimed to elucidate the biosynthesis and metabolism of 15dPGJ2 via conjugation with GSH, to form 15dPGJ2-glutathione (15dPGJ2-GS) and 15dPGJ2-cysteine (15dPGJ2-Cys) conjugates and to characterize the effects of mPGES-1 inhibition on the PGD2/15dPGJ2 pathway in mouse and human immune cells. Our results demonstrate the formation of PGD2, 15dPGJ2, 15dPGJ2-GS, and 15dPGJ2-Cys in RAW264.7 cells after lipopolysaccharide stimulation. Moreover, 15dPGJ2-Cys was found in lipopolysaccharide-activated primary murine macrophages as well as in human mast cells following stimulation of the IgE-receptor. Our results also suggest that the microsomal glutathione S-transferase 3 is essential for the formation of 15dPGJ2 conjugates. In contrast to inhibition of cyclooxygenase, which leads to blockage of the PGD2/15dPGJ2 pathway, we found that inhibition of mPGES-1 preserves PGD2 and its metabolites. Collectively, this study highlights the formation of 15dPGJ2-GS and 15dPGJ2-Cys in mouse and human immune cells, the involvement of microsomal glutathione S-transferase 3 in their biosynthesis, and their unchanged formation following inhibition of mPGES-1. The results encourage further research on their roles as bioactive lipid mediators.

Activation of succinate receptor 1 boosts human mast cell reactivity and allergic bronchoconstriction.

BACKGROUND: SUCNR1 is a sensor of extracellular succinate, a Krebs cycle intermediate generated in excess during oxidative stress and has been linked to metabolic regulation and inflammation. While mast cells express SUCNR1, its role in mast cell reactivity and allergic conditions such as asthma remains to be elucidated. METHODS: Cord blood-derived mast cells and human mast cell line LAD-2 challenged by SUCNR1 ligands were analyzed for the activation and mediator release. Effects on mast cell-dependent bronchoconstriction were assessed in guinea pig trachea and isolated human small bronchi challenged with antigen and anti-IgE, respectively. RESULTS: SUCNR1 is abundantly expressed on human mast cells. Challenge with succinate, or the synthetic non-metabolite agonist cis-epoxysuccinate, renders mast cells hypersensitive to IgE-dependent activation, resulting in augmented degranulation and histamine release, de novo biosynthesis of eicosanoids and cytokine secretion. The succinate-potentiated mast cell reactivity was attenuated by SUCNR1 knockdown and selective SUCNR1 antagonists and could be tuned by pharmacologically targeting protein kinase C and extracellular signal-regulated kinase. Both succinate and cis-epoxysuccinate dose-dependently potentiated antigen-induced contraction in a mast cell-dependent guinea pig airway model, associated with increased generation of cysteinyl-leukotrienes and histamine in trachea. Similarly, cis-epoxysuccinate aggravated IgE-receptor-induced contraction of human bronchi, which was blocked by SUCNR1 antagonism. CONCLUSION: SUCNR1 amplifies IgE-receptor-induced mast cell activation and allergic bronchoconstriction, suggesting a role for this pathway in aggravation of allergic asthma, thus linking metabolic perturbations to mast cell-dependent inflammation.

Changing the threshold-Signals and mechanisms of mast cell priming.

Mast cells play a key role in allergy and other inflammatory diseases involving engagement of multivalent antigen with IgE bound to high-affinity IgE receptors (FcεRIs). Aggregation of FcεRIs on mast cells initiates a cascade of signaling events that eventually lead to degranulation, secretion of leukotrienes and prostaglandins, and cytokine and chemokine production contributing to the inflammatory response. Exposure to pro-inflammatory cytokines, chemokines, bacterial and viral products, as well as some other biological products and drugs, induces mast cell transition from the basal state into a primed one, which leads to enhanced response to IgE-antigen complexes. Mast cell priming changes the threshold for antigen-mediated activation by various mechanisms, depending on the priming agent used, which alone usually do not induce mast cell degranulation. In this review, we describe the priming processes induced in mast cells by various cytokines (stem cell factor, interleukins-4, -6 and -33), chemokines, other agents acting through G protein-coupled receptors (adenosine, prostaglandin E2 , sphingosine-1-phosphate, and ß-2-adrenergic receptor agonists), toll-like receptors, and various drugs affecting the cytoskeleton. We will review the current knowledge about the molecular mechanisms behind priming of mast cells leading to degranulation and cytokine production and discuss the biological effects of mast cell priming induced by several cytokines.

Selective inhibition of prostaglandin D2 biosynthesis in human mast cells to overcome need for multiple receptor antagonists: Biochemical consequences.

BACKGROUND: The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. OBJECTIVES: To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. METHODS: Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. RESULTS: All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. CONCLUSIONS: Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

The Role of Mast Cells in Bacterial Infection.

Mast cells (MCs) are particularly abundant at host-environment interfaces, such as skin and intestinal mucosa. Because of their location, it has been hypothesized that MCs can act as sentinel cells that sense microbial attacks and initiate a protective immune response. Several studies have suggested that animals deficient in MCs exhibit a worsened pathology in various experimental models of bacterial infection. However, other studies have indicated that MCs under certain circumstances may have a detrimental impact on bacterial disease, and there are also recent studies indicating that MCs are dispensable for the clearance of bacterial pathogens. Herein, we review the current knowledge of the role of MCs in bacterial infection.

Active caspase-3 is stored within secretory compartments of viable mast cells.

Caspase-3 is a main executioner of apoptotic cell death. The general notion is that, in viable cells, caspase-3 is found as a cytosolic inactive proenzyme and that caspase-3 activation is largely confined to processes associated with cell death. In this study, we challenge this notion by showing that enzymatically active caspase-3 is stored in viable mast cells. The enzymatically active caspase-3 was undetectable in the cytosol of viable cells, but was recovered in subcellular fractions containing secretory granule-localized proteases. Moreover, active caspase-3 was rapidly released into the cytosolic compartment after permeabilization of the secretory granules. Using a cell-permeable substrate for caspase-3, the presence of active caspase-3-like activity in granule-like compartments close to the plasma membrane was demonstrated. Moreover, it was shown that mast cell activation caused release of the caspase-3 to the cell exterior. During the course of mast cell differentiation from bone marrow cells, procaspase-3 was present in cells of all stages of maturation. In contrast, active caspase-3 was undetectable in bone marrow precursor cells, but increased progressively during the process of mast cell maturation, its accumulation coinciding with that of a mast cell-specific secretory granule marker, mouse mast cell protease 6. Together, the current study suggests that active caspase-3 can be stored within secretory compartments of viable mast cells.

Mast cells are activated by Staphylococcus aureus in vitro but do not influence the outcome of intraperitoneal S. aureus infection in vivo.

Staphylococcus aureus is a major pathogen that can cause a broad spectrum of serious infections including skin infections, pneumonia and sepsis. Peritoneal mast cells have been implicated in the host response towards various bacterial insults and to provide mechanistic insight into the role of mast cells in intraperitoneal bacterial infection we here studied the global effects of S. aureus on mast cell gene expression. After co-culture of peritoneal mast cells with live S. aureus we found by gene array analysis that they up-regulate a number of genes. Many of these corresponded to pro-inflammatory cytokines, including interleukin-3, interleukin-13 and tumour necrosis factor-α. The cytokine induction in response to S. aureus was confirmed by ELISA. To study the role of peritoneal mast cells during in vivo infection with S. aureus we used newly developed Mcpt5-Cre(+) × R-DTA mice in which mast cell deficiency is independent of c-Kit. This is in contrast to previous studies in which an impact of mast cells on bacterial infection has been proposed based on the use of mice whose mast cell deficiency is a consequence of defective c-Kit signalling. Staphylococcus aureus was injected intraperitoneally into mast-cell-deficient Mcpt5-Cre(+) × R-DTA mice using littermate mast-cell-sufficient mice as controls. We did not observe any difference between mast-cell-deficient and control mice with regard to weight loss, bacterial clearance, inflammation or cytokine production. We conclude that, despite peritoneal mast cells being activated by S. aureus in vitro, they do not influence the in vivo manifestations of intraperitoneal S. aureus infection.

Granzyme H is a novel protease expressed by human mast cells.

BACKGROUND: Many of the functions attributed to mast cells depend on the various pro-inflammatory mediators that are secreted upon mast cell activation. These include a panel of mast cell-specific proteases. In addition, recent studies have indicated that murine mast cells also express granzyme D, a protease previously thought to be confined to cytotoxic lymphocytes. Here, we address the human relevance of the latter findings by investigating whether human mast cells express granzyme H, the granzyme that may represent the functional counterpart to murine granzyme D. METHODS: Cord blood-derived mast cells, LAD2 cells and skin mast cells in situ were evaluated for their expression of granzymes using quantitative PCR, Western blot analysis and immunostaining. Mast cells were activated by either calcium ionophore stimulation or IgE receptor cross-linking. RESULTS: Cord blood-derived mast cells and LAD2 cells were shown to express granzyme H and B mRNA, while granzyme A, K and M expression was undetectable. Mast cell activation by either calcium ionophore or IgE receptor cross-linking caused down-regulated expression of granzyme H. In contrast, granzyme B expression was up-regulated by the same stimuli. Granzyme H expression was also confirmed at the protein level, as shown by both Western blot analysis and confocal microscopy. Further, we show that granzyme H is expressed by human skin mast cells in situ. CONCLUSIONS: The present findings implicate granzyme H as a novel protease expressed by human mast cells and support earlier findings obtained in natural killer cells suggesting that granzymes B and H are reciprocally regulated.

An optimized method for IgE-mediated degranulation of human lung mast cells.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

Granzyme D is a novel murine mast cell protease that is highly induced by multiple pathways of mast cell activation.

Granzymes are serine proteases known mostly for their role in the induction of apoptosis. Granzymes A and B have been extensively studied, but relatively little is known about granzymes C to G and K to M. T cells, lymphohematopoietic stromal cells, and granulated metrial gland cells express granzyme D, but the function of granzyme D is unknown. Here we show that granzyme D is expressed by murine mast cells and that its level of expression correlates positively with the extent of mast cell maturation. Coculture of mast cells with live, Gram-positive bacteria caused a profound, Toll-like receptor 2 (TLR2)-dependent induction of granzyme D expression. Granzyme D expression was also induced by isolated bacterial cell wall components, including lipopolysaccharide (LPS) and peptidoglycan, and by stem cell factor, IgE receptor cross-linking, and calcium ionophore stimulation. Granzyme D was released into the medium in response to mast cell activation. Granzyme D induction was dependent on protein kinase C and nuclear factor of activated T cells (NFAT). Together, these findings identify granzyme D as a novel murine mast cell protease and implicate granzyme D in settings where mast cells are activated, such as bacterial infection and allergy.

ADAMTS: novel proteases expressed by activated mast cells.

Here we show that mast cells (MCs) express the metalloproteases of the A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, and that ADAMTS expression is influenced by MC activation. Co-culture of MCs with live Gram-positive bacteria caused a profound induction of ADAMTS-9 and -6, as well as down-regulated expression of ADAMTS-5. Similar patterns were also seen after MC activation with calcium ionophore and by immunoglobulin E receptor crosslinking. Moreover, ADAMTS-5, -6 and -9 were all induced by activation of terminally differentiated murine peritoneal MCs and in a human MC line. ADAMTS-9 up-regulation in response to immunoglobulin E receptor crosslinking was strongly dependent on Gö6976-sensitive protein kinase C and partly dependent on nuclear factor of activated T cells and nuclear factor kappa-light-chain-enhancer of activated B cells, respectively. The expression of ADAMTS-5, -6 and -9 was closely linked to MC maturation, as shown by their strong induction during the differentiation of bone marrow precursor cells into mature MCs. ADAMTS family members have been shown to possess aggrecanase activity. Accordingly, MCs were shown to express aggrecanase activity. Finally, ADAMTS-5 protein was detected in MCs by immunocytochemistry. Taken together, the present study reveals ADAMTS expression by MCs and that MC activation regulates the expression of these proteases, thus implicating the ADAMTS family of proteases in MC function.

Analysis of human lung mast cells by single cell RNA sequencing.

Mast cells are tissue-resident cells playing major roles in homeostasis and disease conditions. Lung mast cells are particularly important in airway inflammatory diseases such as asthma. Human mast cells are classically divided into the subsets MCT and MCTC, where MCT express the mast cell protease tryptase and MCTC in addition express chymase, carboxypeptidase A3 (CPA3) and cathepsin G. Apart from the disctintion of the MCT and MCTC subsets, little is known about the heterogeniety of human lung mast cells and a deep analysis of their heterogeniety has previously not been performed. We therefore performed single cell RNA sequencing on sorted human lung mast cells using SmartSeq2. The mast cells showed high expression of classical mast cell markers. The expression of several individual genes varied considerably among the cells, however, no subpopulations were detected by unbiased clustering. Variable genes included the protease-encoding transcripts CMA1 (chymase) and CTSG (cathepsin G). Human lung mast cells are predominantly of the MCT subset and consistent with this, the expression of CMA1 was only detectable in a small proportion of the cells, and correlated moderately to CTSG. However, in contrast to established data for the protein, CPA3 mRNA was high in all cells and the correlation of CPA3 to CMA1 was weak.

A role for serglycin proteoglycan in mast cell apoptosis induced by a secretory granule-mediated pathway.

Mast cell secretory granules (secretory lysosomes) contain large amounts of fully active proteases bound to serglycin proteoglycan. Damage to the granule membrane will thus lead to the release of serglycin and serglycin-bound proteases into the cytosol, which potentially could lead to proteolytic activation of cytosolic pro-apoptotic compounds. We therefore hypothesized that mast cells are susceptible to apoptosis induced by permeabilization of the granule membrane and that this process is serglycin-dependent. Indeed, we show that wild-type mast cells are highly sensitive to apoptosis induced by granule permeabilization, whereas serglycin-deficient cells are largely resistant. The reduced sensitivity of serglycin(-/-) cells to apoptosis was accompanied by reduced granule damage, reduced release of proteases into the cytosol, and defective caspase-3 activation. Mechanistically, the apoptosis-promoting effect of serglycin involved serglycin-dependent proteases, as indicated by reduced sensitivity to apoptosis and reduced caspase-3 activation in cells lacking individual mast cell-specific proteases. Together, these findings implicate serglycin proteoglycan as a novel player in mast cell apoptosis.

Mast cells express tyrosine hydroxylase and store dopamine in a serglycin-dependent manner.

Here we show that mast cells contain dopamine and that mast cell activation causes dopamine depletion, indicating its presence within secretory granules. Dopamine storage increased during mast cell maturation from bone marrow precursors, and was dependent on the presence of serglycin. Moreover, the expression of tyrosine hydroxylase, the key enzyme in dopamine biosynthesis, was induced during mast cell maturation; histidine decarboxylase and tryptophan hydroxylase 1 were also induced. Mast cell activation caused a robust induction of histidine decarboxylase, but no stimulation of tyrosine hydroxylase or tryptophan hydroxylase 1 expression. The present study points toward a possible role of dopamine in mast cell function.

Mast cell proteases: multifaceted regulators of inflammatory disease.

Mast cells (MCs) are currently receiving increased attention among the scientific community, largely because of the recent identification of crucial functions for MCs in a variety of disorders. However, it is in many cases not clear exactly how MCs contribute in the respective settings. MCs express extraordinarily high levels of a number of proteases of chymase, tryptase, and carboxypeptidase A type, and these are stored in high amounts as active enzymes in the MC secretory granules. Hence, MC degranulation leads to the massive release of fully active MC proteases, which probably have a major impact on any condition in which MC degranulation occurs. Indeed, the recent generation and evaluation of mouse strains lacking individual MC proteases have indicated crucial contributions of these to a number of different disorders. MC proteases may thus account for many of the effects ascribed to MCs and are currently emerging as promising candidates for treatment of MC-driven disease. In this review, we discuss these findings.

Mast cell differentiation and activation is closely linked to expression of genes coding for the serglycin proteoglycan core protein and a distinct set of chondroitin sulfate and heparin sulfotransferases.

Serglycin (SG) proteoglycan consists of a small core protein to which glycosaminoglycans of chondroitin sulfate or heparin type are attached. SG is crucial for maintaining mast cell (MC) granule homeostasis through promoting the storage of various basic granule constituents, where the degree of chondroitin sulfate/heparin sulfation is essential for optimal SG functionality. However, the regulation of the SG core protein expression and of the various chondroitin sulfate/heparin sulfotransferases during MC differentiation and activation are poorly understood. Here we addressed these issues and show that expression of the SG core protein, chondroitin 4-sulfotransferase (C4ST)-1, and GalNAc(4S)-6-O-sulfotransferase (GalNAc4S6ST) are closely linked to MC maturation. In contrast, the expression of chondroitin 6-sulfotransferase correlated negatively with MC maturation. The expression of N-deacetylase/N-sulfotransferase (NDST)-2, a key enzyme in heparin synthesis, also correlated strongly with MC maturation, whereas the expression of the NDST-1 isoform was approximately equal at all stages of maturation. MC activation by either calcium ionophore or IgE ligation caused an up-regulated expression of the SG core protein, C4ST-1, and GalNAc4S6ST, accompanied by increased secretion of chondroitin sulfate as shown by biosynthetic labeling experiments. In contrast, NDST-2 was down-regulated after MC activation, suggesting that MC activation modulates the nature of the glycosaminoglycan chains attached to the SG core protein. Taken together, these data show that MC maturation is associated with the expression of a distinct signature of genes involved in SG proteoglycan synthesis, and that MC activation modulates their expression.

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells.

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Immunoprofiling Reveals Novel Mast Cell Receptors and the Continuous Nature of Human Lung Mast Cell Heterogeneity.

Background: Immunohistochemical analysis of granule-associated proteases has revealed that human lung mast cells constitute a heterogeneous population of cells, with distinct subpopulations identified. However, a systematic and comprehensive analysis of cell-surface markers to study human lung mast cell heterogeneity has yet to be performed. Methods: Human lung mast cells were obtained from lung lobectomies, and the expression of 332 cell-surface markers was analyzed using flow cytometry and the LEGENDScreen™ kit. Markers that exhibited high variance were selected for additional analyses to reveal whether they were correlated and whether discrete mast cell subpopulations were discernable. Results: We identified the expression of 102 surface markers on human lung mast cells, 23 previously not described on mast cells, of which several showed high continuous variation in their expression. Six of these markers were correlated: SUSD2, CD49a, CD326, CD34, CD66 and HLA-DR. The expression of these markers was also correlated with the size and granularity of mast cells. However, no marker produced an expression profile consistent with a bi- or multimodal distribution. Conclusions: LEGENDScreen analysis identified more than 100 cell-surface markers on mast cells, including 23 that, to the best of our knowledge, have not been previously described on human mast cells. The comprehensive expression profiling of the 332 surface markers did not identify distinct mast cell subpopulations. Instead, we demonstrate the continuous nature of human lung mast cell heterogeneity.

Infection of mast cells with live streptococci causes a toll-like receptor 2- and cell-cell contact-dependent cytokine and chemokine response.

Mast cells (MCs) are strongly implicated in immunity toward bacterial infection, but the molecular mechanisms by which MCs contribute to the host response are only partially understood. We addressed this issue by examining the direct effects of a Gram-positive pathogen, Streptococcus equi, on bone marrow-derived MCs (BMMCs). Ultrastructural analysis revealed extensive formation of dilated rough endoplasmic reticulum in response to bacterial infection, indicating strong induction of protein synthesis. However, the BMMCs did not show signs of extensive degranulation, and this was supported by only slow release of histamine in response to infection. Coculture of live bacteria with BMMCs caused a profound secretion of CCL2/MCP-1, CCL7/MCP-3, CXCL2/MIP-2, CCL5/RANTES, interleukin-4 (IL-4), IL-6, IL-12, IL-13, and tumor necrosis factor alpha, as shown by antibody-based cytokine/chemokine arrays and/or enzyme-linked immunosorbent assay. In contrast, heat-inactivated bacteria caused only minimal cytokine/chemokine release. The cytokine/chemokine responses were substantially attenuated in Toll-like receptor 2-deficient BMMCs and were strongly dependent on cell-cell contacts between bacteria and BMMCs. Gene chip microarray analysis confirmed a massively upregulated expression of the genes coding for the secreted cytokines and chemokines and also identified a pronounced upregulation of numerous additional genes, including transcription factors, signaling molecules, and proteases. Together, the present study outlines MC-dependent molecular events associated with Gram-positive infection and thus provides an advancement in our understanding of how MCs may contribute to host defense toward bacterial insults.

Amelioration of Compound 48/80-Mediated Itch and LL-37-Induced Inflammation by a Single-Stranded Oligonucleotide.

Numerous inflammatory skin disorders display a high prevalence of itch. The Mas-related G protein coupled receptor X2 (MRGPRX2) has been shown to modulate itch by inducing non-IgE-mediated mast cell degranulation and the release of endogenous inducers of pruritus. Various substances collectively known as basic secretagogues, which include inflammatory peptides and certain drugs, can trigger MRGPRX2 and thereby induce pseudo-allergic reactions characterized by histamine and protease release as well as inflammation. Here, we investigated the capacity of an immunomodulatory single-stranded oligonucleotide (ssON) to modulate IgE-independent mast cell degranulation and, more specifically, its ability to inhibit the basic secretagogues compound 48/80 (C48/80)-and LL-37 in vitro and in vivo. We examined the effect of ssON on MRGPRX2 activation in vitro by measuring degranulation in a human mast cell line (LAD2) and calcium influx in MRGPRX2-transfected HEK293 cells. To determine the effect of ssON on itch, we performed behavioral studies in established mouse models and collected skin biopsies for histological analysis. Additionally, with the use of a rosacea mouse model and RT-qPCR, we investigated the effect on ssON on LL-37-induced inflammation. We reveal that both mast cell degranulation and calcium influx in MRGPRX2 transfected HEK293 cells, induced by the antimicrobial peptide LL-37 and the basic secretagogue C48/80, are effectively inhibited by ssON in a dose-dependent manner. Further, ssON demonstrates a capability to inhibit LL-37 and C48/80 activation in vivo in two mouse models. We show that intradermal injection of ssON in mice is able to block itch induced via C48/80 in a dose-dependent manner. Histological staining revealed that ssON inhibits acute mast cell degranulation in murine skin treated with C48/80. Lastly, we show that ssON treatment ameliorates LL-37-induced inflammation in a rosacea mouse model. Since there is a need for new therapeutics targeting non-IgE-mediated activation of mast cells, ssON could be used as a prospective drug candidate to resolve itch and inflammation in certain dermatoses.