Antagonism of Protease Activated Receptor-2 by GB88 Reduces Inflammation Triggered by Protease Allergen Tyr-p3.

The occurrence of allergic diseases induced by aeroallergens has increased in the past decades. Among inhalant allergens, mites remain the important causal agent of allergic diseases. Storage mites- Tyrophagus putrescentiae are found in stored products or domestic environments. Major allergen Tyr-p3 plays a significant role in triggering IgE-mediated hypersensitivity. However, its effects on pulmonary inflammation, internalization, and activation in human epithelium remain elusive. Protease-activated receptors (PARs) are activated upon cleavage by proteases. A549 cells were used as an epithelial model to examine the PAR activation by Tyr-p3 and therapeutic potential of PAR-2 antagonist (GB88) in allergic responses. Enzymatic properties and allergen localization of Tyr-p3 were performed. The release of inflammatory mediators, phosphorylation of mitogen-activated protein kinase (MAPK), and cell junction disruptions were evaluated after Tyr-p3 challenge. Enzymatic properties determined by substrate digestion and protease inhibitors indicated that Tyr-p3 processes a trypsin-like serine protease activity. The PAR-2 mRNA levels were significantly increased by nTyr-p3 but inhibited by protease inhibitors or GB88. Protease allergen of nTyr-p3 significantly increased the levels of pro-inflammatory cytokines (IL-6 and TNF-α), chemokine (IL-8), and IL-1ß in epithelial cells. nTyr-p3 markedly increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and MAP kinase. When cells were pretreated with GB88 then added nTyr-p3, the phosphorylated ERK1/2 did not inhibit by GB88. GB88 increased ERK1/2 phosphorylation in human epithelium cells. GB88 is able to block PAR-2-mediated calcium signaling which inhibits the nTyr-p3-induced Ca2+ release. Among the pharmacologic inhibitors, the most effective inhibitor of the nTyr-p3 in the induction of IL-8 or IL-1ß levels was GB88 followed by SBTI, MAPK/ERK, ERK, and p38 inhibitors. Levels of inflammatory mediators, including GM-CSF, VEGF, COX-2, TSLP, and IL-33 were reduced by treatment of GB88 or SBTI. Further, GB88 treatment down-regulated the nTyr-p3-induced PAR-2 expression in allergic patients with asthma or rhinitis. Tight junction and adherens junction were disrupted in epithelial cells by nTyr-p3 exposure; however, this effect was avoided by GB88. Immunostaining with frozen sections of the mite body showed the presence of Tyr-p3 throughout the intestinal digestive system, especially in the hindgut around the excretion site. In conclusion, our findings suggest that Tyr-p3 from domestic mites leads to disruption of the airway epithelial barrier after inhalation. Proteolytic activity of Tyr-p3 causes the PAR-2 mRNA expression, thus leading to the release of numerous inflammatory mediators. Antagonism of PAR2 activity suggests GB88 as the therapeutic potential for anti-inflammation medicine, especially in allergy development triggered by protease allergens.

Macrophage TLR4 and PAR2 Signaling: Role in Regulating Vascular Inflammatory Injury and Repair.

Macrophages play a central role in dictating the tissue response to infection and orchestrating subsequent repair of the damage. In this context, macrophages residing in the lungs continuously sense and discriminate among a wide range of insults to initiate the immune responses important to host-defense. Inflammatory tissue injury also leads to activation of proteases, and thereby the coagulation pathway, to optimize injury and repair post-infection. However, long-lasting inflammatory triggers from macrophages can impair the lung's ability to recover from severe injury, leading to increased lung vascular permeability and neutrophilic injury, hallmarks of Acute Lung Injury (ALI). In this review, we discuss the roles of toll-like receptor 4 (TLR4) and protease activating receptor 2 (PAR2) expressed on the macrophage cell-surface in regulating lung vascular inflammatory signaling.

A role for mast cells and mast cell tryptase in driving neutrophil recruitment in LPS-induced lung inflammation via protease-activated receptor 2 in mice.

OBJECTIVE: This study aims to investigate the role of protease-activated receptor (PAR) 2 and mast cell (MC) tryptase in LPS-induced lung inflammation and neutrophil recruitment in the lungs of C57BL/6 mice. METHODS: C57BL/6 mice were pretreated with the PAR2 antagonist ENMD-1068, compound 48/80 or aprotinin prior to intranasal instillation of MC tryptase or LPS. Blood leukocytes, C-X-C motif chemokine ligand (CXCL) 1 production leukocytes recovered from bronchoalveolar lavage fluid (BALF), and histopathological analysis of the lung were evaluated 4 h later. Furthermore, we performed experiments to determine intracellular calcium signaling in RAW 264.7 cells stimulated with LPS in the presence or absence of a protease inhibitor cocktail or ENMD-1068 and evaluated PAR2 expression in the lungs of LPS-treated mice. RESULTS: Pharmacological blockade of PAR2 or inhibition of proteases reduced neutrophils recovered in BALF and LPS-induced calcium signaling. PAR2 blockade impaired LPS-induced lung inflammation, PAR2 expression in the lung and CXCL1 release in BALF, and increased circulating blood neutrophils. Intranasal instillation of MC tryptase increased the number of neutrophils recovered in BALF, and MC depletion with compound 48/80 impaired LPS-induced neutrophil migration. CONCLUSION: Our study provides, for the first time, evidence of a pivotal role for MCs and MC tryptase in neutrophil migration, lung inflammation and macrophage activation triggered by LPS, by a mechanism dependent on PAR2 activation.

The Transient Receptor Potential Channel Vanilloid 1 Is Critical in Innate Airway Epithelial Responses to Protease Allergens.

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis.

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators. Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel. Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2. Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Aspergillus fumigatus-Secreted Alkaline Protease 1 Mediates Airways Hyperresponsiveness in Severe Asthma.

The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus, can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.

Triggering NETosis via protease-activated receptor (PAR)-2 signaling as a mechanism of hijacking neutrophils function for pathogen benefits.

Neutrophil-derived networks of DNA-composed extracellular fibers covered with antimicrobial molecules, referred to as neutrophil extracellular traps (NETs), are recognized as a physiological microbicidal mechanism of innate immunity. The formation of NETs is also classified as a model of a cell death called NETosis. Despite intensive research on the NETs formation in response to pathogens, the role of specific bacteria-derived virulence factors in this process, although postulated, is still poorly understood. The aim of our study was to determine the role of gingipains, cysteine proteases responsible for the virulence of P. gingivalis, on the NETosis process induced by this major periodontopathogen. We showed that NETosis triggered by P. gingivalis is gingipain dependent since in the stark contrast to the wild-type strain (W83) the gingipain-null mutant strain only slightly induced the NETs formation. Furthermore, the direct effect of proteases on NETosis was documented using purified gingipains. Notably, the induction of NETosis was dependent on the catalytic activity of gingipains, since proteolytically inactive forms of enzymes showed reduced ability to trigger the NETs formation. Mechanistically, gingipain-induced NETosis was dependent on proteolytic activation of protease-activated receptor-2 (PAR-2). Intriguingly, both P. gingivalis and purified Arg-specific gingipains (Rgp) induced NETs that not only lacked bactericidal activity but instead stimulated the growth of bacteria species otherwise susceptible to killing in NETs. This protection was executed by proteolysis of bactericidal components of NETs. Taken together, gingipains play a dual role in NETosis: they are the potent direct inducers of NETs formation but in the same time, their activity prevents P. gingivalis entrapment and subsequent killing. This may explain a paradox that despite the massive accumulation of neutrophils and NETs formation in periodontal pockets periodontal pathogens and associated pathobionts thrive in this environment.

Duodenal bacterial proteolytic activity determines sensitivity to dietary antigen through protease-activated receptor-2.

Microbe-host interactions are generally homeostatic, but when dysfunctional, they can incite food sensitivities and chronic diseases. Celiac disease (CeD) is a food sensitivity characterized by a breakdown of oral tolerance to gluten proteins in genetically predisposed individuals, although the underlying mechanisms are incompletely understood. Here we show that duodenal biopsies from patients with active CeD have increased proteolytic activity against gluten substrates that correlates with increased Proteobacteria abundance, including Pseudomonas. Using Pseudomonas aeruginosa producing elastase as a model, we show gluten-independent, PAR-2 mediated upregulation of inflammatory pathways in C57BL/6 mice without villus blunting. In mice expressing CeD risk genes, P. aeruginosa elastase synergizes with gluten to induce more severe inflammation that is associated with moderate villus blunting. These results demonstrate that proteases expressed by opportunistic pathogens impact host immune responses that are relevant to the development of food sensitivities, independently of the trigger antigen.

Dust mite-derived Der f 3 activates a pro-inflammatory program in airway epithelial cells via PAR-1 and PAR-2.

Protease activity of allergens has been suggested to be involved in the pathogenesis of allergic diseases. The major allergen Der f 3 from Dermatophagoides farinae harbors serine protease activity, but its immunopathogenesis remains unclear. This study aims to explore the effect of Der f 3 on the airway epithelial barrier and on the molecular pathways by which Der f 3 induces inflammation. RNA-seq was performed to identify differentially expressed genes in bronchial airway epithelial cells (AEC) between native Der f 3 and heat-inactivated (H) Der f 3, coupled with real-time PCR (RT-PCR) and ELISA for validation. Unlike other protease allergens such as that induce Th2-promoting alarmins (IL-25, IL-33, TSLP) in AECs, Der f 3 induced pro-inflammatory cytokines and chemokines including IL-6, IL-8 and GM-CSF, which are known to promote Th17 response. These pro-inflammatory mediators were induced by Der f 3 via the MAPK and NF-κB pathways as well as the store-operated calcium signaling. Gene silencing with small interfering RNA in A549 and BEAS-2B cells indicated that activation of AECs by Der f 3 was mainly dependent on protease-activated receptor 2 (PAR-2), while PAR-1 was also required for the full activation of AECs. Double knock-down of PAR-1 and PAR-2 largely impaired Der f 3-inducecd IL-8 production and subsequent signaling pathways. Our data suggest that Der f 3 induces pro-inflammatory mediators in human epithelial cell lines via the PARs-MAPK-NF-κB axis. Our results provide a molecular mechanism by which Der f 3 may trigger the Th17-skewed allergic response toward house dust mites.

PAR2 Pepducin-Based Suppression of Inflammation and Itch in Atopic Dermatitis Models.

PAR2 has been proposed to contribute to lesion formation and intense itch in atopic dermatitis. Here, we tested the ability of a cell-penetrating pepducin, PZ-235, to mitigate the potentially deleterious effects of PAR2 in models of atopic dermatitis. PZ-235 significantly inhibited PAR2-mediated expression of inflammatory factors NF-κB, TSLP, TNF-α, and differentiation marker K10 by 94%-98% (P < 0.001) in human keratinocytes and suppressed IL-4 and IL-13 by 68%-83% (P < 0.05) in mast cells. In delayed pepducin treatment models of oxazolone- and DNFB-induced dermatitis, PZ-235 significantly attenuated skin thickening by 43%-100% (P < 0.01) and leukocyte crusting by 57% (P < 0.05), and it inhibited ex vivo chemotaxis of leukocytes toward PAR2 agonists. Daily PZ-235 treatment of filaggrin-deficient mice exposed to dust mite allergens for 8 weeks significantly suppressed total leukocyte and T-cell infiltration by 50%-68%; epidermal thickness by 60%-77%; and skin thickening, scaling, excoriation, and total lesion severity score by 46%-56%. PZ-235 significantly reduced itching caused by wasp venom peptide degranulation of mast cells in mice by 51% (P < 0.05), which was comparable to the protective effects conferred by PAR2 deficiency. Taken together, these results suggest that a PAR2 pepducin may confer broad therapeutic benefits as a disease-modifying treatment for atopic dermatitis and itch.

Topical administration of EGF suppresses immune response and protects skin barrier in DNCB-induced atopic dermatitis in NC/Nga mice.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

BACKGROUND: Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. OBJECTIVES: To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. METHODS: We have injected recombinant human ßII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. RESULTS: Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. CONCLUSIONS: Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2.

Serine protease allergen favours Th2 responses via PAR-2 and STAT3 activation in murine model.

BACKGROUND: Protease activity of Per a 10 favours Th2 responses by differential regulation of IL-12p70 and IL-23 cytokine subunits. This study aimed to elucidate the underlying mechanism of differential regulation of IL-12p70 and IL-23. METHODS: PAR-2 activation was blocked in murine model by administering SAM11 before each sensitization. CD11c+ p-STAT3+ cells were measured in lungs by flow cytometry. BMDCs were pretreated with SAM11 or isotype control or stattic and stimulated with Per a 10. p-STAT3 levels were measured using Western blot. Transcript levels of IL-12p35, IL-12/23p40 and IL-23p19 were measured using RT-PCR. Cytokine levels were analysed using ELISA. RESULTS: Protease activity of Per a 10 increased p-STAT3 levels in mouse lungs, which was reduced upon PAR-2 blockage. Percentage of p-STAT3+ CD11c+ cells was higher in Per a 10-administered mice and was reduced upon PAR-2 blockage. IL-12p35 and IL-12p70 levels were higher, and IL-23p19 and IL-23 levels were lower in both SAM11-treated mice and BMDCs indicating a role of PAR-2-mediated signalling. IL-4, TSLP, IL-17A, EPO activity, total cell count and specific IgE and IgG1 levels were lower in SAM11-administered mice. Inhibiting STAT3 activation via stattic also leads to lower levels of IL-23p19 and IL-23 and higher levels of IL-12p35. CONCLUSIONS: Per a 10 leads to PAR-2 activation on BMDCs resulting in downstream activation of STAT3 to regulate the balance between IL-12/IL-23 subunits causing a cytokine milieu rich in IL-23 to favour Th2 polarization.

Proteinase-activated receptor-2 blockade inhibits changes seen in a chronic murine asthma model.

BACKGROUND: Proteinase-Activated Receptor-2 (PAR2 ) is a G protein-coupled receptor activated by serine proteinases. We have shown that PAR2 activation in the airways is involved in the development of allergic inflammation and airway hyperresponsiveness (AHR) in acute murine models. We hypothesized that functional inhibition of PAR2 prevents allergic inflammation, AHR and airway remodeling in chronic allergic airway inflammation models. MATERIAL AND METHODS: We developed and used a 12 week model of cockroach extract (CE)-mediated AHR, airway inflammation and remodeling in BALB/c mice. RESULTS: Mice sensitized and challenged with CE for 12 weeks exhibit AHR, increased numbers of eosinophils in bronchoalveolar lavage (BAL) and increased collagen content in the lung tissue compared to saline controls. Administration of an anti-PAR2 antibody, SAM-11, after the initial development of airway inflammation significantly inhibited all these parameters. CONCLUSIONS: Our data demonstrate that PAR2 signaling plays a key role in CE-induced AHR and airway inflammation/remodeling in long term models of allergic airway inflammation. Targeting PAR2 activation may be a successful therapeutic strategy for allergic asthma.

Immediate-type allergic and protease-mediated reactions are involved in scratching behaviour induced by topical application of Dermatophagoides farinae extract in NC/Nga mice.

Atopic dermatitis (AD)-like dermatitis can be induced by repeated topical application of an ointment containing Dermatophagoides farinae body (Dfb) extract in NC/Nga mice. This AD-like murine model also exhibits a biphasic increase in the number of scratching behaviour after topical application of Dfb ointment. In this study, we investigated the possible mechanisms underlying the scratching behaviour in each phase. An increase in the content of mast cell-derived mediators such as histamine and 5-hydroxytryptamine in the lesional skin and increased vascular permeability were observed in the early phase after the Dfb ointment application. Chlorpheniramine (H1 receptor antagonist) and cromoglycate (mast cell stabilizer) reduced the scratching behaviour in the early phase but not that in the later phase. Furthermore, the content of various endogenous pruritogens such as interleukin-31 and thymic stromal lymphopoietin in the lesional skin was increased 1 or 24 hours after the Dfb ointment application. Elevated expression of proteinase-activated receptor-2 (PAR-2) was also observed in the epidermis. Finally, gabexate (serine protease inhibitor) reduced the scratching behaviour in both phases, and anti-PAR2 antibody also showed a tendency to reduce both scratching behaviours. These findings suggest that immediate-type allergic reactions caused by mast cell degranulation and PAR-2 activation by proteases are involved in the scratching behaviour in this AD-like model.

Protease-activated receptor 2 contributes to Toxoplasma gondii-mediated gut inflammation.

Toxoplasma gondii is a widespread intracellular parasite, which naturally enters the organism via the oral route and crosses the intestinal barrier to disseminate. In addition to neuronal and ocular pathologies, this pathogen also causes gut inflammation in a number of animals. This infection-triggered inflammation has been extensively studied in the C57BL/6 mice, highlighting the importance of the immune cells and their mediators in the development of gut pathology. However, despite their importance in inflammation, the role of protease-activated receptors (PAR) was never reported in the context of T.gondii-mediated small intestine inflammation. Using genetically modified mice, we show that PAR2 plays a pathogenic role in the development of gut inflammatory lesions. We find that PAR2 controls the innate inflammatory mediators IL-6, KC/CXCL1, PGE2 as well as neutrophil infiltration in T. gondii-triggered gut damage. These results bring new knowledge on the mechanisms operating in the gut in response to T. gondii infection.

Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells.

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4-CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.

House Dust Mites Induce Production of Endothelin-1 and Matrix Metalloproteinase-9 in Keratinocytes via Proteinase-Activated Receptor-2 Activation.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction and abnormal immune response. House dust mites (HDM) are a major source of allergens, some of which have cysteine and serine protease activities. Keratinocytes stimulated by HDM-derived proteases have been suggested to contribute to the pathogenesis of AD by producing various cytokines. However, whether keratinocytes contribute to the induction of pruritus in AD, especially by producing pruritus-related mediators upon stimulation with HDM-derived proteases, has not been fully elucidated. METHODS: We examined whether the production of endothelin-1 (ET-1), matrix metalloproteinase (MMP)-2, and MMP-9 in keratinocytes can be induced by stimulation with Dermatophagoides farinae extracts, and if so, whether pretreatment with a protease inhibitor or proteinase-activated receptor-2 (PAR-2) antagonist affects the production of these mediators in keratinocytes. RESULTS: Although MMP-2 levels were undetectable in the culture supernatants, the production of ET-1 and MMP-9 was increased upon stimulation with HDM extracts in a concentration- and time-dependent manner and suppressed by pretreatment of HDM extracts with serine protease inhibitor, but not with cysteine protease inhibitor. Mite-derived serine proteases also induced ET-1 and MMP-9 production in a concentration- and time-dependent manner. Moreover, pretreatment with a PAR-2 antagonist inhibited the production of ET-1 and MMP-9 in keratinocytes. CONCLUSION: These results suggest that the activation of PAR-2 on keratinocytes by HDM-derived serine proteases induces the production of ET-1 and MMP-9, and may contribute to the induction of pruritus in AD.

Effect of the house dust mite allergen Der p 1 on tryptase release from human mast cells.

This study aimed to investigate the effects of the house dust mite allergen Der p 1 on the secretion of tryptase from the human mast cell line HMC-1. Flow cytometry was used to determine the expression levels of protease-activated receptor-2 (PAR2) on the surface of HMC-1 cells. HMC-1 cells were treated with Der p 1, SLIGRL-NH2 (PAR2 agonist), LRGILS-NH2 (control peptide for PAR2), or Der p 1 + FSLLRY (PAR2 antagonist), and the tryptase levels were measured using enzyme-linked immunosorbent assay. The biological functions of PAR2 were determined using the calcium green indicator, and intracellular calcium fluorescence intensity in the different groups (Der p 1, SLIGRL-NH2, LRGILS- NH2, Der p 1 + FSLLRY, tryptase, tryptase + FSLLRY, or cell culture medium) was detected by laser scanning confocal microscopy. The mast cells expressed PAR2 receptor on their surfaces. Der p 1 alone induced a significant release of intracellular calcium and tryptase in HMC-1 cells compared with the SLIGRL- NH2 treatment group and the control group. The combination of Der p 1 and FSLLRY partly inhibited intracellular calcium and tryptase release in HMC-1 cells compared with the Der p 1 treatment group. Moreover, tryptase induced a significant release of intracellular calcium in the HMC-1 cells. Der p 1 induced HMC-1 cell degranulation and the release of tryptase by activating the PAR2 receptor on the cell surfaces. Tryptase activated the PAR2 receptor and induced intracellular calcium release from the HMC-1 cells in a positive feedback loop.