The ecotin-like peptidase inhibitor of Trypanosoma cruzi prevents TMPRSS2-PAR2-TLR4 crosstalk downmodulating infection and inflammation.

Trypanosoma cruzi is the causative agent of Chagas disease, a chronic pathology that affects the heart and/or digestive system. This parasite invades and multiplies in virtually all nucleated cells, using a variety of host cell receptors for infection. T. cruzi has a gene that encodes an ecotin-like inhibitor of serine peptidases, ISP2. We generated ISP2-null mutants (Δisp2) in T. cruzi Dm28c using CRISPR/Cas9. Epimastigotes of Δisp2 grew normally in vitro but were more susceptible to lysis by human serum compared to parental and ISP2 add-back lines. Tissue culture trypomastigotes of Δisp2 were more infective to human muscle cells in vitro, which was reverted by the serine peptidase inhibitors aprotinin and camostat, suggesting that host cell epitheliasin/TMPRSS2 is the target of ISP2. Pretreatment of host cells with an antagonist to the protease-activated receptor 2 (PAR2) or an inhibitor of Toll-like receptor 4 (TLR4) selectively counteracted the increased cell invasion by Δisp2, but did not affect invasion by parental and add-back lines. The same was observed following targeted gene silencing of PAR2, TLR4 or TMPRSS2 in host cells by siRNA. Furthermore, Δisp2 caused increased tissue edema in a BALB/c mouse footpad infection model after 3 h differently to that observed following infection with parental and add-back lines. We propose that ISP2 contributes to protect T. cruzi from the anti-microbial effects of human serum and to prevent triggering of PAR2 and TLR4 in host cells, resulting in the modulation of host cell invasion and contributing to decrease inflammation during acute infection.

TF/PAR2 Signaling Axis Supports the Protumor Effect of Neutrophil Extracellular Traps (NETs) on Human Breast Cancer Cells.

Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.

Pharmacological blockade of protease-Activated Receptor 2 improves airway remodeling and lung inflammation in experimental allergic asthma

Abstract Protease-activated receptors (PARs) are metabotropic G-protein-coupled receptors that are activated via proteolytic cleavage of a specific sequence of amino acids in their N-terminal region. PAR2 has been implicated in mediating allergic airway inflammation. This study aims to study the effect of PAR2 antagonist ENMD1068in lung inflammation and airway remodeling in experimental asthma. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with ENMD1068 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs were removed at different time intervals after OVA challenge to analyze inflammation, airway remodeling and airway hyperresponsiveness. Ovalbumin promoted leukocyte infiltration into BALF in a PAR2-dependent manner. ENMD1068 impaired eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity in the lung parenchyma into BALF and reduced the loss of dynamic pulmonary compliance, lung resistance in response to methacholine, mucus production, collagen deposition and chemokine (C-C motif) ligand 5 expression compared to those in OVA-challenged mice. We propose that proteases released after an allergen challenge may be crucial to the development of allergic asthma in mice, and PAR2 blockade may be useful as a new pharmacological approach for the treatment of airway allergic diseases.

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.

Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer.

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Hydrogen sulfide donors alleviate itch secondary to the activation of type-2 protease activated receptors (PAR-2) in mice.

Hydrogen sulfide (H2S) has been highlighted as an endogenous signaling molecule and we have previously found that it can inhibit histamine-mediated itching. Pruritus is the most common symptom of cutaneous diseases and anti-histamines are the usual treatment; however, anti-histamine-resistant pruritus is common in some clinical settings. In this way, the involvement of mediators other than histamine in the context of pruritus requires new therapeutic targets. Considering that the activation of proteinase-activated receptor 2 (PAR-2) is involved in pruritus both in rodents and humans, in this study we investigated the effect of H2S donors on the acute scratching behavior mediated by PAR-2 activation in mice, as well as some of the possible pharmacological mechanisms involved. The intradermal injection of the PAR-2 peptide agonist SLIGRL-NH2 (8-80nmol) caused a dose-dependent scratching that was unaffected by intraperitoneal pre-treatment with the histamine H1 antagonist pyrilamine (30mg/kg). Co-injection of SLIGRL-NH2 (40nmol) with either the slow-release H2S donor GYY4137 (1 and 3nmol) or the spontaneous donor NaHS (1 and 0.3nmol) significantly reduced pruritus. Co-treatment with the KATP channel blocker glibenclamide (200nmol) or the nitric oxide (NO) donor sodium nitroprusside (10nmol) abolished the antipruritic effects of NaHS; however, the specific soluble guanylyl cyclase inhibitor ODQ (30µg) had no significant effects. The transient receptor potential ankyrin type 1 (TRPA1) antagonist HC-030031 (20µg) significantly reduced SLIGRL-NH2-induced pruritus; however pruritus induced by the TRPA1 agonist AITC (1000nmol) was unaffected by NaHS. Based on these data, we conclude that pruritus secondary to PAR-2 activation can be reduced by H2S, which acts through KATP channel opening and involves NO in a cyclic guanosine monophosphate (cGMP)-independent manner. Furthermore, TRPA1 receptors mediate the pruritus induced by activation of PAR-2, but H2S does not interfere with this pathway. These results provide additional support for the development of new therapeutical alternatives, mainly intended for treatment of pruritus in patients unresponsive to anti-histamines.

Gingival crevicular fluid levels of protease-activated receptors type 1 and type 2 in diabetic patients with periodontitis.

BACKGROUND AND OBJECTIVE: Protease activated receptor type 1 (PAR1 ) seems to play a role in periodontal repair, while PAR2 is associated with periodontal inflammation. As diabetes is a known risk factor for periodontal disease, the aim of this study was to evaluate the influence of type 2 diabetes on PAR1 and PAR2 mRNA expression in the gingival crevicular fluid of patients with chronic periodontitis before and after non-surgical periodontal treatment. MATERIAL AND METHODS: Gingival crevicular fluid samples and clinical parameters consisting of measuring probing depth, clinical attachment level, bleeding on probing and plaque index were collected from systemically healthy patients and patients with type 2 diabetes and chronic periodontitis, at baseline and after non-surgical periodontal therapy. PAR1 and PAR2 , as well as the presence of the proteases RgpB gingipain and neutrophil proteinase-3 were assessed by quantitative polymerase chain reaction in the gingival crevicular fluid. RESULTS: The periodontal clinical parameters significantly improved after periodontal therapy (p < 0.01). Diabetes led to increased expression of PAR1 in gingival crevicular fluid, and in the presence of chronic periodontitis, it significantly decreased the expression of PAR1 and PAR2 (p < 0.05). Moreover, non-surgical periodontal treatment in diabetics resulted in increased expression of PAR1 and PAR2 (p < 0.05), and decreased expression of RgpB gingipain and proteinase-3 (p < 0.05). CONCLUSION: The present data demonstrated that diabetes was associated with an altered expression of PAR1 and PAR2 in the gingival crevicular fluid cells of subjects with chronic periodontitis. Future studies are necessary to elucidate the effects of PAR1 upregulation in periodontally healthy sites and PAR2 downregulation in chronic periodontitis sites on the increased susceptibility and severity of periodontitis in diabetes.