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.

Platelet-activating factor and protease-activated receptor 2 cooperate to promote neutrophil recruitment and lung inflammation through nuclear factor-kappa B transactivation.

Although it is well established that platelet-activated receptor (PAF) and protease-activated receptor 2 (PAR2) play a pivotal role in the pathophysiology of lung and airway inflammatory diseases, a role for a PAR2-PAFR cooperation in lung inflammation has not been investigated. Here, we investigated the role of PAR2 in PAF-induced lung inflammation and neutrophil recruitment in lungs of BALB/c mice. Mice were pretreated with the PAR2 antagonist ENMD1068, PAF receptor (PAFR) antagonist WEB2086, or aprotinin prior to intranasal instillation of carbamyl-PAF (C-PAF) or the PAR2 agonist peptide SLIGRL-NH2 (PAR2-AP). Leukocyte infiltration in bronchoalveolar lavage fluid (BALF), C-X-C motif ligand 1 (CXCL)1 and CXCL2 chemokines, myeloperoxidase (MPO), and N-acetyl-glycosaminidase (NAG) levels in BALF, or lung inflammation were evaluated. Intracellular calcium signaling, PAFR/PAR2 physical interaction, and the expression of PAR2 and nuclear factor-kappa B (NF-КB, p65) transcription factor were investigated in RAW 264.7 cells stimulated with C-PAF in the presence or absence of ENMD1068. C-PAF- or PAR2-AP-induced neutrophil recruitment into lungs was inhibited in mice pretreated with ENMD1068 and aprotinin or WEB2086, respectively. PAR2 blockade impaired C-PAF-induced neutrophil rolling and adhesion, lung inflammation, and production of MPO, NAG, CXCL1, and CXCL2 production in lungs of mice. PAFR activation reduced PAR2 expression and physical interaction of PAR2 and PAFR; co-activation is required for PAFR/PAR2 physical interaction. PAR2 blockade impaired C-PAF-induced calcium signal and NF-κB p65 translocation in RAW 264.7 murine macrophages. This study provides the first evidence for a cooperation between PAFR and PAR2 mediating neutrophil recruitment, lung inflammation, and macrophage activation.

Protease-activated receptor type 2 activation downregulates osteogenesis in periodontal ligament stem cells.

Protease-activated receptor-2 (PAR2) is associated with the pathogenesis of many chronic diseases with inflammatory characteristics, including periodontitis. This study aimed to evaluate how the activation of PAR2 can affect the osteogenic activity of human periodontal ligament stem cells (PDLSCs) in vitro. PDLSCs collected from three subjects were treated in osteogenic medium for 2, 7, 14, and 21 days with trypsin (0.1 U/mL), PAR2 specific agonist peptide (SLIGRL-NH2) (100 nM), and PAR2 antagonist peptide (FSLLRY-NH2) (100 nM). Gene (RT-qPCR) expression and protein expression (ELISA) of osteogenic factors, bone metabolism, and inflammatory cytokines, cell proliferation, alkaline phosphatase (ALP) activity, alizarin red S staining, and supernatant concentration were assessed. Statistical analysis of the results with a significance level of 5% was performed. Activation of PAR2 led to decreases in cell proliferation and calcium deposition (p < 0.05), calcium concentration (p < 0.05), and ALP activity (p < 0.05). Additionally, PAR2 activation increased gene and protein expression of receptor activator of nuclear factor kappa-Β ligand (RANKL) (p < 0.05) and significantly decreased the gene and protein expression of osteoprotegerin (p <0. 05). Considering the findings, the present study demonstrated PAR2 activation was able to decrease cell proliferation, decreased osteogenic activity of PDLSCs, and upregulated conditions for bone resorption. PAR2 may be considered a promising target in periodontal regenerative procedures.

Protease-activated receptor 2 enhances innate and inflammatory mechanisms induced by lipopolysaccharide in macrophages from C57BL/6 mice.

OBJECTIVE: This study was conducted to investigate the effects of the synthetic PAR2 agonist peptide (PAR2-AP) SLIGRL-NH2 on LPS-induced inflammatory mechanisms in peritoneal macrophages. METHODS: Peritoneal macrophages obtained from C57BL/6 mice were incubated with PAR2-AP and/or LPS, and the phagocytosis of zymosan fluorescein isothiocyanate (FITC) particles; nitric oxide (NO), reactive oxygen species (ROS), and cytokine production; and inducible NO synthase (iNOS) expression in macrophages co-cultured with PAR-2-AP/LPS were evaluated. RESULTS: Co-incubation of macrophages with PAR2AP (30 µM)/LPS (100 ng/mL) enhanced LPS-induced phagocytosis; production of NO, ROS, and the pro-inflammatory cytokines interleukin (IL)-1ß, tumour necrosis factor (TNF)-α, IL-6, and C-C motif chemokine ligand (CCL)2; and iNOS expression and impaired the release of the anti-inflammatory cytokine IL-10 after 4 h of co-stimulation. In addition, PAR2AP increased the LPS-induced translocation of the p65 subunit of the pro-inflammatory transcription factor nuclear factor kappa B (NF-κB) and reduced the expression of inhibitor of NF-κB. CONCLUSION: This study provides evidence of a role for PAR2 in macrophage response triggered by LPS enhancing the phagocytic activity and NO, ROS, and cytokine production, resulting in the initial and adequate macrophage response required for their innate response mechanisms.

Blockade of protease-activated receptor 2 attenuates allergenmediated acute lung inflammation and leukocyte recruitment in mice.

Protease-activated receptor (PAR)2 has been implicated in mediating allergic airway inflammation.We investigate the role of PAR2 in lung inflammation and neutrophil and eosinophil recruitment into the lungs in amousemodel of shortterm acute allergic inflammation. Allergic lung inflammation was induced in sensitized BALB/c mice through intranasal instillations of ovalbumin (OVA), and mice were pretreated with the PAR2 antagonist ENMD1068 or with the PAR2-activating peptide (PAR2-AP) 1 hour before each OVA challenge. Bronchoalveolar lavage fluid (BALF) was collected, and the lungs, trachea and lymph nodes were removed after the last challenge to analyze the airway inflammation. PAR2 blockade reduced OVA-induced eosinophil and neutrophil counts, CXCL1, CCL5, amphiregulin, and interleukin (IL)-6 and 13 levels.Moreover, PAR2 blockade reduced OVA-induced PAR2 expression in cells present in BALF 2 hour after OVA challenge, and PAR2-AP acted synergistically with OVA promoting eosinophil recruitment intoBALF and increased IL-4 and IL-13 levels in lymph nodes. Conversely, PAR2 blockade increased IL- 10 levels when compared with OVA-treated mice. Our results provide evidence for a mechanism by which PAR2 meditates acute lung inflammation triggered by multiple exposures to allergen through a modulatory role on cytokine production and vascular permeability implicated in the lung diseases such as asthma.

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.

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.(AU)

Proteolytic activity of Triatoma infestans saliva associated with PAR-2 activation and vasodilation

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect and the main vector of Trypanosoma cruzi (Kinetoplastida: Trypanosomatidae). In the present study, the authors investigated whether a serine protease activity from the saliva of T. infestans has a role in vasomotor modulation, and in the insect-blood feeding by cleaving and activating protease-activated receptors (PARs). Methods T. infestans saliva was chromatographed as previously reported for purification of triapsin, a serine protease. The cleavage activity of triapsin on PAR peptides was investigated based on FRET technology. Mass spectrometry was used to analyze the sites of PAR-2 peptide cleaved by triapsin. NO measurements were performed using the DAN assay (2,3-diaminonapthalene). The vasorelaxant activity of triapsin was measured in vessels with or without functional endothelium pre-contracted with phenylephrine (3 µM). Intravital microscopy was used to assess the effect of triapsin on mouse skin microcirculation. Results Triapsin was able to induce hydrolysis of PAR peptides and showed a higher preference for cleavage of the PAR-2 peptide. Analysis by mass spectrometry confirmed a single cleavage site, which corresponds to the activation site of the PAR-2 receptor. Triapsin induced dose-dependent NO release in cultured human umbilical vein endothelial cells (HUVECs), reaching a maximum effect at 17.58 nM. Triapsin purified by gel-filtration chromatography (10-16 to 10-9 M) was applied cumulatively to mouse mesenteric artery rings and showed a potent endothelium-dependent vasodilator effect (EC30 = 10-12 M). Nitric oxide seems to be partially responsible for this vasodilator effect because L-NAME (L-NG-nitroarginine methyl ester 300 µM), a nitric oxide synthetase inhibitor, did not abrogate the vasodilation activated by triapsin. Anti-PAR-2 antibody completely inhibited vasodilation observed in the presence of triapsin activity. Triapsin activity also induced an increase in the mouse ear venular diameter. Conclusion Data from this study suggest a plausible association between triapsin activity mediated PAR-2 activation and vasodilation caused by T. infestans saliva.(AU)

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.

Triggering of protease-activated receptors (PARs) induces alternative M2 macrophage polarization with impaired plasticity.

Protease-activated receptors (PARs) have been described in a wide diversity of vertebrate cells, including human immune cells. Macrophages are pivotal cells in the host-pathogen interaction and their polarization in M1 or M2 cells has been described as a new central paradigm in the immune response to pathogens. In this context, we explored the involvement of PAR activation by serine proteases on M1/M2 macrophage differentiation and their impact on the Th1/Th2 cytokine profile in response to Mycobacterium tuberculosis antigen. Our results demonstrate that the serine proteases, thrombin and trypsin, induce interleukin (IL)-4 release from human monocytes, together with upregulation of the macrophage mannose receptor (CD206) in the same way that alternative M2a differentiated cells with M-CSF/IL-4. Protease stimulation of monocytes in the presence of PAR-1 (SCH-79797) or PAR-2 (FSLLRY-NH2) antagonists abolished IL-4 release from monocytes, whereas the use of the peptide agonist for PAR-1 (SFLLRNPNDKYEPF-NH2) or PAR-2 (SLIGKV-NH2) induced the secretion of IL-4 at a level comparable to thrombin or trypsin. When these protease-induced M2 macrophages from healthy human PPD + donors were co-cultured with autologous lymphocyte population in the presence of Mycobacterium tuberculosis antigen, we found a consistent inhibition of IFN-γ/IL-12 release together with persistent IL-4 expression, in contrast to the expected Th1 profile obtained with M2a macrophages. To our knowledge, this is the first observation that proteolytic activation of PAR1/2 receptors in monocytes induces M2-like macrophages with impaired plasticity and their implication in the driving of the Th1/Th2 cytokine profile.

Protease-activated receptor 2 (PAR2) upregulates granulocyte colony stimulating factor (G-CSF) expression in breast cancer cells.

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor which is activated upon cleavage of its N-terminal region. PAR2 has been associated with many aspects regarding tumor progression, such as the production of pro-tumoral cytokines. Granulocyte colony-stimulating factor (G-CSF) is a cytokine essential to neutrophil production and maturation, and it is often overexpressed in tumors. In this study, we evaluated the ability of PAR2 to modulate G-CSF expression. PAR2 and G-CSF were significantly more expressed in metastatic (4T1 and MDA-MB-231) as compared to non-metastatic (67NR and MCF7) breast cancer cell lines. In addition, PAR2 stimulation by a synthetic agonist peptide significantly increased G-CSF gene expression in the metastatic cell lines. Knockdown of PAR2 in 4T1 cells decreased G-CSF expression and secretion. In addition, treatment of 4T1 with the commercial PAR2 antagonist, ENMD-1068, significantly decreased G-CSF expression. cBioPortal analyses of the TCGA database showed a significant co-occurrence of G-CSF and PAR2 gene overexpression in breast cancer samples. In conclusion, our data suggest that PAR2 contributes to G-CSF expression in breast cancer cells, possibly favoring tumor progression.

Mast cell-nerve interaction in the colon of Trypanosoma cruzi-infected individuals with chagasic megacolon.

Chagas disease is an infection caused by the parasite Trypanosoma cruzi that affects millions of people worldwide and is endemic in Latin America. Megacolon is the most frequent complication of the digestive chronic form and happens due to lesions of the enteric nervous system. The neuronal lesions seem to initiate in the acute phase and persist during the chronic phase, albeit the mechanisms involved in this process are still debated. Among the cells of the immune system possibly involved in this pathological process is the mast cell (MC) due to its well-known role in the bi-directional communication between the immune and nervous systems. Using ultrastructural analysis, we found an increased number of degranulated MCs in close proximity to nerve fibers in infected patients when compared with uninfected controls. We also immunostained MCs for the two pro-inflammatory molecules tryptase and chymase, the first being also important in neuronal death. The number of MCs immunostained for tryptase or chymase was increased in patients with megacolon, whereas increased tryptase staining was additionally observed in patients without megacolon. Moreover, we detected the expression of the tryptase receptor PAR2 in neurons of the enteric nervous system, which correlated to the tryptase staining results. Altogether, the data presented herein point to the participation of MCs on the denervation process that occurs in the development of T. cruzi-induced megacolon.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells.

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS42↓F43LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR20↓S21LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity.

Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

PAR-2 expression in the gingival crevicular fluid reflects chronic periodontitis severity

Abstract Recent studies investigating protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. It is known that gingipain, a bacterial protease secreted by the important periodontopathogen Porphyromonas gingivalis can activate PAR-2. Previous studies by our group found that PAR-2 is overexpressed in the gingival crevicular fluid (GCF) of patients with moderate chronic periodontitis (MP). The present study aimed at evaluating whether PAR-2 expression is associated with chronic periodontitis severity. GCF samples and clinical parameters, including plaque and bleeding on probing indices, probing pocket depth and clinical attachment level, were collected from the control group (n = 19) at baseline, and from MP patients (n = 19) and severe chronic periodontitis (SP) (n = 19) patients before and 6 weeks after periodontal non-surgical treatment. PAR-2 and gingipain messenger RNA (mRNA) in the GCF of 4 periodontal sites per patient were evaluated by Reverse Transcription Polymerase Chain Reaction (RT-qPCR). PAR-2 and gingipain expressions were greater in periodontitis patients than in control group patients. In addition, the SP group presented increased PAR-2 and gingipain mRNA levels, compared with the MP group. Furthermore, periodontal treatment significantly reduced (p <0.05) PAR-2 expression in patients with periodontitis. In conclusion, PAR-2 is associated with chronic periodontitis severity and with gingipain levels in the periodontal pocket, thus suggesting that PAR-2 expression in the GCF reflects the severity of destruction during periodontal infection.

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.

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.

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.

Downregulation of Proteinase-Activated Receptor-2, Interleukin-17, and Other Proinflammatory Genes by Subantimicrobial Doxycycline Dose in a Rat Periodontitis Model.

BACKGROUND: Subantimicrobial dose doxycycline (SDD) has been used as an adjunct in periodontal treatment because of its matrix metalloproteinase inhibition properties. Although the benefits of SDD therapy, such as improvement in the parameters of periodontal probing depth and clinical attachment level, have been proven in multiple clinical studies, the comprehension of other biologic mechanisms of action on periodontitis remains poorly investigated. Therefore, this animal-model study evaluated the effects of SDD monotherapy on the expressions of the following key proinflammatory genes: proteinase-activated receptor-2 (PAR2), tumor necrosis factor (TNF)-α, interleukin (IL)-17, and IL-1ß. METHODS: Male Wistar rats were assigned randomly to the following: 1) control group: no ligature-induced periodontitis and no treatment; 2) ligature group: ligature-induced periodontitis and placebo treatment; and 3) ligature + doxycycline group: ligature-induced periodontitis and SDD treatment. After the experimental time, animals were sacrificed, and reverse transcription-polymerase chain reaction was performed to analyze the mRNA expression of IL-1ß, IL-17, TNF-α, and PAR2 in gingival tissue samples. Histologic analyses were performed on the furcation region and mesial gingiva of mandibular first molars to measure periodontal bone loss and collagen content. RESULTS: SDD administration significantly downregulated PAR2, IL-17, TNF-α, and IL-1ß mRNA expressions (P <0.05). In addition, SDD treatment was accompanied by lower rates of alveolar bone loss (P <0.05) and maintenance of the amount of gingival collagen fibers. CONCLUSION: These findings reveal new perspectives regarding SDD efficacy because it can be partially related to proinflammatory gene expression modulation, even considering PAR2 and IL-17, which has not been investigated thus far.

Expression of protease activated receptor-1 in chronic periodontitis.

BACKGROUND: Protease activated receptor-1 (PAR1) activation by thrombin may play a role in repair and homeostasis of periodontal tissues. The main objective of this study is to investigate PAR1 expression in patients with periodontitis, before and after non-surgical periodontal treatment, and to associate its expression with the presence of inflammatory biomarkers and PAR2 expression. METHODS: Gingival crevicular fluid (GCF) samples and clinical parameters, including probing depth, clinical attachment level, bleeding on probing, and gingival and plaque indices, were collected from periodontally healthy individuals and patients with moderate chronic periodontitis (CP) before and 6 weeks after periodontal non-surgical treatment. PAR1 and PAR2 messenger RNA (mRNA) at the GCF were evaluated by quantitative polymerase chain reaction (qPCR). Flow cytometry analysis identified the GCF PAR1-expressing cells. GCF inflammatory biomarkers were also determined. RESULTS: Clinical parameters were significantly improved after therapy (P <0.01). The qPCR analysis showed that, before therapy, PAR1 mRNA levels in CP were similar to controls. Periodontal treatment led to increased PAR1 expression in CP (P <0.05). PAR1 expression was inversely correlated to PAR2 expression and with interleukins 6 and 8, tumor necrosis factor-α, interferon-γ, and matrix metalloproteinase-2 levels. CONCLUSIONS: Periodontal treatment results in PAR1 overexpression in the GCF, and PAR1 expression is associated with decreased expression of inflammatory biomarkers and inversely correlated to PAR2 expression in the GCF. Therefore, the data suggest the importance of PAR1 mediating the known anabolic actions of thrombin in the periodontium.