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

Abstract

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-1β 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-1β 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.

Resolving the conflicts around Par2 opposing roles in regeneration by comparing immune-mediated and toxic-induced injuries

“…On the other hand, Par2 activation protected against myocardial ischemia-reperfusion [ 41. ], as well as aggravated epithelial inflammation [ 42. ].…”

Protease allergens as initiators–regulators of allergic inflammation

“…7,26 For mites, junctional complexes in the airway epithelium also undergo indirect proteolysis which may be PAR-dependent. 6,7,15,34 A recent study showed that the fungal allergen Asp f 13 damaged airway epithelial TJ and E-Cadherin (AJ).…”

“…[3][4][5][6] However, this process is prevented by specific 6,7 and non-specific protease inhibitors, [6][7][8][9][10][11][12] or inhibitors of signaling pathways activated by protease allergens. [13][14][15][16][17][18] Furthermore, the addition of a protease to an enzymatically inactive protein can render that protein allergenic. 4,10,[19][20][21] Thus, not only does protease activity endow an allergen with its adjuvant activity, but it also acts as an exogenous allergic adjuvant for other inert or otherwise not allergenic proteins.…”

Zonulin as a Potential Therapeutic Target in Microbiota-Gut-Brain Axis Disorders: Encouraging Results and Emerging Questions

“…Similarly, a small molecule antagonist, GB83, exerted harmful effects on colon epithelial cells by decreasing the expression of autophagy- and TJ-related factors and increased permeability [ 234 ]. In contrast, inhibition of the PAR 2 pathway by GB88 in lung epithelial cells [ 235 ] or using I-191 in arterial endothelial cells [ 236 ] moderated actin rearrangement and TJ disruption and reduced the permeability of the cellular monolayers. Moreover, a non-peptidic PAR 2 ligand, the full agonist AC-55541, ameliorated the IL-17-induced loss of epithelial resistance in brain microvascular endothelial cells [ 237 ].…”