Abstract Title

PROTEASE-ACTIVATED RECEPTOR 2 (PAR2) IS UPREGULATED BY ACANTHAMOEBA PLASMINOGEN ACTIVATOR (APA) AND INDUCES PROINFLAMMATORY CYTOKINE IN HUMAN CORNEAL EPITHELIAL CELLS

Presenter Name

trivendra tripathi

Abstract

Acanthamoeba keratitis (AK) is a vision-threatening disease caused by pathogenic strains of Acanthamoeba. The main risk factors for AK are wearing eye contact lenses, corneal injuries, and contact with contaminated water. Diagnosis of AK is not straightforward and treatment is very demanding because of Acanthamoeba cysts resist to most antimicrobial agent. Our project is based on the observations that the innate immune system plays an important role in AK, and exploring the sequential pathogenic cascades of AK and targeting the therapeutic approaches to determine new remedies to prevent and treat AK. We have shown that Acanthamoeba trophozoites secrete cytopathic serine proteases, MIP-133 and aPA. Role of MIP-133 in the pathogenesis of AK via cPLA2α pathway has been explored; however, aPA interaction to corneal epithelial cells in disease progression is still unknown. In the present study, we have shown that aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK.

Purpose (a):

Acanthamoeba plasminogen activator (aPA), is a serine protease elaborated by Acanthamoeba trophozoites, facilitates invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA induces proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptor PAR2 pathway.

Methods (b):

A. castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system and aPA activity was determined by zymography assays. HCE cells were incubated with or without aPA (100µg/ml), PAR1-agonists (Thrombin, 10µM; TRAP-6, 10µM), and PAR2-agonists (SLIGRL-NH2, 100µM; AC55541, 10µM) for 24 hours. Inhibition of PAR1 and PAR2 involved pre-incubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH79797, 60μM) and PAR2 (FSLLRY-NH2, 100μM) and then incubated with or without aPA, Thrombin, TRAP-6, SLIGRL-NH2, and AC55541 for 24 hours. Expression of PAR1 and PAR2 was examined by qRT-PCR, flow cytometry, and immunocytochemistry. IL-8 expression was quantified by qRT-PCR and by ELISA.

Results (c):

PAR1 and PAR2 surface protein were expressed in HCE cells. aPA and PAR2-agonists significantly upregulated PAR2 expression (~1-2 times) (P<0.05). PAR2-antagonist significantly inhibited aPA and PAR2-agonists-induced PAR2 expression (~2-5 times) (P<0.5) in HCE cells. PAR1-agonists, but not aPA, significantly upregulated PAR1 expression, which was significantly inhibited by PAR1-antagonist (~30-45 times) in HCE cells. aPA and PAR2-agonists, but not PAR1-agonists, stimulated IL-8 production, which is significantly diminished by PAR2-antagonist (~2-10 times) (P<0.5). PAR1-antagonist did not diminish aPA-induced IL-8 production in HCE cells.

Conclusions (d):

aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK.

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PROTEASE-ACTIVATED RECEPTOR 2 (PAR2) IS UPREGULATED BY ACANTHAMOEBA PLASMINOGEN ACTIVATOR (APA) AND INDUCES PROINFLAMMATORY CYTOKINE IN HUMAN CORNEAL EPITHELIAL CELLS

Acanthamoeba keratitis (AK) is a vision-threatening disease caused by pathogenic strains of Acanthamoeba. The main risk factors for AK are wearing eye contact lenses, corneal injuries, and contact with contaminated water. Diagnosis of AK is not straightforward and treatment is very demanding because of Acanthamoeba cysts resist to most antimicrobial agent. Our project is based on the observations that the innate immune system plays an important role in AK, and exploring the sequential pathogenic cascades of AK and targeting the therapeutic approaches to determine new remedies to prevent and treat AK. We have shown that Acanthamoeba trophozoites secrete cytopathic serine proteases, MIP-133 and aPA. Role of MIP-133 in the pathogenesis of AK via cPLA2α pathway has been explored; however, aPA interaction to corneal epithelial cells in disease progression is still unknown. In the present study, we have shown that aPA specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway and PAR2-antagonists may be a therapeutic target in AK.