Role of protease-activated receptors 2 ( PAR 2 ) in ocular infections and inflammation

Protease-activated receptors (PARs) belong to a unique family of G protein-coupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by a variety of proteinases, essentially of the serine (Ser) proteinase family. After cleavage, the new N-terminal sequence functions as a tethered ligand, which binds intramolecularly to activate the receptor and initiate signaling. Cell signals induced through the activation of PARs appear to play a significant role in innate and adoptive immune responses of the cornea, which is constantly exposed to proteinases under physiological or pathophysiological conditions. Activation of PARs interferes with all aspects of the corneal physiology such as barrier function, transports, innate and adoptive immune responses, and functions of corneal nerves. It is not known whether the proteinase released from the microorganism can activate PARs and triggers the inflammatory responses. The role of PAR2 expressed by the corneal epithelial cells and activation by serine protease released from microorganism is discussed here. Recent evidences suggest that activation of PAR2, by the serine proteinases, play an important role in innate and inflammatory responses of the corneal infection.


Introduction
Protease-activated receptors (PARs) belong to a distinctive family of the seven transmembrane G proteincoupled receptors (GPCRs) that are cleaved at an activation site within the N-terminal exodomain by the serine (Ser) proteinase (or serine endopeptidases) family [1-5]   .The cleaved new N-terminal sequence acts as a tethered ligand which interacts with the receptor within extracellular loop-2 to activate the receptor and initiates signaling [1][2][3][4][5] .To date, four members of the PARs family have been recognized: PAR1-PAR4 [1][2][3][4][5] .PAR1, PAR3, and PAR4 can be stimulated by thrombin and PAR2 can be activated by trypsin, tissue factor/factor VIIa/factor Xa, neutrophil proteinase 3, mast cell tryptase, and membranetethered serine proteinase-1 [1][2][3][4][5] .Activation mechanism of PAR1 through PAR4 by serine proteases, coupling to intracellular signaling cascades, and their important physiological and pathophysiological functions in chemotaxis, inflammation, platelet aggregation, cell REVIEW growth and division, endothelial barrier dysfunction, neuronal cell survival, gastrointestinal, cardiovascular responses, regulation of skin function, thrombosis and vascular remodeling, genetic disorders, cancer, inflammatory diseases, and neurological disorders, have been extensively reviewed [6] .Lang et al. [7] first time demonstrated the PAR1 and PAR2 expression and their functional activity in proinflammatory cytokines secretion in human corneal epithelial (HCE) cells.Expression of PAR1 and PAR2 in HCE cells is a first step to investigate corneal biology by targeting PARs signaling in corneal inflammatory diseases (Acanthamoeba keratitis, bacterial keratitis, and fungal keratitis).Recently, we have demonstrated that Acanthamoeba castellanii trophozoites' secreted serine protease, Acanthamoeba plasminogen activator (aPA) [8] , induces proinflammatory cytokine IL-8 by the activation of PAR2 signaling in HCE cells [9] .This signaling is the first illustration of PAR2 activation by microbial serine proteinase in corneal epithelial cells that triggers the inflammatory response; also this might be a novel mechanistic approach of aPA-induced pathogenesis of Acanthamoeba keratitis.This review briefly focuses the role of Acanthamoeba trophozoites' secreted serine protease, aPA, in the production of inflammatory mediator by the activation of PAR2 pathway in HCE cells.

Acanthamoeba Keratitis and Pathogenesis
Acanthamoeba keratitis is known as a rare but potentially sight-threatening and painful infectious corneal disease worldwide [10,11] .It is caused by the ubiquitous free-living pathogenic species of Acanthamoeba [10,[12][13][14][15] .Acanthamoeba can be found commonly in soil, air, water, cooling towers, sewage systems, and heating, ventilating, air conditioning (HVAC) systems [16,17] .Since the first case of Acanthamoeba keratitis reported by Naginton et al. [18] in United Kingdom and shortly thereafter by Jones et al. [19] in United States, the incidence of this disease has been augmented with increasing the number of contact lens (CL) wearers [20] (Table ).Recently, the preliminary investigations of Centers for Disease Control and Prevention (CDC) of a national Acanthamoeba keratitis outbreak of 105 patients from 30 states of United States, 2005-2007, showed that enrolled patients had percent corneal symptoms of pain 74, redness 74, sensitivity to light 72, sensation of foreign body 68, increased tearing 56, blurred vision 54, and discharge from eye 19 [33] .Thus, severe uneven ocular pain to the clinical signs has long been known as one hallmark of Acanthamoeba keratitis.
Many studies have been conducted on the pathogenesis of Acanthamoeba keratitis [9, ; however, biology and pathogenic mechanisms of Acanthamoeba are still beginning to emerge. The naional outbreak of Acanthamoeba keratitis reported that Acanthamoeba primarily affects contact lens users [33] ; however, other risk factors and environmental exposures associated with Acanthamoeba keratitis should be considered.Moreover, an increased incidence of Acanthamoeba keratitis is known as an important cause of amoebic keratitis in noncontact lens wearers.A three-year clinical study conducted between 1999 to 2002 in South India diagnosed thirty three patients with positive Acanthamoeba infection (approximate 1%) out of 3183 enrolled patients with corneal infections [63] .They observed that twenty six out of thirty three patients diagnosed with Acanthamoeba infection were peasants from countryside areas and got corneal injury from mud [63] .Treatments of Acanthamoeba keratitis exist with hourly applications of brolene, polyhexamethylene biguanide (PHMB), and chlorhexidine for several weeks.Even with such therapies, Acanthamoeba species can cause severe damage to the corneal epithelium and stroma, resulting in the need for corneal grafting [12] .Topical steroids are often used to control corneal inflammation and uveitis or is administered after surgery, to prevent the rejection of corneal transplant; however, in vitro exposure of Acanthamoeba cysts to dexamethasone increased trophozoite's number through excystment and growth [64] . Thu, reactivation of Acanthamoeba keratitis may occur after corneal grafting if residual Acanthamoeba cysts in the host peripheral cornea are exposed to steroids after surgery.Although the biology of Acanthamoeba is not fully explored, Acanthamoeba trophozoites' secreted serine proteases, mannose-induced protein (MIP-133) by contact-dependent mechanism [14,15,52,65] and Acanthamoeba plasminogen activator (aPA) by contactindependent mechanism [8] .(a) Contact-dependent mechanism of the Acanthamoeba keratitis pathogenesis begins when Acanthamoeba trophozoites interact to the corneal surface by mannose binding protein (MBP) [11,56] .This interaction releases the MIP-133 from A. castellanii trophozoites [15] , which interacts with membrane phospholipids on corneal epithelium and triggers arachidonic acid production, pro-inflammatory cytokines (IL-8, IL-6, IL-1β, IFNγ, and CXCL2), apoptosis, and polymorphonuclear neutrophils (PMNs) infiltration that leads to corneal lesion by the activation of cytosolic phospholipase A2α (cPLA2α) pathway; cPLA2α inhibitors (AACOCF3, CAY10650, and MAFP) therapeutically in vitro and in vivo mitigate inflammation and resolved the Acanthamoeba keratitis [60,61] .(b) Contact-independent mechanism of the Acanthamoeba keratitis pathogenesis involves in the secretion of the Acanthamoeba plasminogen activator (aPA) which has been characterized a serine protease [8] .Acanthamoeba plasminogen activator recently has been revealed to be involved in stimulation of pro-inflammatory cytokine in HCE cells via PAR2 pathway [9] .

Activation of PARs and Role of Pathogen-Derived Proteases in Inflammatory Response by PAR2 Pathway
Proteases cleave PARs' molecules at a specific activation site on the extracellular N-terminal domain.The released new N-terminal domain of the receptor acts as a tethered ligand which binds to the second extracellular loop of the receptor and triggers intracellular signaling [5] .Stimulation of PAR2 leads to a variety of responses such as activating the secretion of cytokines, prostanoids, and metalloproteases which involves to play critical pathophysiological roles in the inflammatory response in joints, skin and kidney, allergic inflammation of airways, and leukocyte migration [2] .Importantly, P. aeruginosa secrets elastase B (EPa) which potentially silences the role of PAR2 in the respiratory tract and alters the host's innate defense mechanisms and respiratory tract functions, thus EPa by PAR2 signaling contributes a significant role in the pathogenesis of a disease like cystic fibrosis [66] .Other microorganisms such as Serratia marcescens (gramnegative enteric bacterium), and Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans (gram-negative bacterium causes periodontitis), activate PAR2 and induce proinflammatory cytokines secretion [67- 69] and stimulate inflammatory responses in vitro and in vivo [70,71] .
In addition, Wang et al. [75] observed that normal corneal epithelial cells' cultures and non-ulcerating corneas have PA molecular weight (MW) species of 72 kDa and 46 kDa, and ulcer corneas, species of 72 kDa, 46kDa, and 35 kDa MW.They showed that rabbit epithelial cells, fibroblasts, and ulcer corneas release uPAlike PA; and human cornea extracts and tears also have PA immunoreactivity with anti-uPA antibodies.Noteworthy, the tPA expressed by the corneal epithelium, endothelium, stroma, and the lens, as well as the aqueous and vitreous humors, dog, calf, and monkey eyes.It suggests that tPA plays an important role in non-fibrinolytic processes, mitosis and cell migration, as well as in the destructive remodeling of the extracellular matrix such as the turnover of collagen and glycoproteins in the corneal stroma and vitreous body [76] .However, Tervo et al. [77] observed a robust immunoreaction for uPA, a weaker reaction for PAI-1, and a very weak tPA-like immunoreaction in the anterior stroma of the wounded cornea.Mirshahi et al. [78] support the findings of Tervo et al. [77] and showed that bovine corneal epithelial cells secrete tPA, uPA, and their inhibitor (PAI-1).This study first time demonstrated the role of PAI-1 in the regulation of active protease which is plasmin, may be involved in the destructive processes or tissue remodeling such as the normal turnover of stromal collagens and glycoproteins.
Berk et al . [79]observed the overexpression of PA system (uPA, the receptor of uPA (uPAR), tPA, and both inhibitors, PAI-1 and PAI-2) in mice corneas during the inflammatory response intracorneally infected with P. aeruginosa.Urokinase PA activity also investigated in the epithelial cell migration during corneal epithelial wound healing, which was significantly inhibited by antibodies to uPA.Thus, the activity of uPA in corneal epithelial wound healing may be facilitated in part by the expression of uPAR on epithelial cell surface [80] .Recently, Sugioka et al. [81] investigated the role of uPA in a murine model of lipopolysaccharide (LPS)-induced corneal inflammation.Their results suggest that uPA promotes LPS-induced leukocyte infiltration in cornea, and that uPA is an important component in LPS-induced corneal inflammatory responses.Thus, PA released from all layers of cornea may play an important role in corneal biology, as for example, in the regulation of vessel permeability, angiogenesis, and in the initiation of PMNs chemotaxis.
Interestingly, PARs are specific targets to initiate protease mediated inflammation [1][2][3]82] . Ziesk and Bukusoglu [83] demonstrated that protease inhibitors inhibit corneal epithelial migration.Moreover, Lang et al. [7] and Nickel et al. [84] demonstrated that human corneal and conjunctival epithelial cells express functional PAR1 and PAR2.Activation of PAR1 and PAR2 on HCE cells by thrombin and trypsin resulted in the production of proinflammatory cytokines such as IL-6, IL-8, and TNFα [7]   . In his regard, PAR1 and PAR2 may be important in inflammatory eye diseases.
The Role of aPA in Recognition of PAR2, But not and nonpathogenic (A.hatchetti, A. astronyxis, and A. castellanii neff) strains of Acanthamoeba.aPA activity of the purified aPA (1 µg) from Acanthamoeba trophozoites was measured by radial diffusion in the fibrin agarose (1%) clot.Clots lacking plasminogen were included in all experiments for plasminogenindependent fibrinolysis.aPA activity was expressed in international units per milligram relative to human tPA standard.(B) Fibrin plasminogen zymography of purified aPA from A. castellanii.Purified protein (10 µg) was electrophoresed in 4% to 15% SDS-PAGE-ready gels and then was washed three times in PBS to remove SDS.Gels were then overlaid onto fibrinogen containing plasminogen in agarose clots for 2 hours.Lane 1: untreated aPA protein; lanes 2 and 3: pretreatment with serine proteinase inhibitors 1 mM PMSF and 1 mM 1, 10phenanthroline (1,10-ph); lane 4: pretreatment with 10 µM cysteine proteinase inhibitor E6; lane 5: human standard urokinase (UK).kDa, molecular mass standard.Reprinted with permission [65] .Copyright held by The Association for Research in Vision and Ophthalmology (ARVO).

PAR1 in HCE Cells
We have shown that PAR1 and PAR2 transcripts were constitutively expressed in HCE cells and aPA induced up regulation of PAR2, but not PAR1 transcript in the HCE cells.HCE cells stimulated with PAR2 agonists (SLIGRL-NH2 and AC 55541) and PAR1 agonists (thrombin and TRAP-6) upregulate PAR2 and PAR1 mRNA, respectively.PAR2 antagonist (FSLLRY-NH2), but not PAR1 antagonist (SCH 79797), effectively inhibits PAR2 mRNA expression induced by aPA, suggested that aPA activates PAR2 similar to PAR2 agonist.Likewise, upregulated transcripts of PAR1 and PAR2 by their specific agonists were inhibited by SCH 79797 and FSLLRY-NH2, respectively (Figure 3A and 3B).Furthermore, immunolocalization studies demonstrated that HCE cells stimulated with specific PAR1 and PAR2 agonist activate PAR1 and PAR2 on HCE cell surface.aPA specifically activates PAR2 expression on HCE cell surface, but not PAR1 [9] .Thus, taken together, the flow cytometry and immunocytochemistry assessments demonstrated that aPA is a PAR2 activator, but not a PAR1 activator (Figure 4A and 4B) [9] .Immunolocalization findings of our study [9] agree with surface protein expression of PAR1 and PAR2 in whole human cornea [7] ; however, in our study PAR1 and PAR2 cell surface expression observed upon stimulation by specific agonists [9] while whole human cornea expressed PAR1 and PAR2 immunoreactivity on the apical cell surface of the most superficial corneal epithelial cells in normal condition [7] .Gene expression and cell surface expression of PAR1 and PAR2, and specific activation of PAR2 by aPA seems to play a critical role in pathophysiology of Acanthamoeba keratitis and fascinates to use PAR2 blockers in vivo to determine therapeutic approach in management of Acanthamoeba keratitis.
NH2 (PAR2 agonist) specifically induced production of pro-inflammatory cytokines (IL-6, IL-8, and TNFα) protein in HCE cells.Our study suggests that aPA is an inflammatory protein which plays a critical role in pathogenesis of Acanthamoeba keratitis through PAR2 pathway [9] , and suggests further in vivo studies to To immunocytochemistry assessment, cells were stained with polyclonal rabbit anti-PAR2 antibody and Alexa Fluor 488-conjugated anti-rabbit antibody.Cells without primary antibody incubation were used as a negative control.Rat PAR2 (368-382) peptide were used as absorption control.4, 6-diamidino-2-phenylindole (DAPI) counterstaining was used to visualize cell location and morphology.Three slides in each group were viewed using fluorescence microscopy.Images were captured with an Olympus AX70 Upright Compound Microscope.Reprinted with permission [9] .Copyright held by The Association for Research in Vision and Ophthalmology (ARVO).
delineate the PAR2 pathway as a specific target to approach Acanthamoeba keratitis related therapy.

Summary: Potential Role of PAR2 Antagonists to Attenuate aPA Induced Proinflammatory Mediators
The potential role of PAR2 antagonist in attenuating proinflammatory cytokine IL-8 induced by Acanthamoeba plasminogen activator, aPA, in human corneal epithelial (HCE) cells, is summarized in figure 6.It is possible that other chemokines/cytokines are involved in inflammatory responses in Acanthamoeba keratitis.We have shown that CXCL2 (IL-8 equivalent in rodents) is the major chemokines that plays a major role in attracting antagonist, but not by PAR1 antagonist.HCE cells were incubated with aPA (100 µg/ml) and PAR1 agonists (thrombin, 10 µM; TRAP-6, 10 µM) for 48 hours.Inhibition of PAR1 involved pre-incubating the HCE cells for 1 hour with the PAR1 antagonist (SCH 79797, 60 μM) and then incubated with or without aPA, PAR1 agonists for 48 hours (5A).HCE cells were incubated with aPA (100 µg/ml) and PAR2 agonists (SLIGRL-NH2, 100 µM; AC 55541, 10 µM) for 24 hours.Inhibition of PAR2 involved pre-incubating the HCE cells for 1 hour with the PAR2 antagonist (FSLLRY-NH2, 100 μM) and then incubated with or without aPA and PAR2 agonists for 24 hours (5B).Supernatants were collected from harvested cells and subjected to IL-8 ELISA.The data are mean ± SEM of three independent experiments (*P < 0.05).P values were obtained by unpaired Student's t-test.Reprinted with permission [9] .Copyright held by The Association for Research in Vision and Ophthalmology (ARVO).
inflammatory cells such as polymorphonuclear neutrophils (PMNs) at the site of infection in Chinese hamster model of Acanthamoeba keratitis [61,62] .Therefore, to determine PAR2 implication in Acanthamoeba keratitis using in vitro model of human corneal epithelial cells, we focused on IL-8 production to explore the functional activity of aPA which effectively inhibited by the use of PAR2 antagonist [9]   .Likewise the agonists of PAR2 including trypsin and SLIGRL-NH2, modulate IL-6, IL-8, TNFα in HCE cells [7]   , aPA can induce other chemokines/cytokines in corneal epithelial cells, warranting further study.
In conclusion, increased expression and activation of PAR2 in HCE cells by aPA exposure is closely associated with inflammatory conditions, suggesting that PAR2 is relatively novel receptor to contribute critical role in inflammatory process during contact-independent mechanism of Acanthamoeba infection.Acanthamoeba plasminogen activator induced PAR2 expression and IL-8 modulation at gene and protein levels is specifically inhibited by PAR2 antagonist [9] .Thus, PAR2 antagonists may be therapeutic target to cure Acanthamoeba keratitis.

Future Work
In order to further evaluate the potential roles of PAR2 in Acanthamoeba keratitis, the following experiments should be considered: (i) investigating further expression and production of various inflammatory mediators mediated by aPA through PAR2 pathway; (ii) investigating the effects of various antagonists of PAR2 associated G protein and anti-aPA antibody on expression and function of inflammatory mediators during aPA exposures; (iii) investigating PAR2 associated downstream signaling pathway induced by aPA exposures.More importantly, future studies are warranted to investigate pharmacological approach using the selective PAR2 antagonists and proteinase inhibitors to determine the role of PAR2 in corneal infections and inflammation induced by Acanthamoeba infection.

Figure 1 .
Figure 1.(A) Levels of aPA activity produced by pathogenic (A.castellanii, A. polyphaga, A. rhysodes, and A. culbertsoni)and nonpathogenic (A.hatchetti, A. astronyxis, and A. castellanii neff) strains of Acanthamoeba.aPA activity of the purified aPA (1 µg) from Acanthamoeba trophozoites was measured by radial diffusion in the fibrin agarose (1%) clot.Clots lacking plasminogen were included in all experiments for plasminogenindependent fibrinolysis.aPA activity was expressed in international units per milligram relative to human tPA standard.(B) Fibrin plasminogen zymography of purified aPA from A. castellanii.Purified protein (10 µg) was electrophoresed in 4% to 15% SDS-PAGE-ready gels and then was washed three times in PBS to remove SDS.Gels were then overlaid onto fibrinogen containing plasminogen in agarose clots for 2 hours.Lane 1: untreated aPA protein; lanes 2 and 3: pretreatment with serine proteinase inhibitors 1 mM PMSF and 1 mM 1, 10phenanthroline (1,10-ph); lane 4: pretreatment with 10 µM cysteine proteinase inhibitor E6; lane 5: human standard urokinase (UK).kDa, molecular mass standard.Reprinted with permission[65] .Copyright held by The Association for Research in Vision and Ophthalmology (ARVO).

Figure 2 .
Figure 2. Schematic representation of Acanthamoeba plasminogen activator (aPA) characterization.aPA produces a single band of lysis on fibrinogen-agarose zymographs in the presence of plasminogen.Activity of aPA is completely inhibited by the treatment with diisopropylfluorophosphate (DIFP), phenylmethylsulfonyl fluoride (PMSF), and 1,10-phenanthroline (1,10-ph) indicating that it is a serine protease.aPA activity is not inhibited by amiloride (a strong inhibitor of uPA) and plasminogen activator inhibitor-l (PAI-1, an inhibitor of uPA and tPA).

Figure 4 .
Figure 4. PAR2 surface protein expression is upregulated by aPA in HCE cells.HCE cells were incubated with or without aPA (100 µg/ml) and PAR2-agonist (SLIGRL-NH2, 100 µM) for 24 hours.PAR2 surface protein expression in HCE cells were examined by flow cytometry and immunocytochemistry. (4A) Briefly, to flow cytometry assessment cells were incubated with PE-labeled mouse IgG2a anti-human PAR2 and isotype control (PE-labeled mouse IgG2b) antibody.PAR2 expression in untreated HCE cells were compared with treated HCE cells.The results were expressed as normalized median fluorescence intensity (nMFI) units of positively stained HCE cells with PE-labeled antibody subtracted from MFI of unstained HCE cells as, (MFIPositive -MFINegative).The data are mean ± SEM of three independent experiments (*P < 0.05).P values were obtained by unpaired Student's t-test.(4B)To immunocytochemistry assessment, cells were stained with polyclonal rabbit anti-PAR2 antibody and Alexa Fluor 488-conjugated anti-rabbit antibody.Cells without primary antibody incubation were used as a negative control.Rat PAR2 (368-382) peptide were used as absorption control.4, 6-diamidino-2-phenylindole (DAPI) counterstaining was used to visualize cell location and morphology.Three slides in each group were viewed using fluorescence microscopy.Images were captured with an Olympus AX70 Upright Compound Microscope.Reprinted with permission[9] .Copyright held by The Association for Research in Vision and Ophthalmology (ARVO).