Conjunctival Fluid Secretion Impairment via CaCC-CFTR Dysfunction Is the Key Mechanism in Environmental Dry Eye

Dry eye disease (DED) is a multifactorial disease with an incidence of approximately 50% worldwide. DED seriously affects quality of life and work. The prevalence of environmental DED (eDED) ranges from 35 to 48%. Conjunctival fluid secretion dysfunction may be one of the major causes of DED. Notably, the Cl– flux corresponds to the conjunctival fluid secretion and could be affected by ATP. Both the cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+-activated Cl– channel (CaCC) are Cl– channels involved in epithelial fluid secretion. Conjunctival fluid secretion could be increased by activating P2Y2R (an ATP receptor) in DED. However, the role of the CaCC and CFTR channels regulated by P2Y2R in eDED remains unclear. In this study, we established a rabbit eDED model using a controlled drying system. A Ussing chamber was used to perform a conjunctival short-circuit current induced by ATP to evaluate the reactivity of the ion channels to the ATP. Our results revealed that eDED accompanied by conjunctival fluid secretion impairment was caused by a P2Y2R dysfunction, which is related to CaCC-CFTR signaling in the conjunctiva epithelium. Notably, the coupling effect of the ATP-induced CaCC-CFTR activation and intracellular Ca2+ may represent a promising therapeutic target for treating eDED.


Introduction
More than one billion people suffer from dry eye disease (DED) worldwide. Severe DED can cause visual impairment, which severely affects the quality of life of patients [1,2]. DED is a multifactorial disease of the ocular surface, characterized by a loss of homeostasis of the tear film [3]. The predisposing factors for DED include environmental risk, androgen deficiency, contact lenses, and aging [4]. A mild DED, environmental dry eye disease (eDED), has become increasingly common, with an incidence of 35-48% [1]. Notably, tear production has been shown to be significantly decreased in eDED patients [5]. In tear production, the secretory rate of the conjunctival fluid exceeds the basal tear production [6]. Conjunctival secretion has been shown to contribute approximately one-fourth of the total tear production [7], indicating that conjunctival secretion could play an important role in tear production. Whether conjunctival secretion is impaired in eDED and the role it plays in the pathogenesis of eDED are still unknown.
The secretion of water from conjunctival epithelial cells was evaluated using chloride ion (Cl -) fluid. The outflow of the Clflux corresponded to the conjunctival fluid secretion [8]. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMPdependent Clchannel that facilitates fluid secretion in the apical side of the conjunctiva [9]. The CFTR agonist could increase the tear volume in clinical DED patients [8]. However, no

eDED Has Ocular Surface Injury and Conjunctival Fluid Secretion Dysfunction
To investigate an ocular surface injury and the tear secretion in eDED, a controlled drying system (CDS) was established. In this study, the disruptions of the conjunctiva and cornea epithelium were stained with lissamine green and fluorescein, respectively. The staining score was positively correlated with the staining area. Compared with the control group, the lissamine green score increased from day 7 to day 14 in the dry eye group (day 7: 0.15 ± 0.08 vs. 1.30 ± 0.15; day 14: 0.15 ± 0.08 vs. 1.90 ± 0.23, all n = 10-11, p < 0.001) (Figure 1a). Similarly, the fluorescein staining score significantly increased from day 7 to day 14 in the dry eye group (day 7: 1.00 ± 0.26 vs. 5.00 ± 0.49; day 14: 1.10 ± 0.18 vs. 7.27 ± 0.65, all n =10-11, p < 0.001) (Figure 1b). The fluid secretion from the conjunctiva is one of the most vital components of tear production. As expected, the tear production also declined from day 7 to day 14 in the dry eye group (9.45 ± 0.47 vs. 5.80 ± 0.39 mm at day 7; 9.63 ± 0.51 vs. 3.20± 0.24 mm at day 14; all n = 10-11, p < 0.001) (Figure 1c). Consistent with the above results, the ocular epithelium combined with the fluid reduction in the conjunctiva was impaired in eDED.

P2Y 2 R Plays a Key Role in eDED
Activating ATP receptors increases the intracellular Ca 2+ and cAMP [19,20]. To confirm the changes in the intracellular Ca 2+ in eDED, we digested the eDED conjunctival epithelium and loaded it with an intracellular calcium fluorescence probe (Fluo-3/AM). The results showed that the intensity of the intracellular Ca 2+ was decreased in the eDED conjunctival epithelium (10.80 ± 1.02 vs. 4.85 ± 0.9 AU, n = 6, p < 0.001) (Figure 2a). In addition, the concentration of the cAMP in the conjunctiva epithelium was reduced in the dry eye group (5.08 ± 0.26 vs. 4.30 ± 0.21 nmol/mL, n = 9, p < 0.05) (Figure 2b). Subsequently, the ATP did not evoke an intracellular Ca 2+ release of the same amplitude in the conjunctival epithelium in the dry eye group as in the control group (Figure 2c). The ATP-dependent Isc reflects the reactivity of the ion channels to the ATP, which plays a role via P2Y 2 R [13]. The conjunctival fluid secretion is closely related to the function of the anion channel [8]. Notably, the initial peak (first peak) and sustained peak (second peak) of the conjunctival epithelium Isc were markedly decreased after the ATP stimulation in the dry eye group (first peak: 27.19 ± 3.06 vs. 7.08 ± 1.79, n = 6, p < 0.001; second peak:

P2Y2R Plays a Key Role in eDED
Activating ATP receptors increases the intracellular Ca 2+ and cAMP [19,20]. To confirm the changes in the intracellular Ca 2+ in eDED, we digested the eDED conjunctival (c) Bar graph showing the tear volume in the control (n = 11) and eDED groups (n = 10). Data are represented as mean ± SEM. *** p < 0.001. [13]. The conjunctival fluid secretion is closely related to the function of the anion channel [8]. Notably, the initial peak (first peak) and sustained peak (second peak) of the conjunctival epithelium Isc were markedly decreased after the ATP stimulation in the dry eye group (first peak: 27.19 ± 3.06 vs. 7.08 ± 1.79, n = 6, p < 0.001; second peak: 28.84 ± 2.92 vs. 6.32 ± 1.28, n = 6, p < 0.05) (Figure 3a). These results demonstrated that the ATP receptor dysfunction in the conjunctiva epithelium plays a key role in eDED. To elucidate the relationship between the P2Y2R and Isc variation, a P2Y2R inhibitor (suramin) was applied before the conjunctival ATP stimulation. The results showed that treatment with the P2Y2R inhibitor led to a decrease of 50% in the ATP-induced conjunctival Isc (first peak: 24.06 ± 3.24 vs. 11.98 ± 2.34; second peak: 27.27 ± 2.50 vs. 5.14 ± 2.10, all n = 6-8, p < 0.01) (Figure 3b). The ATP-induced conjunctival Isc decreased by more than half in the P2Y2R-siRNA treatment group compared with the control group (first peak: 16.67 ± 2.10 vs. 6.79 ± 3.17; second peak:15.84 ± 2.60 vs. 6.50 ± 2.84, all n = 4-5, p < 0.05) (Figure 3c). The rabbit ocular surfaces were also impaired using the P2Y2R-siRNA eyedrop treatment ( Figure 3d). These findings showed that the ATP-induced conjunctival Isc appropriately showed conjunctival fluid secretion. The ATP-induced conjunctival Isc deficiency and subsequent decrease in the conjunctival fluid secretion in eDED contributed to the conjunctival P2Y2R dysfunction. To elucidate the relationship between the P2Y 2 R and Isc variation, a P2Y 2 R inhibitor (suramin) was applied before the conjunctival ATP stimulation. The results showed that treatment with the P2Y 2 R inhibitor led to a decrease of 50% in the ATP-induced conjunctival Isc (first peak: 24.06 ± 3.24 vs. 11.98 ± 2.34; second peak: 27.27 ± 2.50 vs. 5.14 ± 2.10, all n = 6-8, p < 0.01) (Figure 3b). The ATP-induced conjunctival Isc decreased by more than half in the P2Y 2 R-siRNA treatment group compared with the control group (first peak: 16.67 ± 2.10 vs. 6.79 ± 3.17; second peak:15.84 ± 2.60 vs. 6.50 ± 2.84, all n = 4-5, p < 0.05) (Figure 3c). The rabbit ocular surfaces were also impaired using the P2Y 2 R-siRNA eyedrop treatment (Figure 3d). These findings showed that the ATP-induced conjunctival Isc appropriately showed conjunctival fluid secretion. The ATP-induced conjunctival Isc deficiency and subsequent decrease in the conjunctival fluid secretion in eDED contributed to the conjunctival P2Y 2 R dysfunction.

Discussion
In this study, P2Y2R dysfunction combined with the decreased intracellular cAMP and cytosolic Ca 2+ in the conjunctival epithelium was demonstrated in an eDED rabbit model. The P2Y2R dysfunction caused a conjunctival fluid secretion impairment, which was derived from the ATP through the CaCC-CFTR signaling. The release of the intracellular Ca 2+ plays a core cross-pivotal role in the CaCC-CFTR signaling and is indispensable for the ATP-induced conjunctival fluid secretion. Therefore, the impairment of the conjunctival fluid secretion via the CaCC-CFTR pathway dysfunction could be a key mechanism in eDED.
The previous studies have shown that fluid in the conjunctiva epithelium is transported from the stromal (basolateral) side to the mucosal (apical) side. Conjunctival fluid secretion is dependent on the outflow of the anion flux [8]. There are various ion channels , PLC inhibitor group, IP3R inhibitor group, and Ca 2+ chelator group (n = 6 in each group). Data are represented as mean ± SEM. PLC, phospholipase C; IP3R, inositol (1,4,5) trisphosphate receptor. All results were compared with those of the control group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Discussion
In this study, P2Y 2 R dysfunction combined with the decreased intracellular cAMP and cytosolic Ca 2+ in the conjunctival epithelium was demonstrated in an eDED rabbit model. The P2Y 2 R dysfunction caused a conjunctival fluid secretion impairment, which was derived from the ATP through the CaCC-CFTR signaling. The release of the intracellular Ca 2+ plays a core cross-pivotal role in the CaCC-CFTR signaling and is indispensable for the ATP-induced conjunctival fluid secretion. Therefore, the impairment of the conjunctival fluid secretion via the CaCC-CFTR pathway dysfunction could be a key mechanism in eDED.
The previous studies have shown that fluid in the conjunctiva epithelium is transported from the stromal (basolateral) side to the mucosal (apical) side. Conjunctival fluid secretion is dependent on the outflow of the anion flux [8]. There are various ion channels on the conjunctival epithelium, among which aquaporins, CFTR, ENaC, and CaCC are closely related to tear secretion [8,23]. Isc was found to present cellular ion channel reactivity on different activators. The ATP-induced Isc reflects the reactivity of the ion channels to the ATP [24]. As shown in this study, more than 90% of the ATP-induced Isc amplitude responses from the apical side of the conjunctival epithelium were produced by the anion secretion, suggesting that the ATP-induced Isc response could well represent the conjunctival fluid. The secretion of Cl − from the conjunctival basal side to the apical side via an exchange with the sodium ions was demonstrated to account for approximately 60% of the Isc response [25,26]. Therefore, the outfluxes of HCO 3 − and other anions could occupy more than one-third of the ATP-induced Isc response. A similar fluid secretion was also detected in the cortical collecting duct cells [27] and the caudal epididymal epithelium [28].
To date, the activation of the P2Y receptors has been considered to stimulate the fluid secretion across the conjunctiva [24]. The levels of the intracellular cAMP and Ca 2+ are elevated after the P2Y receptor activation [19,20]. The intrinsic process of activating P2Y 2 R is interrelated with the increase in the PLC and the transient increase in the intracellular Ca 2+ concentration [11], suggesting that the cytosolic Ca 2+ may regulate the conjunctival fluid secretion. Moreover, a release of the intracellular Ca 2+ by another way, but not the PLC pathway, could have the same impact [29]. These results indicate that P2Y 2 R is a key factor in the conjunctival fluid secretion. However, the concentration of the ATP in tears increased in dry eye patients [30]. The concurrent cellular apoptosis of the conjunctiva epithelium, which resulted in P2Y 2 R dysfunction, was believed to cause increased tear ATP. The results of the present study showed that the P2Y 2 R dysfunction with decreased intracellular cAMP and Ca 2+ existed in the conjunctiva of eDED, suggesting that P2Y 2 R plays a key role in eDED. In contrast, silencing P2Y 2 R led to a dry eye condition, and the inhibition of P2Y 2 R effectively suppressed the ATP-induced conjunctival Isc.
The CFTR is a cAMP-dependent Cl − channel existing in both the corneal and conjunctival epithelium, which increased the basal tear volume after activation [8]. The present study also demonstrated that the ATP-induced conjunctival Isc was significantly suppressed by applying a CFTR inhibitor. Hence, the CFTR contributed to the ATP-induced conjunctival fluid secretion. However, the intracellular cAMP in the conjunctiva was decreased in the eDED model, indicating that the CFTR signaling was impaired in the dry eye condition.
In this study, the CaCC inhibitor decreased the ATP-induced conjunctival Isc, indicating that the CaCC signaling may be the main contributor to the ATP-induced conjunctival fluid secretion. The CaCC, another Cl − channel, exists in the apical surface of the conjunctiva epithelium [31]. The CaCC is a Ca 2+ dependent Clchannel that transports the fluid secretion from the airway epithelium [32]. Our results showed that increased intracellular Ca 2+ activated a transient opening of the CaCC and then a persistent CFTR channel for the Cl − secretion from the conjunctival epithelial cells. This study is the first in the literature to verify the combination of the CaCC signaling and CFTR in the Cloutflow across the conjunctival epithelium.
Our results showed that the content of the intracellular Ca 2+ in the conjunctiva epithelium was decreased in eDED and did not respond to the ATP activation. The level of the intracellular Ca 2+ was shown to be closely related to the function of the epithelial secretion, such as in the airway epithelium [13], caudal epididymal epithelium [28], and conjunctiva epithelium [29]. The intracellular Ca 2+ in the conjunctiva epithelium can be regulated via different mechanisms. The activation of the transient receptor potential melastatin 8 channel increased the intracellular Ca 2+ and then conducted sense information to the higher brain center [33]. In this study, P2Y 2 R dysfunction was found in the conjunctiva epithelium in eDED, which may be an intrinsic cause of the decreased intracellular Ca 2+ . Except for P2Y 2 R, the imbalance in the intracellular Ca 2+ could be affected by endoplasmic reticulum stress and a mitochondrial matrix Ca 2+ overload, which triggered cell apoptosis [34,35]. Endoplasmic reticulum stress-induced ocular surface epithelial cell apoptosis was shown in the pathogenesis of DED. Thus, further work is needed to explore the detailed mechanism of intracellular Ca 2+ in eDED.

Animals
Male New Zealand white rabbits weighing 2.0-2.5 kg (purchased from Guangdong Provincial Center for Animal Research, Guangzhou, China) were chosen for the study. The rabbits were quarantined and acclimatized for one week before the experiments. All experimental procedures were approved by the Institutional Animal Care and Use Committee at Zhongshan Ophthalmic Center at Sun Yat-sen University and complied with all relevant ethical regulations for animal testing and research.

Environmental Dry Eye Disease Model
The rabbit eDED model was established using a controlled drying system (CDS). A detailed system for dry eye models was published in our previous report [36]. In brief, the rabbits in the dry eye group were reared in the CDS at a relative humidity of 22 ± 4%, an air flow of 3-4 m/s, and temperatures from 23 to 25 • C. The control group remained in a normal environment. Each rabbit was lissamine green/fluorescein stained as normal [9,37] and evaluated for tear production on days 0, 7, and 14.

Intervention of Rabbit Eyes with P2Y 2 R siRNA
Small interfering RNA (siRNA) sequences for silencing P2Y 2 R were applied topically four times per day to the ocular surfaces of the rabbits. The nucleotide sequence of the siRNA target site chosen to silence the expression of the rabbit P2Y 2 R-coding sequence (GenBank EU886321) was as follows: 5 -AAC CTG TAC TGC AGC ATC CTC-3 [38,39]. The siRNA molecule was synthesized by RiboBio company (Guangzhou, China). The control group was given a normal saline as siRNA vehicle. The measurement of corneal fluorescein staining was in accordance with the above regulations.

Short-Circuit Current (Isc) Measurements
The Isc measurement was performed following the modified procedure described in our previous reports [40]. The superior conjunctival tissues were carefully isolated from the rabbits and clamped vertically between two halves of a Ussing chamber (EM-CSYS-2 Ussing Chamber Systems, Physiologic Instruments, San Diego, CA, USA) with an inner area of 0.45 cm 2 . The side of the epithelium was placed in a fixed direction facing the positive electrode. Then, the conjunctiva was bathed on both sides with bicarbonate Ringer's solution composed of the following (in mM): 116.4 NaCl, 5.4 KCl, 5.4 NaHCO3, 0.78 NaH2PO4, 1.8 CaCl2, 0.81 MgCl2, 5.55 D-glucose, and 10 HEPES. It was then gassed with 95% O 2 /5% CO 2 at 37 • C to maintain a pH of 7.4. The epithelium exhibited a basal transepithelial potential difference, which was measured by Ag/AgCl electrodes with KCl/Agar bridges connected to a voltage-clamp amplifier (VCC MC6, Physiologic Instruments, San Diego, CA, USA). The transepithelial potential difference in the conjunctiva epithelial layer was clamped at 0 mV for the Isc measurement.
The change in Isc was defined as the difference between the value at baseline and that at a peak following compound addition, displayed via a signal collection and analysis system (BL-420E + Systerm, Chengdu TME Technology Co., Ltd., Chengdu, China). When the current changed to the 1 mV pulse, it was used to estimate transepithelial resistance according to Ohm's law [40]. Ten minutes after the Isc plateau, ATP was applied to the apical side, and then the ATP-induced Isc was recorded.
In the Cl --free BRS, NaCl, KCl, and CaCl 2 were replaced by the respective salts of gluconate. In the HCO 3 --free solution, HCO 3 − was substituted by 10 mM HEPES and bathed with 100% O 2 to maintain pH at 7.4. In the Cl --and HCO 3 --free solution, the components were the same as in the Cl − -free solution, except that NaHCO 3 was replaced by 20 mM Na-gluconate and 10 mM HEPES. Inhibitors or a vehicle (0.5% DMSO, D2650, Sigma, Burlington, MA, USA) were added to the buffer medium for 20 min before ATP was applied to the apical side. The dosages of inhibitors were as follows: 100 uM U73122

Measurement of Intracellular cAMP
The concentration of intracellular cAMP in the conjunctival epithelium was measured using a cAMP assays kit (E-EL-0056c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol. Optical density readings were measured using a SynergyTM H1 multifunctional microplate reader (BioTek Instruments, Inc., Winooski, VT, USA). Concentrations were determined using a standard curve as a reference.

Measurement of Intracellular Ca 2+
Intracellular Ca 2+ was measured using the fluorescence dye Fluo-3/AM according to a modified procedure described in a previous report [41]. The conjunctival epithelium from the rabbits was digested and harvested with 1 mg/mL dispase-II solution for approximately 1 h, seeded in DMEM/F12 medium on glass-bottom cell culture dishes (20 mm, NEST, Wuxi, China), and cultured at 37 • C. After 10 min of cell adhesion, the cells were prepared and loaded with the fluorescent dye Fluo-3/AM (Molecular Probes, Eugene, OR, USA). The fluorescence signal was monitored and recorded using a laser-scanning confocal imaging system (TCS SP2; Leica Microsystems, Wetzlar, Germany). The change in fluorescence intensity after ATP treatment was normalized to the initial intensity.

Statistical Analysis
All statistical analyses were performed using SPSS version 22.0. All results were expressed as mean ± SEM. Statistical significances were determined using a Student's t-test (two groups), and a repeated measures ANOVA, followed by a Least significant difference post hoc test (groups with repeated measurements). The differences between the groups were considered statistically significant if p was less than 0.05.

Conclusions
The results of this study indicate that the Ca 2+ -activated CaCC-CFTR pathway plays a key role in conjunctival secretion. The impairment of the P2Y 2 R-initiated CaCC-CFTR may be a key mechanism of the conjunctival secretion dysfunction in eDED.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.