Effects of fine particulate matter on the ocular surface: An in vitro and in vivo study

Biomedicine & Pharmacotherapy 117 (2019) 109177

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Effects of fine particulate matter on the ocular surface: An in vitro and in vivo study Qian Yanga,c,1, Kunke Lia,1, Dai Lib, Yafang Zhangb, Xiuping Liua, Kaili Wua,

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Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-Sen University, Guangzhou 510060, China Department of Ophthalmology, Hubei University of Science and Technology, Xianning, China c Department of Ophthalmology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China b

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Keywords: Fine particulate matter Ocular surface Conjunctiva Corneal epithelial cell Tear film break-up time

Exposure to ambient fine particulate matter (fine PM) pollution has been previously associated with ocular surface diseases. But, to the best of our knowledge, the in vivo long-term effects of fine PM on the ocular surface have not been investigated. We aimed to evaluate the effects of fine PM on cultured human corneal epithelial (HCE) cells and on the ocular surfaces of mice, with standard reference material of fine PM(SRM 2786). We applied fine PM suspension to the eyes of C57BL/6 mice for up to 6 months. In vivo examinations, including tear secretion, tear film break-up time (TBUT) and corneal fluorescein staining, were performed in the 3rd and 6th month. At the end of the in vivo study, the corneal histological changes and conjunctival goblet cells were examined by staining, and cytokines in tissue were also detected. In addition, HCE cells were treated with fine PM for 12 h and 24 h. Then, cell apoptosis and reactive oxygen species (ROS) formation was detected. We found that fine PM damages the mouse eye in a dose- and time-dependent manner. In mice, the tear secretion and tear film break-up time were significantly reduced, along with the development of corneal epithelial damage, apoptosis of conjunctival epithelial cells and hypoplasia of conjunctival goblet cells. In addition, IL-18, IL-22, IL23 and MCP-1 were increased in both conjunctiva and cornea of the fine PM-treated animals. Furthermore, increased apoptosis and ROS production were observed in time- and dose-dependent manner in HCE cells after fine PM exposure for 12 h and 24 h. Our results indicate that fine PM is cytotoxic to both HCE cells and the ocular surface. Long-term topical application of fine PM suspension in mice results in ocular surface changes that are similar to those observed with dry eye.

1. Introduction With the development of modern society, environmental pollution, especially air pollution, is seriously increasing. Air pollutants mainly consist of thousands of solid particles, gases, and liquid droplets in the air, of which particulate matter (PM) is an important component [1]. According to the aerodynamic diameter, PM10 represents the particle mass that enters the respiratory tract and, moreover, it includes both the coarse (particle size between 2.5 and 10 μm) and fine particles (measuring less than 2.5 μm, PM2.5) that are considered to contribute to the health effects [2]. Both PM 10 and PM2.5 have recently garnered wide interest and is thought to greatly endanger people’s health. Increases in a number of diseases, such as cardiovascular and cerebrovascular diseases [3,4], respiratory disorders [5] as well as an increase in neonatal mortality [6] have been attributed to PM.

The eye is one of the few organs that are constantly exposed to the external environment. Some studies have indicated that air pollution may have harmful effects on the eye [7,8]. For example, epidemiological investigations have found that people with short- or long-term exposure to severe air pollution often experienced eye irritation (jealous, itchy eyes, irritation, tearing, etc.) [9,10]. Other studies have reported that outpatient visits for conjunctivitis are significantly associated with air pollution [11–13]. In addition, the number of patients with allergic conjunctivitis has been shown to increase with increasing levels of PM during the pollen-free season [14,15]. Most of these studies are based on epidemiological data, in vitro cell models or other indirect evidence. To date, since there have been no in vivo studies of the longterm effects of PM on the ocular surface, an in-depth exploration of the direct effects of PM on the ocular surface is needed. Most of the studies evaluating the effects of air pollution on the eye

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Corresponding author at: Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, China. E-mail address: [email protected] (K. Wu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109177 Received 10 April 2019; Received in revised form 12 June 2019; Accepted 25 June 2019 0753-3322/ © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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2.3. Examination of ocular surface and the tear fluids

have focused on the conditions of the eye surface. Dry eye, a common ocular surface disease with multiple risk factors, has increased rapidly in recent years, and air pollution is thought to be associated with this rise [16,17]. An epidemiological investigation reported that exposure to air pollution reduced tear film stability and influenced the tear osmolarity [1]. Nitrogen dioxide (NO2) exposure has also been shown to lead to goblet-cell hyperplasia [18]. Recently, Cui and colleagues reported that PM2.5 can delay corneal epithelium wound healing by inhibiting cell migration [19]. Li reported that a dry eye model can be induced by topical administration of PM10 for two weeks in BALB/c mice [20]. Although these studies suggesting that various particulate matters can damage the ocular surface, the long-term effects of PM on the occurrence and development of dry eye are presently lacking. In addition, various mimetic substances have been used in previous studies. For example, self-collected PM2.5 from the local atmosphere [19,21], a single substance of a substitute of PM [22], and other sources of PM [20,23], have been investigated. Researches on the toxic effects of PM frequently disregard the differences in particle composition among different sources of the fine PM employed [24]. It has been suggested that individual PM concentrations are less important than its components in determining its damaging effects [21,25]. We should therefore consider these different sources of PM when comparing and explaining the results from different studies. Therefore, the aim of this study was to explore the long-term effect of fine PM on the ocular surfaces of mice, with Standard Reference Material (SRM 2786, mean diameter < 4 μm) of PM powder containing organic/inorganic constituents, and particle-size characteristic of atmospheric particulate material and similar matrices, which is somewhat different from those fine PM used in most previous studies [21,24,26].

Examinations were conducted before and 3 and 6 months posttreatment. The examination technique and the environment were kept as consistent as possible. All mice were anesthetized with intraperitoneal injection of 10% chloral hydrate (300 mg/kg) before the measurements. The ocular surface of each animal was examined routinely with or without fluorescein staining and photographed [27]. The tear secretions of each mouse were measured using phenol red thread (Tianjin JingMing Tech Co., Ltd) test (PRT-test). One end of the thread was hung on the lower lateral 1/3 of the eyelid for 15 s. The length of the wetted cotton thread was determined. Each mouse was independently measured 3 times, and the average of the values was calculated for further analysis. To test the tear film break-up time (TBUT), 2 μl of 0.5% sodium fluorescein was instilled into the conjunctival sac [27]. After manually closing the eyelids three times, the TBUT was recorded using a slit-lamp biomicroscope with a cobalt blue filter. The right eye of each mouse was measured 3 times, and the average value was used for analysis. 2.4. Morphological assessment After using the PM eye drops for 6 months, eight mice in each group were sacrificed by overdose anesthesia, and the eyeballs were gently dissected. Four ocular specimens in each group were fixed in 10% formalin, embedded in paraffin and, then sectioned into 4 μm vertical slices. Hematoxylin and eosin (H&E) staining was conducted as previously reported [27]. Briefly, the tissue sections were washed in distilled water for 5 min and treated with 0.1% hematoxylin buffer (Sigma-Aldrich Co., St. Louis, MO, USA) for 8 min at room temperature. Then, the sections were washed and dipped in 1% Eosin Y (Sigma-Aldrich Co., St. Louis, MO, USA) solution for 1 min. Histological analyses of corneal tissues were conducted under a light microscope (Olympus, Tokyo, Japan). Periodic acid-Schiff (PAS) staining was performed with a commercially available kit (395B-1KT, Sigma-Aldrich Co.) following the manufacturer’s instructions as previously reported [27]. The number of goblet cells in the same area of conjunctivas in each animal was counted under a microscope. Three different specimen of each eye were randomly selected for counting, and the average was calculated (cells/ high-power visual field, 400x).

2. Materials and methods 2.1. Animals The experimental animals consisted of 6-8-week-old female C57BL/ 6 mice. The mice were housed in plastic cages lined with soft wood chips and placed in an air-conditioned room at 23 °C ± 1 °C with 60% ± 5% humidity and a light/dark (12 h/12 h) cycle. All experiments and animal care procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research under the supervision of a health authority-accredited staff member for animal care and management. The research protocol was approved by the Animal Care and Use Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University. Animals were quarantined before the experiment and adapted to the environment for 1 week. All participating mice had a normal ocular surface under slit lamp biomicroscope.

2.5. Detection of inflammatory cytokines Cornea or conjunctiva tissues from each mouse were dissected and used to extract proteins for measuring inflammatory cytokines as previous described [27]. The dissected corneal and conjunctival tissues from each eye (n = 4) were homogenized and centrifuged (15,000×g, 4 °C for 15 min) to extract proteins using the cold CelLytic™ MT Cell Lysis Reagent (Sigma-Aldrich Co.) according to the manufacturer’s instructions. The total protein concentration was determined using the bicinchoninic acid method (Shanghai Shengzheng Biot. Co., Ltd, Shanghai, China) [28]. Ten cytokines/chemokines (IL4, IL6, IL18, IL22, IL23, MCP-1, MIP-1, GROa, TNFalpha, INFgamma) were tested in this study. The concentration of cytokines in the supernatant (50 μL) was analyzed using the ProcartaPlex® Multiplex Immunoassay (Affymetrix, eBioscience, Santa Clara, CA, USA), which uses Luminex technology (multianalyte profiling beads) to simultaneously detect and quantify multiple protein targets in a sample.

2.2. Intervention with fine PM eye drops Fine PM powder (SRM 2786 - Fine Atmospheric Particulate Matter < 4 μm, NIST, Gaithersburg, MD, USA)(https://www-s.nist.gov/ ) [26] was dissolved in phosphate buffered saline (PBS) to prepare three different concentrations (0.5, 1.0, 5.0 mg/mL) of PM eye drops. Then, the suspension was bottled in plastic bottles and stored at 4 °C and used within one month. Thirty-two female C57BL/6 mice were randomly divided into a control group and three experimental groups, with 8 mice per group. The mice in each group were topically administered 5 μl PM eye drops (0, 0.5, 1.0 and 5.0 mg/mL, respectively). The eye drops were applied 4 times per day (8 A.M., 12 A.M., 4 P.M. and 8 P.M.) for 6 months. The right eye of each mouse was used for the experiment.

2.6. Cell culture and exposure to fine PM Human Corneal Epithelial-Transformed (HCE) cells (RRID: CVCL_1272) were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 (Invitrogen, Waltham, MA, USA) with 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA), 10 ng/mL human 2

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3.2. Effects of fine PM on tear fluids in mice

epidermal growth factor (hEGF) (Sigma-Aldrich Co.), 5 μm/mL insulin transferrin selenium supplement (Sigma-Aldrich Co.), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Invitrogen). The HCE cells were seeded at a density of 1.5 × 105 cells/mL onto 6-well plates with 2 mL of medium per well overnight. Subsequently, all the culture medium was replaced with fresh basic medium containing fine PM (0.1 mg/mL and 0.2 mg/mL) and treated for 12 h and 24 h.

The effect of fine PM on tear secretion was investigated using the PRT-test. The PRT-test data in control mice were 4.60 ± 0.45 mm and 4.64 ± 0.34 mm in the 3rd and 6th month, respectively. After topical fine PM treatment for 3 and 6 months, PRT-test values significantly decreased compared to those of animals in the control group (p < 0.01, Fig. 2A). Furthermore, with increased fine PM concentration, PRT-test levels decreased in a dose-dependent manner. However, the PRT-test values in the 3rd and 6th month in mice that were treated with the same doses of fine PM did not show significant differences. TBUT was measured to evaluate the influence of PM on the stability of tear film. TBUT values in control mice were similar at the 3rd (4.59 ± 0.22 s) and the 6th month(4.49 ± 0.14 s) (p > 0.05, Fig. 2B). In mice treated with fine PM eye drops, the results showed that, at two checked time points, there were significant decreases in TBUT compared with the control group. The higher the fine PM dosage, the shorter the TBUT (p < 0.01). When comparing fine PM effects at the same concentration, the TBUT decreased more significantly at the 6th month than at the 3rd month.

2.7. Examination of cell apoptosis with TUNEL staining Apoptosis was assessed using a kit of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Roche, Kanton BaselStadt, Switzerland) according to the manufacturer's protocol. The airdried cell climbing slides of cultured cells were fixed with 4% paraformaldehyde solution for 30 min at room temperature. The slices were then rinsed with PBS and incubated in permeabilization solution (0.5% Trion X-100) for 20 min at room temperature, followed by rinsing with PBS and air-dried. Then, after pre-treatment of climbing slides of cultured cells or tissue slides, the slices were incubated with 50 μL terminal digoxigenin-labeled dUTP in a humidified chamber for 60 min at 37 °C and then restained with 100 μL DAPI for 5 min at room temperature. Finally, the slices were photographed using a fluorescence microscope (Zeiss, Oberkochen, Germany). Each experiment was repeated 3 times.

3.3. Morphological changes induced by fine PM The tissular alteration was examined by H&E staining. After application of fine PM at different concentrations on the ocular surface for 6 months, the corneal epithelial layer became thinner, and the number of epithelial cells significantly decreased when fine PM levels increased (Fig. 3A). The corneal stroma and endothelial cells exhibited no discernible change. There was no obvious inflammatory cell infiltration or corneal neovascularization. The conjunctival goblet cells were investigated by PAS staining (Fig. 3B and C). It can be seen that with the increase in fine PM concentration, the number of conjunctival goblet cells in mouse conjunctiva decreased significantly compared with that in the PBS control group (p < 0.01).

2.8. Reactive oxygen species analysis The reactive oxygen species (ROS) within cultured cells were measured using an ROS assay Kit (Abcam, Cambridge, MA, USA), by which a fluorescent dye, DCFDA, is deacetylated by intracellular esterases to dichlorodihydrofluorescein (DCFH) that can be oxidized ROS-dose-dependently to the highly fluorescent 2′, 7′-dichlorodifluorescein (DCF) [29]. Briefly, the cells were collected and washed with PBS solution and incubated with DCFDA (5 μM) at 37 °C for 10 min in the dark. Then the ROS levels were analyzed using a Flow Cytometer (BD Biosciences, San Jose, CA, USA). Ten thousand cells were analyzed for each sample. Experiments containing an normal cell group (NC), positive control group, and experimental groups were performed at the same time, both 12 h and 24 h after treatment. Three independent experiments were conducted.

3.4. Cytokine expression in corneal and conjunctival tissues After applying drops of fine PM suspension on the ocular surface of C57BL/6 mice for 6 months, 4 out of 10 cytokines/chemokines show significant changes. The levels of IL-18, IL-22, IL-23 and MCP-1 in the conjunctiva (p < 0.05 for IL-23 and MCP-1; p < 0.01 for IL-18 and IL22) and IL-22 in the cornea (p < 0.05) increased significantly in 5.0 mg/mL PM treated mice (Fig. 4). Meanwhile, IL-22 increased significantly in the conjunctiva that was exposed to 1.0 mg/mL PM (p < 0.01). Additionally, expression of these four cytokines tended to be more enhanced in the conjunctiva than in the cornea, though this difference did not reach significance.

2.9. Statistical analysis All data are expressed as the mean ± standard deviation. Statistical analysis was performed with SPSS software (version 18.0, SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used for comparisons between groups. A p-value less than 0.05 was considered statistically significant. 3. Results

3.5. Fine PM induced apoptosis in mouse conjunctiva and in cultured HCE cells

3.1. Ocular surface changes induced by fine PM Fine PM exposure results in cellular apoptosis as examined by TUNEL staining in ocular surface tissues of mouse and in cultured human cornea cells. Fig. 5A shows the staining of mouse conjunctival sections that were exposed to fine PM over 6 months. A number of TUNEL-positive cells were observed in fine PM-treated superficial conjunctival cells. Furthermore, with the higher fine PM concentration, the TUNEL staining of cells was dramatically increased. In addition, human cornea cells were exposed to 0.1 mg/mL and 0.2 mg/mL of fine PM over 12 h and 24 h. The cells displayed uneven cell sizes, a fuzzy cellular boundary, and sparse cell density, which was more apparent in cells treated with 0.2 mg/mL than 0.1 mg/mL of fine PM (Fig. 5C). TUNEL staining showed that few control cells stained positive at 12 or 24 h. In contrast, a much greater number of TUNELpositive cells were observed in fine PM-treated cells. Furthermore, with

Generally, there were no signs of infection of the ocular surface, conjunctival congestion or cornea neovascularization in any of the animals from each group. When the corneas were examined by sodium fluorescein staining under a slit-lamp biomicroscope in the 3rd and 6th month, the fluorescein staining that indicates damages on the ocular surfaces was gradually aggravated in the fine PM groups (Fig. 1). In the 0.5 mg/mL group, the fluorescein staining of the cornea appeared as scattered spots. These spots then became clusters at 1.0 mg/mL group. When the fine PM concentration increased to 5.0 mg/mL, diffuse fluorescein staining was seen on the entire cornea. In addition, with the same concentration of fine PM, ocular surface damage increased with the time extension. There was no fluorescein staining on the ocular surface of the control animals in the 3rd and 6th month. 3

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Fig. 1. Topical application of fine PM suspension effects on the cornea in C57BL/6 mice. Fine PM suspensions at 0 (NC), 0.5, 1.0 and 5.0 mg/mL were applied topically to mice over the course of 6 months. Images of mouse cornea stained with sodium fluorescein were taken at the 3rd and 6th months.

4. Discussion There are many studies that have demonstrated the adverse impact of PM on human health. PM plays a harmful role in cardiovascular diseases and increases chronic cardiopulmonary morbidity and mortality [3,30,31]. Several epidemiologic studies and controlled human exposure clinical studies have reported that exposure to PM is harmful to the ocular surface and causes various types of discomfort [7,9,14,32]. In the current study, we applied fine PM suspension to the eyes of C57BL/6 mice over 6 months to explore the long-term effects of fine PM on the ocular surface. We found that fine PM damages the mouse eye in a dose- and time-dependent manner. In mice, fine PM leads to shortening of the TBUT, reduces tear production, thins the corneal epithelial layer with a cellular defect, and decreases conjunctival goblet cells. Furthermore, using multiplex immunoassays, we found that after the local use of fine PM, there are increased contents of IL-18, IL-22, IL-23 and MCP-1 at different levels in mouse conjunctiva and cornea, with the most significant increases in 5.0 mg/mL PM treated conjunctiva. In addition, increasing ROS generation and cellular apoptosis were identified in HCE cultured in fine PM. To the best of our knowledge, this study is the first to directly demonstrate that topical application of fine PM over 6 months damages the ocular surface in vivo. The most common eye condition due to air pollution that has been described in previous studies is the dry eye syndrome. Our in vivo manifestations of fine PM effects are consistent with the clinical characteristic of dry eye [32,33], the diagnosis of which in clinical practice is widely based on the TBUT, the tear secretion test and goblet cell counting. In consistent with other studies that presented TBUT in normal mouse less than 10 s [34,35], which is the minimum human normal value, we found that TBUT of mice was 4.59 ± 0.22 s at 3rd and 4.49 ± 0.14 s at 6th month. The lower value of mice may be attributed to materials, animals, handling techniques and protocols. Nevertheless, we found that in PM-treated mice, the values of PRT and TBUT were significantly reduced. Cui and colleagues have also found thinning of the corneal epithelial layer of patients with dry eye [36]. Although there have been several epidemiological studies suggesting that PM damages the ocular surface, the evidence that fine PM causes dry eye are presently lacking. Recently, Cui and colleagues showed that PM2.5 delays wound healing in the murine cornea [19]. Li reported that a dry eye model can be induced by topical administration of PM10 for two weeks in BALB/c mice [20]. When compared with large PMs, small PMs are less likely to directly impact tear secretion and epithelial barrier function [1]. As we demonstrated in the present study, after 6 months application of fine PM, we only observed mild to moderate surface abnormalities in the epithelial layer and tear fluids. Microscopic examination did not reveal any obvious inflammation as evidenced by cell infiltration and neovascularization. There were no significant

Fig. 2. Effects of fine PM on tear secretion and tear break-up time (TBUT) in C57BL/6 mice. A: Phonel red thread test (PRT-test) for mice that treated with different concentrations of fine PM eye drops. With the increase of fine PM concentration, the PRT-test values decreased significantly. *: p < 0.01 (experimental groups VS. control group); B: Changes of TBUT in mice administered topically with fine PM. *: p < 0.01 (experimental groups VS. control group at the same assayed time); #: p < 0.01 (3rd month VS. 6th month at the same fine PM levels). Two-way repeated measures ANOVA was used to evaluate the changes.

the higher fine PM concentration and/or the longer exposure, the TUNEL staining of HCE cells was dramatically enhanced (Fig. 5B & D).

3.6. Fine PM increases ROS production in cultured HCE cells After being cultured in media contained fine PM, ROS levels in HCE cells were analyzed by flow cytometry (Fig. 6). After 12 h of exposure to fine PM, ROS with higher fluorescence intensity accounted for 16.5% of control cells, while it increased in the 0.1 mg/mL (21.5%) and 0.2 mg/ mL (35.5%) PM-treated cells (p < 0.01; vs. NC). At 24 h, high ROS cells in NC, 0.1 and 0.2 mg/mL PM groups accounted for 39.6%, 48.3% and 69.7%, respectively. A large amount of ROS was produced in PMtreated HCT cells compared with that in NC cells (p < 0.01, for 0.2 mg/mL vs. NC).

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Fig. 3. Morphological changes of mouse ocular surfaces after topical application of fine PM suspension over 6 months. A: H&E staining shows corneal alteration, with a thinning of the epithelial layer. All pictures are at the same magnification (400x). B: Changes of conjunctival PAS-stained goblet cells in three groups of mice treated with fine PM at different concentrations. C: Comparing the amount of conjunctival goblet cells in B. cells/high-power visual field. **: p < 0.01 (each experimental group vs. NC, n=4).

increases in cytokine expression in low-dose fine PM-treated animals. Similarly, the majority of patients with superficial injury to the corneal or conjunctival epithelium from dry eye have no evidence of stromal haze, unstable refractive error, or other changes [37]. Thus, the main finding of our in vivo study is that long-term exposure to fine PM results in dry eye changes on the ocular surfaces in mice. The occurrence of apoptosis and ROS generation in PM-associated studies has been widely reported in cell models, animals as well as human beings [20,32,38–40]. Apoptosis of corneal and conjunctival epithelia is an essential pathological feature of dry eye [41–43]. Furthermore, cellular ROS generation and apoptosis are associated with the pathogenesis of dry eye [42,44,45]. Our in vivo experiments demonstrated that fine PM results in the features of a dry eye in mouse model. We also found fine PM enhanced ROS synthesis and apoptosis in fine PM-treated HCE cells, which suggested that the cytotoxic effect of fine PM leads to the apoptosis by means of cellular oxidative stress [44,46]. However, the apoptosis of the conjunctival epithelia may also be a consequence of the pathology associated with dry eye rather than a

direct consequence of the PM, since apoptosis frequently occurrs in dry eye [42,43]. In addition, time- and dose-dependent changes were found in the in vivo and in vitro studies of fine PM exposure. These results are in agreement with those studies previously reported [21,40,47–49]. Gao and colleagues reported that PM2.5 leads to senescence and DNA damage in corneal cells and promotes ROS formation in cultured HCE cells [40]. Another study performed by Fu et al found that PM2.5 has a time- and dose-dependent effect on cytotoxicity in HCE cells [49]. Yoon found that PM2.5 induces nitric oxide production and interleukin 8 expression in cultured human corneal epithelial cells [21]. These results are similar to our in vitro findings that PM enhances ROS generation and apoptosis in HCE cells. These data further support results of our in vivo study In addition, changes in various cytokines have been reported in in vitro or in vivo studies and may be responsible for the mechanism underlying PM-induced damage. Prolonged exposure to PM2.5 results in elevated levels of IL-6 in the serum of patients with coronary artery disease [50]. Additionally, a high level of PM2.5 exposure is associated Fig. 4. Changes in the expression level of cytokines in corneal and conjunctival tissues. C57BL/6 mice were topically administered fine PM suspension at 0, 0.5, 1.0 and 5.0 mg/mL, separately, for 6 months. IL-18, IL-22, IL-23 and MCP-1 were determined by using the ProcartaPlex® Multiplex Immunoassay with Luminex technology. Data are presented as the mean ± standard deviation (n = 4). *: p < 0.05; **: p < 0.01. (experiment vs. NC, ANOVA).

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Fig. 5. Fine PM inducing apoptosis of mouse conjunctiva and human cornea cells. A: TUNEL staining shows that positive cells (red) were found in fine PM-treated superficial conjunctival cells, being more obvious in 5.0 mg/mL of fine PM (scale bar: 50 μm). B: TUNEL staining of cultured human cornea cells that were exposed to 0.1 and 0.2 mg/mL of fine PM for 12 and 24 h. TUNEL positive (red) and DAPI stained cells (green) are displayed. C: Images of cultured human cornea cells that were exposed to 0, 0.1 and 0.2 mg/mL of fine PM 24 h. D: Comparison of the relative intensity of apoptotic cells with red staining in B. * p < 0.01 (experimental groups VS. control group at the same assayed time, ANOVA). Samples without PM exposure were used as contorl (NC). The cellular experiments were repeated in triplicate. (Scale bar: 50 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

more significantly in conjunctival tissues and in high level of fine PMtreated mice. The change in the expression of inflammatory cytokines in the corneal and conjunctival tissues indicates that there may be a possible modulatory action of fine PM on the ocular surface immune

with decreases in IL-5 and IL-10 levels in human tear fluids [51]. In our current study, we detected the expression of cytokines in mice corneal and conjunctival tissues using multiplex immunoassay and found significant increases of IL-18, IL-22, IL-23 and MCP-1 in both corneal and

Fig. 6. Flow cytometry analysis of ROS production in HCE cells treated with fine PM. HCE cells were exposed to 0.1 and 0.2 mg/mL of fine PM for 12 and 24 h. Cells without PM exposure were used as contorl (NC). The ROS, represented by fluorescence intensity, within cells were determined by a flow cytometer. A is a set of recording data of one measurement. B: The results in A are reported as fluorescence arbitrary units based on 3 independent experiments (mean ± sd). *: p < 0.01 (experiment vs. NC, ANOVA). 6

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response. We find increases of cytokines/chemokines but without significant tissue/cells alteration under microscope, which may be due to the weak effects of long term PM exposure. However, it needs further experiments to clarify. We demonstrated a time- and dose-dependent fine PM effect in both our in vivo and in vitro studies. However, compared with the in vitro study in which ≤0.2 mg/mL fine PM was used, we applied up to 5 mg/ mL fine PM suspension 4 times a day for 3 or more months to produce an obvious ocular damage. We assumed that, even with 5 mg/mL applied 4 times a day, fine PM was less likely to remain in the eye and interact with the epithelia of cornea and conjunctiva because of eye blinking and tearing. In our pilot study, the application of a lower concentration (0.1 and 0.2 mg/mL) of fine PM suspension to the eyes for one month did not cause any significant changes in vivo. Thus, in our in vivo study, we used an increased dose of fine PM and extended the duration of the time of applying drops to the eyes. In the 3rd month, we recorded the changes in the TBUT and the PRT-test. However, in future studies, time dependent changes in ocular surface upon exposure to fine PM should be monitored to determine short-term and long-term effects of exposure of various PM. It has been suggested that individual PM concentrations are less important than its components in determining its damaging effects [21,25]. Furthermore, research on the toxic effects of PM frequently disregards the differences in particle composition among different sources of the fine particulate matters employed [24]. In the present study, we used the fine PM powder, a standard reference material (SRM 2786) containing organic/inorganic constituents, and particle-size characteristic of atmospheric particulate material and similar matrices, which is somewhat different from those fine particulate matters used in most previous studies [21,24]. It would be more convincing if we use eye drops of natural PM, or even treat animal eyes under natural exposure. In addition, WHO established the annual exposure standard at 10 μg/m3 for PM2.5 and 50 μg/m3 for PM10 [2], which is difficult to be compared with the PM levels in eye drops we used for half a year in mice. Thus, we should consider different PM exposure when comparing and explaining the results from different studies. In conclusion, fine PM is cytotoxic to both ocular surface and corneal epithelial cells. After topical treatment with fine PM suspension, corneal epithelial cells and conjunctival goblet cells in C57BL/6 mice were greatly decreased, and tear film stability was significantly reduced. The long-term effects of fine PM on the mouse ocular surface are similar to the changes observed in dry eye.

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