Abstract Dry eye (DE) is a multifactorial ocular surface disease whose incidence continues to rise. Various environmental stresses such as low air humidity and pollution are known to be involved in epithelial alterations inducing ocular discomfort. However, no experimental study assessing the combined effects of dry air and polluted atmospheres has been conducted so far. Formaldehyde (FA) is a ubiquitous pollutant present in the living spaces where humans spend most of their time. Using an in vitro DE model, we evaluated the cytotoxic and inflammatory responses of a conjunctival cell line exposed at the air-liquid interface (ALI) conditions to various controlled atmospheres combining low humidity (LH), airflow (AF), and formaldehyde gas (FG). Conjunctiva-derived cells grown onto transwell inserts were directly exposed to LH conditions without AF, with AF or with FG flow at 100 or 1200 µg/m3 for 15–30 min. Cell viability assays revealed an increase in cell death after a 15-min exposure to FG at 100 or 1200 µg/m3, whatever the recovery period. After a 1-h recovery period, an increase in IL-6 and CXCL8/IL-8 gene expression was observed with the 15-min exposure at 100 µg/m3 FG and with 30 min of exposure at 1200 µg/m3 FG. After 24 h of recovery, we also noted increased secretion of the proinflammatory cytokine MIF with 100 µg/m3 FG exposure and CXCL8/IL-8 at 1200 µg/m3, for both exposure periods. Together, these findings suggest that the exposure to FG at environmental levels aggravates cell death and inflammation observed in dry air conditions. This in vitro model of DE seems to be a relevant tool to study and explain the inflammatory responses observed in dry eye patients when exposed to combined environmental disturbances such as LH, airflow, and the presence of airborne pollutants. ocular toxicity, exposure/environmental, volatile organic compounds, cytokines, inflammation, cell culture Today’s populations, mainly in developed countries, spend most of their time in enclosed environments (home, office, school, transport, etc.), leading to exposure to invisible and omnipresent airborne pollution. Indoor air pollution is a major public health concern even more disturbing than outdoor air pollution. Among the most widespread air pollutants, volatile organic compounds (VOCs) are emitted by some furniture, wood products, glues, varnishes, paints, etc., and are responsible for adverse effects on short- and long-term health. Formaldehyde (FA) is considered as the major ubiquitous pollutant present in the living environment. Indeed, because of its physicochemical properties, FA has many industrial applications, and is used, eg, as a biocide, preservative and fixative. It is also naturally emitted during any combustion phenomenon (fires, cigarette smoke) and by human activities (cooking, wood stoves). FA levels found in indoor air range in some homes from 10.7 to 47.7 μg/m3 in bedrooms, from 9.65 to 37.2 μg/m3 in living rooms and from 5.86 to 40.4 μg/m3 in workplaces (Rovira et al., 2016). Comparatively, FA levels found in outdoor air are lower (0.96–3.37 μg/m3). Thus, the levels of FA in the indoor air are on average close to 50 μg/m3 or less in Europe and North America except for new housing or buildings with extensive wooden surfaces, where the level may exceed 100 μg/m3 (Wolkoff and Nielsen, 2010). Known for its irritant effects, FA is an important environmental toxic substance, inducing both genotoxic and clastogenic effects, DNA adducts, leading to nasopharyngeal cancer and leukemia (Nielsen et al., 2013). The World Health Organization (WHO) recommends a guide value of 100 μg/m3 for a 30-min period of exposure, a level respecting indoor air quality (Nielsen et al., 2017) and preventing irritation and sensitization in the general population. The ocular surface is a functional unit composed of a set of structures, which protect the eye against environmental stress. Allergy and dry eye disease (DED) are 2 major examples of ocular surface pathologies, affecting 10%–20% and 20%–35% of the general population, respectively (International Dry Eye WorkShop, DEWS, 2007). According to an updated definition adopted by the recently published Dry Eye WorkShop II (DEWS II), “Dry eye is a multifactorial disease of the ocular surface characterized by a loss of homeostasis of the tear film, and accompanied by ocular symptoms, in which tear film instability and hyperosmolarity, ocular surface inflammation and damage, and neurosensory abnormalities play etiological roles.” (Craig et al., 2017). The impact on the ocular surface of the atmospheric environment in which an individual lives and biological effects of pollutants still need to be determined. Very few studies have assessed the impact of FA on the ocular surface. Two in vivo studies explored the effect of FA in liquid form directly applied to the cornea in rabbits (Lai et al., 2013; Maurer et al., 2001), but concentrations were too high to mimic usual FA exposures (125 000 μg/m3 and 37%, respectively). Furthermore, although tear film covers the ocular epithelium, the exposure model used in this study is far from the real conditions of eye exposure to a gaseous pollutant like FA. Salem et al. (2016) evaluated the consequences on the ocular surface of rats exposed for 2 weeks, 2 h/day, 5 days a week, to gaseous FA released from a piece of cotton soaked with 10% FA and placed 15 cm away from the rats. This in vivo model revealed corneal injury with marked necrosis of epithelial cells. However, these in vivo studies raise the question about the respect of the “Three Rs” (3Rs) guiding principles for more ethical use of animals in testing. Therefore, any alternative method that could replace animal experimentation has to be encouraged and explored. That is what Lai et al. (2013) did in testing the in vitro exposure of corneal epithelial cells to FA in liquid form, showing increased cell death. But once again, the exposure conditions used by these authors did not reflect the real condition of exposure to gaseous pollutants such as FA. The main in vitro studies assessing the impact of gaseous FA on the modulation of biological activity after exposure were carried out on nasal epithelial cells (Bardet et al., 2014; Wang et al., 2014b), tracheal/bronchial epithelial cells (Sexton et al., 2008), alveolar cells (Persoz et al., 2011, 2012), or lung epithelial cells (Persoz et al., 2010; Rager et al., 2010) and use an air-liquid interface (ALI) exposure system. However, despite significant anatomical and histological similarities existing between the ocular surface and airway epithelial tissues, no study has been conducted to date in an in vitro gaseous exposure model concerning ocular epithelial tissues for either pollutants in general or FA in particular. Moreover, to our knowledge, no experimental study assessing the combined effects of dry and polluted atmospheres has ever been undertaken. In this study, we proposed to use an in vitro ALI exposure system to study the combined effects of environmental stresses, namely low humidity, airflow, and the presence of gaseous FA, on the inflammatory responses and cellular death of a conjunctiva-derived cell line. This experimental model could help better understand the impact of pollution on the inflammatory responses observed in the conjunctiva of patients suffering from DED. MATERIALS AND METHODS Cell line and culture conditions The human conjunctiva-derived epithelial cell line WKD (Wong-Kilbourne derivative cells) was obtained from ATCC (The Wong-Kilbourne derivative of Chang conjunctival epithelial cell line, WKD; clone 1–5c-4, American Type Culture Collection [ATCC, Manassas, Virginia] certified cell line [CCL], 20.2) and grown in culture flasks using DMEM (Dulbecco’s Modified Eagle Medium) + Glutamax culture medium (Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% FBS (fetal bovine serum) (Sigma-Aldrich, Darmstadt, Germany), and 1% penicillin/streptomycin, hereafter referred to as DMEM complete medium. Culture was carried out in an incubator (5% CO2, 37°C and maximal relative humidity [RH]). At confluence every 2 days, cells were released with trypsin (0.25% in PBS [phosphate-buffered saline]), counted and transferred to inserts (ThinCert PET cell culture inserts for 12-well plates with transparent membrane, 0.4-µm pores and 2 × 106 pores/cm2, Greiner Bio-One, Frickenhausen, Germany) for experiments. The cells used in this study were from passages 15–25. Seeding was performed with 0.5 ml of a cell suspension per insert, at a density of 1.5 × 105 cells/ml. DMEM complete medium was put into the wells under the inserts. After 24 h in an incubator, the inserts underwent experiments. For each experimental condition, the exposures were conducted between 5 and 10 times (n = 5–10) with cells of different passages each time. This cell line had been used previously for toxicological in vitro studies despite the presence of a small amount of HeLa cells and was shown to respond similarly to the IOBA-NHC conjunctival cell line (Brasnu et al., 2008; Clouzeau et al., 2012; Warcoin et al., 2016, 2017). Design of the exposure system The generation of FA gas atmosphere was performed according to a technique adapted from (Saltzman, 2003) and commonly used in the Public Health and Environment Laboratory of the Paris School of Pharmacy (Bardet et al., 2014; Persoz et al., 2010, 2011, 2012). According to the desired final gaseous concentration, 5 μl of a defined FA solution (FA solution F-1635, Sigma-Aldrich, Saint-Quentin-Fallavier, France) was sprayed into a 4.5 L glass chamber previously put under vacuum, and then completed with the air from a controlled air creation device made up of a compressor coupled to a desiccator, manometers and a hygrometry supply system using a water bath. At the end of the device, a flow meter displayed about 4 L/min. Gaseous FA at the desired concentration could thus be generated with a controlled temperature (T) and relative humidity (RH), ie, T: 23°C and RH: 20%. The atmosphere was stabilized for about 15 min before the first exposure. It was then connected to the high part of the Vitrocell Systems dynamic ALI exposure module. The atmospheres generated at concentrations of 100 and 1200 μg/m3 were prepared according to the description already published by Bardet et al. (2014) (Appendix). The concentration of 100 μg/m3 is considered as the threshold of odor detection (Golden, 2011; Nielsen et al., 2013) and 1200 μg/m3 corresponds to 1 ppm. The Vitrocell exposure module contains 3 chambers, each one with 1 insert and 1 trumpet. A pump downstream of the device allowed an airflow rate at 5 ml/min (controlled using a flow meter), creating an airflow sent (air interface) through the trumpet on the apical side of the cell layer. The trumpets were previously adjusted 3 mm above the bottom of the insert on the apical side of the cell layer. A water bath was connected to the exposure module to maintain the chamber containing the medium (DMEM + Glutamax without FBS) at 37°C (liquid interface). For this study, we had 2 devices with 3 trumpets connected together, so that we could combine the following different exposure conditions, the 2 exposure periods, the 2 recovery periods, and the different assays evaluated (cell viability, cytotoxicity, reverse transcription-polymerase chain reaction [RT-qPCR], enzyme-linked immunosorbent assay [ELISA]). Exposure conditions Five exposure conditions were applied: One/Cell culture control in “submerged” classical conditions at the liquid-liquid interface (LLI) in the incubator; 2/the cell layer at the ALI without airflow (AIR_F−); 3/the cell layer at the ALI with airflow (AIR_F+); 4/the cell layer at the ALI with gaseous FA exposure at 100 μg/m3 (HCHO100_F+); 5/the cell layer at the ALI with gaseous FA at 1200 μg/m3 (HCHO1200_F+). To test the exposure of the cells in the ALI condition, the cells were seeded onto insert membranes and allowed to grow in LLI conditions. At cell confluence, and after withdrawal of the medium from the apical side of the cell culture, the inserts were placed in the Vitrocell chambers. Then the ALI exposures to normal conditions (temperature: 22°C–24°C and dry conditions: RH: 15%–25%) were tested. Cells were exposed for 15 or 30 min and then put back into the incubator in the classical culture (LLI) with fresh DMEM complete medium with 2.5% FBS (to limit cellular proliferation) for 1 or 24 h. Just before exposure, the supernatant of each culture (on the basal side) was collected, centrifuged at 150 g for 5 min and aliquoted in 2 samples. These “pre-exposure” supernatants were considered as basal conditions for the cytotoxicity assay and the cytokine immunoassays. Metabolism and cell viability Cell viability was assessed using the CellTiter-Glo Luminescent Cell Viability Assay Kit, according to the manufacturer’s protocol (Promega, Madison, Wisconsin). This test quantifies the intracellular adenosine triphosphate (ATP), and therefore the presence of metabolically active cells. After the recovery times (1 or 24 h), on exposed cells in the inserts, the cell culture medium was replaced with a solution composed of 100 µl of CellTiter-Glo Reagent and 100 µl of cell culture medium containing 2.5% FBS. After 2 min on an orbital shaker to promote cell lysis, cells were incubated at room temperature (RT) for 20 min to stabilize the luminescent signal generated by the luciferase-catalyzed reaction of luciferin and ATP. Cell lysates were transferred into black 96-well plates with clear bottoms to measure the signal using a VICTOR X Light Luminescence Plate Reader (PerkinElmer, Waltham, Massachusetts). The cell viability was expressed as a ratio versus the LLI baseline group corresponding to the 100% cell viability control that was carried out at each exposure time (15 and 30 min) and each recovery time (1 and 24 h). Cell membrane integrity and cell viability After recovery (1 or 24 h), all supernatants were collected, centrifuged for 5 min at 150 g and stored at RT. Cell death was assessed by measuring the release of cytoplasmic lactate dehydrogenase (LDH) isoenzymes from damaged cells using the Siemens Dimension LDI Flex Reagent Cartridge, according to the manufacturer’s instructions (Siemens, Erlangen, Germany). This test was performed with the Dimension Xpand Plus integrated chemistry system (Siemens). LDH oxidizes the L-lactate substrate buffered at pH 9.4 in presence of NAD+ (nicotinamide adenine dinucleotide) to yield pyruvate and NADH that absorbs light at 340 nm. LDH activity is measured as a rate reaction at 340/700 nm, proportional to the amount of LDH in the sample. The results were expressed as a ratio versus the maximum LDH activity obtained for a total cell lysis induced by 1% Triton X100 for 2 h and then converted into relative cell viability. Negative and positive controls were carried out for each exposure time (15 and 30 min) and each recovery time (1 and 24 h). RNA isolation and (RT-PCR) After exposure and at the end of the 1 h recovery time (1hR), one-half of the cell cultures were taken and the monolayers were rinsed twice with 600 μl of PBS. Then the insert membrane containing the cell monolayers were cut and placed in tubes containing 100 μL of lysis buffer RA1 (Macherey Nagel, Düren, Germany), vortexed and stored at −80°C. Total RNA was extracted from the cells using NucleoSpin RNA kits (Macherey Nagel). Reverse transcription was carried out with 400 ng RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, Massachusetts); 5 μl of the RT products was amplified with a TaqMan Universal PCR Master Mix, no AmpErase UNG (Life Technologies, Carlsbad, California) with primers and probes from TaqMan Gene Expression Assay (Life Technologies) for Interleukin (IL)-6, Macrophage migration inhibitory factor (MIF) and chemokine (C-X-C motif) ligand 8 (CXCL8)/IL-8. These cytokines and chemokines are commonly involved in the inflammatory processes of immune cell recruitment and activation. Relative expression levels were determined using the ddCt method and normalized to endogenous control (GAPDH, glyceraldehyde 3-phosphate dehydrogenase). Relative expressions of IL-6, MIF, and CXCL8/IL-8 mRNAs were related to the respective negative controls (LLI baseline group). Negative controls were carried out for each exposure time (15 and 30 min). Proinflammatory mediator quantification After exposure and at the end of the 24-h recovery time (24hR), the supernatant of each cell culture was collected, centrifuged for 5 min at 150 × g and stored at −80°C. The cytokines IL-6, MIF, and CXCL8/IL-8 secreted by epithelial cells were quantified in the basal side of the cell culture (supernatants) using ELISA kits (DuoSet ELISA Development Systems, R&D Systems, Minneapolis, Minnesota), according to the manufacturer’s instructions. Absorbance was measured at 450 nm with a correction at 570 nm using a microplate reader (Infinite M1000 PRO, Tecan, Männedorf, Switzerland). The Four Parameter Logistic calibration curves were used to calculate the cytokine concentrations (in pg/mL). The results are expressed as ratios of cell viability obtained by ATP measurement. Negative controls were carried out for each exposure time (15 min and 30 min). Statistical analyses The results are expressed as arithmetic means ± SEM of 5 to ten (n = 5–10) independent experiments performed in monoplicates. Outliers were excluded by the Robust regression and Outlier removal (ROUT) method using the GraphPad Prism 7 software. Stress condition groups were analyzed by ordinary 1-way analysis of variance (ANOVA) followed by the Tukey’s or Holm-Sidak’s multiple comparisons posttest. Exposure period or recovery period groups were analyzed using an unpaired t-test. If some results did not respond to normality or homogeneity of variances, we used nonparametric tests (Kruskal-Wallis test and Mann-Whitney test, respectively). Data were considered as statistically different when p < .05. RESULTS Cell Viability Assessed With CellTiter-Glo Assay A short stress of 15 min of exposure did not alter cell metabolism (ATP) in conditions of dry air + airflow, compared with dry air alone, irrespective of the recovery time. After a longer exposure (30 min), adding the airflow to the classical ALI culture after 30 min tended to aggravate the cellular metabolism alteration, although this reduction remained nonsignificant. Concerning FA exposure, a trend of cell viability reduction in the 15-min condition was seen regardless of the recovery time, particularly at a high concentration (1200 μg/m3) after 1 h of recovery and at a low concentration (100 μg/m3) after 24 h of recovery. For the 30-min exposure, the loss of viability reached a plateau after adding the airflow, independently of the recovery time. Thus, FA at low or high concentrations, for the 30-min exposure, did not seem to worsen the cellular suffering caused by the presence of airflow. A significant exposure time-dependent cell viability decrease was observed after 1hR, for AIR_F+ (p < .001), HCHO100_F+ (p < .05), and HCHO1200_F+ (p < .05) and after 24hR for AIR_F− (p < .01), AIR_F+ (p < .001) and HCHO1200_F+ (p < .05) (Figure 1A). Figure 1. View largeDownload slide Cell viability assays; (A) CellTiter-Glo assay; (B) LDH cytotoxicity assay. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). Relative ATP measurement in cell lysates and relative LDH enzyme activity in supernatants collected after 1 or 24 h of recovery. Outliers were detected by the ROUT method. Mean ± SEM. Ordinary 1-way ANOVA with Holm-Sidak's multiple comparisons test (between stress conditions); unpaired t test (between exposure periods or recovery periods) (n = 6–10). *p < .05; **p < .01; ***p < .001. Figure 1. View largeDownload slide Cell viability assays; (A) CellTiter-Glo assay; (B) LDH cytotoxicity assay. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). Relative ATP measurement in cell lysates and relative LDH enzyme activity in supernatants collected after 1 or 24 h of recovery. Outliers were detected by the ROUT method. Mean ± SEM. Ordinary 1-way ANOVA with Holm-Sidak's multiple comparisons test (between stress conditions); unpaired t test (between exposure periods or recovery periods) (n = 6–10). *p < .05; **p < .01; ***p < .001. Cell Viability Assessed With LDH Cytotoxicity Assay The cytotoxicity assessed by the release of the LDH cytoplasmic enzyme showed consistent results with those obtained for cell viability by measuring ATP. However, the global trend of an impact of FA after the 15-min exposure and an impact of airflow after the 30-min exposure was less pronounced. Alteration of cell metabolism (ATP) indeed precedes alteration of the cell membrane. A significant exposure time-dependent cell death increase was observed after 1hR for AIR_F+ (p < .001) and HCHO100_F+ (p < .01) and after 24hR for AIR_F− (p < 0.05), AIR_F+ (p < .001), HCHO100_F+ (p < 0.05) and HCHO1200_F+ (p < .01) (Figure 1B). RNA Levels Assessed With qRT-PCR Assay The IL6 gene was stimulated in a stress-dependent manner in the 15-min condition (except at the highest dose of FA, where we observed a decrease most likely due to major cytotoxic conditions). In addition, the gradual increase of IL6 expression was less significant for 30 min (Figure 2A). Figure 2. View largeDownload slide mRNA relative expression assessed by RT-qPCR test; (A–C): relative expressions of IL6, MIF, and CXCL8 mRNAs, respectively. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). mRNA relative expression of IL6, MIF and CXCL8 genes in cell lysates collected after 1 h of recovery. Outliers were detected by the ROUT method. Mean +/− SEM. Ordinary 1-way ANOVA with Tukey's multiple comparisons test (between stress conditions); unpaired t test (between exposure periods) (n = 6–9). *p < .05; **p < .01; ***p < .001. Figure 2. View largeDownload slide mRNA relative expression assessed by RT-qPCR test; (A–C): relative expressions of IL6, MIF, and CXCL8 mRNAs, respectively. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). mRNA relative expression of IL6, MIF and CXCL8 genes in cell lysates collected after 1 h of recovery. Outliers were detected by the ROUT method. Mean +/− SEM. Ordinary 1-way ANOVA with Tukey's multiple comparisons test (between stress conditions); unpaired t test (between exposure periods) (n = 6–9). *p < .05; **p < .01; ***p < .001. The CXCL8 gene was stimulated at 15 min and this stimulation increased gradually from pure dry air to a flow of FA (except at the highest dose of FA, where we observed a decrease). The same behavior was observed after 30 min in a more pronounced way, without the decrease for the high dose of FA. A significant increase of gene expression was observed after 15 min between HCHO100_F+ and AIR_F− (p < .05) and after 30 min between HCHO1200_F+ and HCHO100_F+ (p < .05), AIR_F+ (p < .05) and AIR_F- (p < .001) (Figure 2C). It was noteworthy that neither physical nor chemical factors induced gene expression of MIF, irrespective of the exposure time (Figure 2B). Protein Levels Assessed With ELISA Assay Regarding the release of cytokines in the cell supernatant collected after the 24-h recovery, a slight increase of IL-6 was observed for the 15-min exposure with airflow but no additional effect for FA conditions. IL-6 secretion was kept at the same level for the 30-min exposure, regardless of the stimulus (Figure 3A). Figure 3. View largeDownload slide Cytokine releases assessed by ELISA test; (A–C): secretions of IL-6, MIF, and CXCL8/IL-8 proteins, respectively. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). Secretion levels (in pg/ml) of IL-6, MIF and CXCL8/IL-8 proteins in supernatants collected after 24 h of recovery. outliers were detected by the ROUT method. Mean ± SEM. Ordinary 1-way ANOVA with Holm-Sidak’s multiple comparisons test (between stress conditions); unpaired t-test (between exposure periods) (n = 5–9). *p < .05; **p < .01; ***p <.001. Figure 3. View largeDownload slide Cytokine releases assessed by ELISA test; (A–C): secretions of IL-6, MIF, and CXCL8/IL-8 proteins, respectively. WKD cells exposed for 15–30 min in LLI at 37°C, 5% CO2 (dotted baseline), in ALI at normal temperature (22°C–24°C) to low-humidity air (15%–25%) without air flow (AIR_F−), with 5 ml/min air flow (AIR_F+) or with 5 ml/min FA gas flow at 100 or 1200 µg/m3 (HCHO100_F+, HCHO1200_F+). Secretion levels (in pg/ml) of IL-6, MIF and CXCL8/IL-8 proteins in supernatants collected after 24 h of recovery. outliers were detected by the ROUT method. Mean ± SEM. Ordinary 1-way ANOVA with Holm-Sidak’s multiple comparisons test (between stress conditions); unpaired t-test (between exposure periods) (n = 5–9). *p < .05; **p < .01; ***p <.001. The chemokine CXCL8/IL-8- was released quite prematurely since there was a gradual secretion increase from pure dry air for 15 min, with the higher value obtained for the highest dose of FA. For the 30-min exposure the results showed a more or less precise plateau with increasing stresses, and a peak with HCHO1200_F+ (Figure 3C). A significant increase of MIF release was observed after 15 min between HCHO100_F+ and AIR_F− (p < .01) and after 30 min between AIR_F− and AIR_F+ (p < .05), HCHO100_F+ (p < .01) and HCHO1200_F+ (p < .05). A significant exposure time-dependent MIF release increase was observed for AIR_F−, AIR_F+, HCHO100_F+ (p < .01 each) and HCHO1200_F+ (p < .001), probably corresponding to a MIF preformed stock (Figure 3B). DISCUSSION FA was incriminated as the most widely distributed pollutant in indoor environments where we spend the majority of our lives, as a result of many emission sources (furniture, paints, human activities, etc.). However, to our knowledge, no experimental study assessing the combined effects of dry and polluted atmospheres has been assessed to date. Using a sophisticated in vitro ALI cell exposure system, we attempted to investigate the biological responses of conjunctiva-derived cells exposed to different atmospheric conditions. We used conjunctival epithelial cells because conjunctiva is a mucosal tissue, rich in immune cells and vessels; it covers most of the ocular surface and acts as a defense tissue to protect the cornea that must remain totally clear, exempt of any inflammation. Moreover, conjunctival epithelial cells were already reported to be able to secrete inflammatory chemokines and cytokines in response to toxic challenge or in DED (Warcoin et al., 2017). Therefore, we evaluated the impact of growing stress conditions with various physical and chemical factors (air without airflow, air with airflow with or without gaseous FA at 2 concentrations). Two exposure durations (15 min and 30 min), followed by 2 periods of cell recovery, 1 h for gene expression analysis and 24 h for protein secretion, were applied. We tested 2 sufficiently different gaseous FA concentrations to evaluate a possible concentration-dependent biological effect. The 2 concentrations of gaseous FA were reported to induce irritation symptoms in different exposure situations: the concentration of 100 μg/m3 is the average concentration found in some modern domestic environments (Liu et al., 2005), while the concentration of 1200 μg/m3 corresponds to an exposure close to that encountered in professional environments (medical students, foundry workers, health workers, scientific researchers, etc.) (Lakchayapakorn and Watchalayarn, 2010; Löfstedt et al., 2011; Mori et al., 2013, 2016; Raja and Sultana, 2012). The 2 exposure time points were used to evaluate a possible time-dependent biological effect. Fifteen and 30 min of exposure were decided in accordance with previous studies in our team on xenobiotics contained in eyedrops, most particularly preservatives that are known to remain 15–30 min on the ocular surface (Debbasch et al., 2001). We also experimented 24-h exposure in classical culture conditions, but this duration is not feasible in this ALI in vitro system, because it is overly toxic and difficult to maintain the gaseous Fa concentration. Finally, we wished to investigate a combination of parameters (exposure time and airflow) that moderately impact cell viability in order to allow the cells to express their biological responses, after 1 h of recovery for gene expression and 24 h of recovery for protein secretions. Very little information is available on the influence of environmental factors on the eye, including the impact of air pollutants on dry eye syndrome (Lois et al., 2008; Rozanova et al., 2009). However, many epidemiological studies showed a positive association between air pollution, whether outdoor or indoor air, and the prevalence of certain respiratory diseases (asthma, rhinitis, bronchitis). Much less attention has been focused on the adverse effects of airborne pollutants on the ocular surface. Like the respiratory tract, the eyes are chronically exposed to air pollution and pollutants enter directly in contact with the thin tear film separating both corneal and conjunctival epithelia (Torricelli et al., 2011). Some studies underlined that air pollutants or adverse indoor and/or outdoor environmental conditions could reduce tear film stability, influence tear film osmolarity, and affect ocular surface components (Berra et al., 2015; Torricelli et al., 2013). These effects could be mediated by selective binding of the environmental agents to ocular surface membrane receptors, leading to activation of proinflammatory signaling pathways and modulation of immune response (Alves et al., 2014; Matsuda et al., 2015). Moreover, growing evidence shows hyperosmolar stress to be a potent inflammatory stimulus by triggering the release of proinflammatory cytokines such as IL-6 or CXCL8/IL-8 (Clouzeau et al., 2012). Hyperosmolarity causes morphological changes such as apoptosis of conjunctival and corneal cells, and triggers inflammatory cascades that promote cell death, including the loss of mucin-producing goblet cells (Baudouin et al., 2013). This exacerbates tear film instability and leads to a vicious circle of events that perpetuate the pathological condition (Baudouin et al., 2013, 2016). The critical effects of gaseous FA in humans are irritation of the eyes and the respiratory tract, observed for both acute and chronic exposure. Liu et al. (2005) discovered that FA (170 ± 75 µg/m3) present in some decorated houses caused eye symptoms in residents. Furthermore, the exposure of medical students, workers or researchers to low levels of FA in anatomy laboratories has been well documented. Lakchayapakorn and Watchalayarn determined that FA concentrations in the indoor air and in the breathing zone of individuals during cadaver dissection ranged from 501 to 726 µg/m3 and from 590 to 1059 µg/m3, respectively, and caused burning eye sensations (Lakchayapakorn and Watchalayarn, 2010). In the same environment, other studies highlighted ocular irritation and tear overflow (Raja and Sultana, 2012), eye fatigue, and dry eyes (Mori et al., 2013, 2016). Löfstedt et al. (2011) demonstrated that FA exposure in foundry workers has a prolonged influence on the high prevalence of ocular symptoms despite reduced exposure levels at follow-up, 4 years after an initial assessment. Dry eye is a multifactorial ocular surface disease whose incidence continues to rise. Its prevalence around the world varies from 5% to 34% (Messmer, 2015). This pathology has already been related to air pollution (Hwang et al., 2016). Moreover, the combination of pollutants with physical factors, such as air velocity and high temperature, have been reported to be able to increase eye discomfort symptoms (Tesón et al., 2013; Wolkoff, 2017) and chronic environmental stress conditions are known to be involved in epithelial alterations related to ocular discomfort (De Kluizenaar et al., 2016). Many studies were conducted to understand the pathophysiological mechanisms involved in DED, but very few have evaluated the relationships between dry eye and pollutants (Galor et al., 2014; Hwang et al., 2016; Li et al., 2017; Wolkoff and Nielsen, 2010). In vitro models are recognized tools allowing deep mechanistic molecular studies, avoiding the use of animal experimental models according to the 3Rs ethical animal experimentation guidelines: Replacing, Reducing, and Refining animal experimentation. But very few in vitro models have been adapted to evaluate the impact of dry air and gaseous pollutants. To this aim, 2 major in vitro exposure systems exist on the market, namely Cultex™ and Vitrocell. These tools allow one to apply controlled environmental conditions such as temperature, RH, and air flow to cell cultures. These systems are adapted to the evaluation of many airborne pollutants found in indoor or outdoor atmospheres. This approach is innovative and useful to assess the impact of chemicals, tested alone or in combination with other environmental factors, in the outcome of DED as a primary stress or as supplementary/additive stress conditions. Since the eye is the most sensitive organ in the body, eye irritation is the most sensitive effect reported in human volunteers exposed to FA, but it has been mentioned only occasionally in the studies used by the European Commission (Arts et al., 2008). According to Wolkoff and Nielsen, subjective eye irritation is reported at levels close to 300–500 µg/m3, higher than reported odor thresholds, while the objective ocular effects occur around 600–1000 µg/m3. Lang et al. (2008) demonstrated that the no observed adverse-effect level for both subjective and objective eye irritation (blinking frequency and conjunctival redness) due to FA exposure in human volunteers was 615 µg/m3 at a constant exposure level, and 369 µg/m3 with peaks at 738 µg/m3 in case of short-term exposure. An indoor air quality guideline of 100 µg/m3 is recommended for all individuals (hypo- and hypersensitive) for odor detection, and protection from both acute and chronic sensory irritation (Golden, 2011; Nielsen et al., 2013). In this study, dryness induced by ALI culture alone was a condition that greatly reduced cell viability and increased the inflammatory responses of conjunctival cells. This condition of ALI culture made it possible to mimic the pathophysiological conditions of desiccative stress as observed in DED (Coursey et al., 2014). The addition of airflow confirms these results in most of the tests. Therefore, this model at the ALI seems to be of great interest for evaluating the impact of gaseous environmental pollutants in patients who have already installed DED. These results are corroborated by published data, showing that prevalence of DED is correlated with environmental factors such as lower humidity levels in outdoor air (Hwang et al., 2016; Tesón et al., 2013; Um et al., 2014). Xiao et al. (2016) demonstrated that inflammation occurred by culturing human conjunctival tissues in ALI conditions (Xiao et al., 2016). In the same way, air exposure to conjunctival tissues, in an airlifted situation, induced symptoms of dry eye with apoptosis and upregulation of proinflammatory cytokine, IL-1beta, TNF, and metalloproteinase MMP9 expression (Lin et al., 2014). In a dry eye in vitro model, CCL2 was found to be stimulated by hyperosmolar conditions (Warcoin et al., 2016, 2017). In a dry eye in vivo model, MIF was found increased (Park et al., 2007) and was recognized as playing a role in the pathophysiology of the disease (Willeke et al., 2007). In DED patients, inflammatory mediators were reported in both tears and conjunctival tissues (Acera et al., 2008; Pisella et al., 2000; Pflugfelder and de Paiva, 2017). Several inflammatory cytokines/chemokines (such as IL-6, IL-8, and CCL2) have been found to be significantly increased in tears from DED patients (Enríquez-de-Salamanca et al., 2010; Na et al., 2012; Tishler et al., 1998). Our study suggests that FA, at 100 or 1200 µg/m3, aggravates cell death and inflammation in dry conditions, but these results need to be further explored to confirm the additive effects of dry and polluted conditions. In particular, after a 15-min exposure period, IL6 and IL8 gene expressions were higher for 100 μg/m3 than for 1200 μg/m3 of FA. In fact, these results and the high variability observed for the other gene expressions, depending on the cytotoxic effect, appeared quite difficult to interpret at a single recovery time point. This could have benefited from a gene expression time course with different recovery periods, so that the biological impacts stemming from hormesis or transcription inhibition could be distinguished. Persoz et al. (2012) studied the exposure of alveolar and bronchial cell lines to FA at the environmental level (50 μg/m3) for 30 min in ALI conditions. These authors showed that by interacting with the respiratory epithelium, FA seems to exacerbate airway inflammation, a phenomenon occurring in severe asthma. Similarly, they showed a significant increase in CXCL8/IL-8 after TNFα sensitization, without decreasing cellular viability 24 h after exposure (Persoz et al., 2010). Rager et al. (2010) studied human lung epithelial cells grown in ALI conditions and exposed to gaseous FA at 1249 μg/m3 for 4 h. These authors revealed the modulation of inflammatory gene expression and the increased secretion of the chemokine CXCL8/IL-8 after FA exposure of the cells. To mimic the realistic conditions of exposure, it would be interesting to study the effect of environmental pollutant mixtures. Wang et al. (2012, 2014a) studied the subchronic exposure of mice to a low-dose VOC mixture (FA, benzene, toluene, and xylene) in a whole-body exposure chamber. Kastner et al. (2013) attested that repeated exposure of a human bronchial epithelial cell line to FA and NO2, at concentrations found in indoor spaces, in LLI culture conditions, triggered a significant decrease in cell metabolism and an increase in CXCL8/IL-8 secretion. To get closer to the real conditions of eye irritancy caused by chemicals, some studies experimented the use of a 3D model with stratified corneal epithelia (Matsuda et al., 2009a,b,c; Postnikoff et al., 2014; Takezawa et al., 2011) or with a corneal explant, both models being cultured and maintained in ALI conditions (Jester et al., 2001; Zheng et al., 2013). From there, it would be interesting to expose conjunctival cells in a 3D model to gaseous FA at ALI. To conclude, these results demonstrate that gaseous FA (at 100 or 1200 µg/m3) aggravates the cytotoxic and proinflammatory effects caused by dry air conditions. This study suggests the feasibility and sensitivity of this original ALI exposure system for testing inflammatory responses of gaseous FA directly deposited onto the apical side of human conjunctival cells. This model remains a proof of concept, and additional studies are required to validate it under other conditions and using various analysis approaches. Although this sophisticated and original system is fastidious and time-consuming, it is a valuable tool that can help better understand the toxic mechanisms induced by gaseous FA or any other environmental pollutant in conjunction with desiccative stress, a hallmark condition used in DED models (Coursey et al., 2014). It could therefore be advantgeous to enhance the model by increasing the number of exposure parameters: different toxicants, exposure duration, number of exposures in a repeated manner, recovery times, broader range of concentrations, and by studying signaling pathways related, for example, to inflammation, oxidative stress or apoptosis (TNF-α, NF-κB, MAP kinases, Keap1-Nrf2, caspases, etc.). Despite the constraints of this exposure system, it opens a major field of research on the relations between ocular surface epithelial cells and the environment. Indeed, the environment plays a major role among the factors that trigger the vicious circle of the pathophysiology of DED. This is a promising system that can improve our understanding of the responses of ocular surface epithelial cells to toxic environmental stresses. In the future, this exposure system should help us better characterize the impact of indoor air pollution on the ocular surface and its interactions with DED. FUNDING This study was supported in part by the Institut National pour la Santé et la Recherche Médicale (INSERM) and by Horus Pharma, Saint-Laurent-du-Var, France. ACKNOWLEDGMENTS The authors thank the core facilities of the Institut de la vision, Région Ile-de-France and Ville de Paris. They also thank Linda Northrup for editorial assistance with the article. APPENDIX According to Bardet et al., the FA concentration in the glass vacuum desiccator jar dryer is equal to 50% of its theoretical concentration. Therefore, to obtain concentrations of 100 and 1200 μg/m3, it was necessary to prepare the initial FA solutions at concentrations of 200 and 2400 μg/m3, according to the protocol below. The molar concentration c(HCHO) of FA is 13.3 mol/L, the molecular weight M(HCHO) of FA being 30 g/mol, the mass concentration ρ(HCHO) (ρ = c × M) of the stock solution (SS) is therefore 13.3 × 30.0 = 399 g/L. 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Toxicological Sciences – Oxford University Press
Published: Sep 1, 2018
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