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The effects of short-term and long-term air pollution exposure on meibomian gland dysfunction

We aim to assess the effects of different air pollutants on meibomian gland dysfunction (MGD). As a prospective multicenter study, 864 patients were recruited from four different regions (i.e., coal, oil, steel, and living). The oil region had a significantly lower temperature and higher O3 and SO2 concentrations than other regions. Notably, participants in oil region presented with more frequent and serious MGD signs and higher cytokine levels (median interleukin 6 [IL-6] in oil: 2.66, steel: 0.96, coal: 0.38, living: 0.56; IL-8 in oil: 117.52, steel: 46.94, coal: 26.89, living: 33; vascular endothelial growth factor [VEGF] in oil: 25.09, steel: 14.02, coal: 14.02, living: 28.47). The short-term fluctuations of cytokine levels were associated with the changes in gas levels (PM2.5 and IL-8: β = 0.016 [0.004–0.029]; O3 and IL-6: β = 0.576 [0.386–0.702]; O3 and IL-8: β = 0.479 [0.369–0.890]; SO2 and VEGF: β = 0.021 [0.001–0.047]). After long-term exposure, lid margin neovascularization (r = 0.402), meibomian gland (MG) expression (r = 0.377), MG secretion (r = 0.303), MG loss (r = 0.404), and tear meniscus height (r = − 0.345) were moderately correlated with air quality index (AQI). Individuals in oil region had more serious MGD signs and higher cytokine levels. MGD is susceptible to long-term exposure to high AQI.


Materials and methods
Study design and participants. In this multicenter prospective cohort study, individuals were recruited from five provinces across China, including 11 hospitals in Beijing, Hebei, Heilongjiang, Anhui, and Inner Mongolia from February 2020 to February 2021, covering four different regions where factories mainly dedicated to steel, coal or oil production, and densely population (living) are predominant in the area. Participants aged 20-80 years who spend 3-4 h outdoor activities per day (average) in the corresponding zone and met diagnostic criteria of MGD 16,17 were recruited in the study. Individuals with histories of another ocular surface abnormality, contact lens use, ocular surgeries, glaucoma medications use were excluded. Participants in each hospital were examined by the same trained doctors. Only first visit participants were included and divided into four groups according to their sampling regions (i.e., steel, coal, oil, and living). The study was performed in accordance with the Declaration of Helsinki and was approved by the Peking University Third Hospital Ethics Committee (Research Ethics Number M2019101). Informed consent was obtained from all participants.
Outdoor air pollutants and meteorology data. During the observation period, the daily mean temperature, relative humidity, and wind speed were provided by the meteorological administrations of Beijing, Hebei, Heilongjiang, Anhui, and Inner Mongolia. The concentrations of daily air quality index (AQI), PM 2.5 , PM 10 , O 3 , NO 2 , and SO 2 were obtained from open-access government air-quality monitoring data. Since the air pollutants exposure are constant, the 24-h average concentrations of PM 2.5 , PM 10 , NO 2 , and SO 2 as well as the 8-h maximum value of O 3 were collected as daily exposures according to previous studies [18][19][20][21] . AQI is determined by monitoring the five major air pollutants, i.e., PM, O 3 , NO 2 , SO 2 and carbon monoxide (CO), and the maximum value among each of the pollutants is assigned as the date-and location-specific AQI value 20,21 . As Gope et al. 20 and Mirabelli et al. 21 reports, we recorded 24-h average AQI as daily AQI. According to different AQI levels (level I: 0-50; level II: 51-100; level III: 101-150), participants were divided into three groups. The daily exposure is considered as short-term exposure. And we recorded the 1-month average AQI (AQI mean) before the first observation as long-term exposure.
Tear film collection and cytokine measurement. Tear samples were collected from the right eyes of participants. Without any anesthetic, non-irritating tear collection was conducted using 5-μL capillary pipettes. A plastic head was used to squeeze tears into 0.2 mL Eppendorf tubes, and tears were immediately frozen at − 80 °C. The cytokine levels in the tear samples were measured by a flow cytometer (BD FACS Canto ll, Becton Dickinson, Franklin Lakes, NJ, USA) and a bead array system (BD Cytometric Bead Array system, Becton Dickinson) following the instructions of the manufacturer. At least 50 μL undiluted tear samples were analyzed for cytokines including interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 17 (IL-17), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), vascular endothelial growth factor (VEGF), and B-cell activating factor (BAFF).

Individuals in four regions presented different ocular surface status and cytokine levels.
The ocular surface status and cytokine levels of individuals according to the four regions are shown in Table 1 and Fig. 1. More than half of the participants demonstrated a variety of lid margin abnormalities. Individuals in coal and oil regions exhibited a high proportion of lid margin debris (coal: 27.6%, oil: 13.8%, living: 8.0%, steel: 4.5%, P = 0.000). Individuals in oil and steel regions exhibited a high proportion of eyelid neovascularization (oil: 33.0%, steel: 7.6%, living: 1.1%, coal: 0%, P = 0.000). Participants in steel region had a relatively high proportion of lid edema/thickening (38.4%, P = 0.000) and irregularity (15.2%, P = 0.026). Individuals in living region presented a low proportion of eyelid hyperemia (living: 26.8%, coal: 34.6%, oil: 36.7%, steel: 42.7%, P = 0.028).
Air quality index monthly average was related to MGD. Participants were divided into three groups according to the average AQI 1 month before the study, resulting in 144 individuals in level I, 574 in level II, and 146 in level III. The 1-month average AQI (AQI mean) of levels I, II, and III were 38.95 ± 8.86, 77.34 ± 10.59, and 108.99 ± 8.76, respectively. The ocular surface status and cytokine levels of the three groups, and the correlations between the ocular surface and AQI mean during long-term exposure are shown in Table 5 and Fig. 2.

Discussion
In this study, MGD signs (eyelid neovascularization, MG plugging, MG expression, MG secretion, and MG loss) in the participants from oil region were more frequent and severe compared with other regions. At the same time, individuals in oil region showed a significant reduction in the TMH, ST, and TBUT, and a significant increase in CFS, IL-1β, IL-6, IL-8, and VEGF concentration. In the long term, individuals exposed to higher AQI levels presented with MGD more commonly. , VEGF vascular endothelial growth factor. # Crosstab analyses were compared and results were expressed as number (percentage); $ Kruskal-Wallis tests were compared among four groups, independent t tests between two groups and results were shown as mean ± standard deviation (SD); & Kruskal-Wallis tests were compared among four groups, Mann-Whitney nonparametric U tests between two groups and results were shown as median (25% quantile, 75% quantile); Chi-square tests were used for comparing gender among four regions. *P < 0.05.  29 . NO 2 and PM 2.5 were also found to cause tear film stability and osmolality reduction 30 . Another study by Um et al. found that only SO 2 was associated with DED in South Korea, while NO 2 , O 3 , CO, and PM 10 were not 31 . Nevertheless, the impact of air pollutants on ocular surface remains controversial. In our study, lower temperature, higher O 3 , and higher SO 2 levels in oil region are theorized to be contributing factors. Our result suggested that reduced temperatures might have favored the lower TBUT, more serious lid margin neovascularization, MG plugging and MG secretion. A possible reasonable explanation is that low temperatures may cool down the MG orifice surface, which results in the transient blockages of orifices, premature condensation of secretions and an increase in the viscosity after being discharged from MG orifices, leading to tear film instability and serious MGD signs 32 . However, the  www.nature.com/scientificreports/ more serious lid margin hyperemia, debris and edema were seen in a relatively higher temperatures, suggested that elevated temperatures might favor the lid margin abnormality and inflammation. The results seemed to be a paradox. A human study was similar with our results that showing increased lipid layer thickness and TBUT but a high tear evaporation rate at a higher temperature 32 . Therefore, appropriate temperature is recommended for ocular surface health. O 3 possesses the property of powerful oxidation and could affect the eye, skin, and     29 . According to Lee et al., long-term exposure to a high concentration of O 3 induces oxidative damage, enhances inflammatory cytokine levels in tears, and decreases tear production and conjunctival goblet cell density, known to be via the NF-κB pathway in vitro 33,34 . Additionally, in SOD1 (−/−) mice, increased periglandular inflammatory infiltrates, increased fibrosis, decreased glandular acinar density, and apoptosis were found in MGs 35 . Those previous researches in agreement with our results, the increased O 3 concentration might contribute to the elevated Table 5. Correlations between the ocular surface status and air quality index (AQI) during long-term exposure. OSDI ocular surface disease index, TBUT tear film break-up time, ST Schirmer's I test, CFS corneal fluorescein staining, TMH tear meniscus height, IL-1β interleukin 1 beta, IL-6 interleukin 6, IL-8 interleukin 8, VEGF vascular endothelial growth factor. # Crosstab analyses were compared and results were expressed as number (percentage); $ Kruskal-Wallis tests were compared among three groups, independent t tests between two groups and results were shown as mean ± standard deviation (SD); & Kruskal-Wallis tests were compared among three groups, Mann-Whitney nonparametric U tests between two groups and results were shown as median (25% quantile, 75% quantile); Spearman correlation analysis was conducted to assess the associations between different atmospheric environments, various indices of ocular surface, and cytokine levels. *P < 0.05.  www.nature.com/scientificreports/ cytokine levels (IL-1β, IL-6, IL-8), decreased ST, TMH and TBUT, higher OSDI and CFS scores, and serious MGD signs (MG loss and margin vascularization). Ozone is such a small molecule that it may approach the ocular surface and lacrimal glands, then induce inflammation and tear secretion abnormality. According to the studies of Wolkoff et al., aggressive aerosols and combustion products, such as SO 2 , could alter the structural composition of the outermost lipid layer of the precorneal tear film and cause eye burning, drying, and itching, resulting in DED in most cases 15 . The results of this study demonstrated SO 2 concentrations were associated with more serious MGD (lid vascularization), lower TBUT, higher OSDI and CFS scores, and higher VEGF concentration, which is in accordance with the previously reported findings. Therefore, it is speculated that lower temperature induces temporary blockages of orifices, SO 2 alters the lipid layer of the tear film, and excessive oxidative stress exacerbates inflammation and the chance of microbial infection. In this study, individuals in oil regions presented more frequent and severe MGD, along with an increased level of cytokines, including IL-1β, IL-6, IL-8, and VEGF. Cytokine fluctuations were significant associated with air pollutants, such as PM, O 3 , and SO 2 levels. The fluctuations in the cytokine concentrations showed a strong correlation with ocular surface changes, including conjunctivochalasis, TBUT and CFS, among others. These indicated that the expression of proinflammatory cytokines was sensitive to air pollutant changes, especially gases. Solomon et al. have detected an increase in the proinflammatory forms of IL-1 (IL-1α and mature IL-1β) in the tear fluid of patients with DED, suggesting that IL-1 may play a key role in the pathogenesis of keratoconjunctivitis sicca 36 . Barton et al. demonstrated an increased expression of IL-1, IL-6, and IL-8, and decreased epidermal growth factor levels in eyes with Sjögren's syndrome 37 . Lee et al. demonstrated that the expression of IL-6, IL-8, IL-17, and IFN-γ increased after exposure to O 3 in cultured human conjunctival epithelial cells not pretreated with IL-1α 34 . Generally, IL-1β, IL-6, and IL-8 are considered to induce an oxidative stress response 34 . IL-1 and IL-6 jointly promote Th-17 cell differentiation, and IL-6 is also associated with the B-cell activation, proliferation, and differentiation 34 . The chronic inflammation is associated with high tear cytokines concentrations [38][39][40] , and both of them are important in the pathogenesis of MGD 41 . Therefore, the tear cytokines may play key roles in the pathogenesis of MGD exposed on air pollution.
According to the WHO, PM concentration in China have reached "bad" or even "very bad" levels, particularly in those industrial and densely populated areas. PM is identified as a crucial indicator of environment pollution and should draw public attention 19,42 . Previous studies demonstrated that high levels of PM 2.5 and PM 10 were associated with decreased TBUT and conjunctival goblet cells density and increased pro-inflammatory cytokines in mice 30,43 , which were in accordance with our results. There were some correlations between PM concentration fluctuations and MG morphology/function and tear quantity/stability. These results suggested that PM 2.5 and PM 10 were positively related to the severity of ocular surface pathologies and MGD, we suspect that the PM may first affect the MG, leading to abnormal morphology and secretory function, thus affecting the tear film stability and ocular surface, resulting in ocular surface discomfort. Fu et al. reported that PM 2.5 has a time-and dose-dependent effect on cytotoxicity in cultured human corneal epithelial cells 44 . Thus, it may be difficult to detect ocular surface differences when the short-term changes in PM are not obvious. Yoon et al. have found that the effects of collected road dust on cellular responses are strongly dependent on their concentration and solubility 45 , indicating that atmospheric PM concentrations cannot be equal to PM concentrations in tears. Rather than PM concentrations, the components of PM are more important 45,46 . The specific compositions of PM, such as polyaromatic hydrocarbons, elemental/organic carbon, heavy metals, nitrate and sulfate 47 , change according to different zones and time. And the chemical characteristics of the compounds adsorbed to the particle surface will definite affect the PM toxic effects. In our study, the concentrations of PM 2.5 and PM 10 in the oil region were higher than those in the living and steel regions but lower than those in the coal region. However, the ocular surface presented more serious signs, and cytokine concentrations are higher than the three regions. We suspect that the air pollutants in the oil region are more soluble and complex, especially when affected by weather conditions, atmospheric chemistry, and complicated interactions with multiple air pollutants (such as O 3 and SO 2 ) 47 .
This study attempts to reflect the long-term pollution status by using the average preliminary AQI. Previous studies reported dose-response relationships in the constant concentration of air pollutants 48,49 . However, the exposure on the ocular surface is continuous, and we used the mean AQI in the present study inevitably. Symptoms and signs of MGD gradually increased with the 1-month average AQI, especially the lid margin neovascularization, MG expression, MG secretion, and MG loss. A certain transition period between the onset of DED and MGD signs is suspected, and long-term air quality likely plays an important role in the process. However, the AQI reflects the pollution levels of the most influential pollutants daily, which the 1-month AQI may not have captured.
The study had several limitations. First, the study's sample size was not large enough, making it difficult to stratify the differences in temperature and relative humidity for further analysis. Second, the study was a prospective cohort study, so the results did not definitively provide causal evidence between MGD and air pollutants. Third, the air pollutants exposure on the ocular surface is continuous. However, it is hard to monitor air quality personally and constantly in a large amount of people. We used the daily basis in the present study inevitably. There were differences between the indoor and outdoor activities of individual. However, our participants were asked to do 3-4 h outdoor activities to eliminate this difference. Despite some limitations, we believe this study is meaningful because it is a well-designed multicenter prospective study with organized statistical analysis. We investigate different air pollutants, ocular surface parameters and inflammation cytokines in different areas across China, and found it necessary to make a protocol to handle those air pollutants as much as possible to decrease their harmful effects.
In conclusion, individuals in the oil region presented more frequent and severe MGD, along with higher cytokine levels. MGD is likely in long-term exposure to relatively high AQI. The significant association between air pollution and ocular surface discomfort indicates the urgency to accelerate the environmental protection.