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FEATURE ARTICLE – PUBLIC ACCESS

Impact of Low Humidity on Damage-associated Molecular Patterns at the Ocular Surface during Dry Eye Disease

Alven, Alyce OD, MS1; Lema, Carolina PhD1; Redfern, Rachel L. OD, PhD, FAAO1∗

Author Information
doi: 10.1097/OPX.0000000000001802
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Abstract

FU1

Inflammation is a known component of dry eye; however, the mechanisms that drive the chronic inflammatory response are not fully understood. In the United States, dry eye affects about 14.4% of the population,1 and a study in Canada reported that 25% of patients at ophthalmic clinics suffer from dry eye,2 making it the leading reason for patients to seek ophthalmic care.1,3 It is well known that the tear film in dry eye becomes hyperosmotic because of insufficient tear production or excessive tear evaporation.4 Although the exact mechanism remains unknown, ocular surface desiccation stimulates the production of inflammatory cytokines and metalloproteinases,5–7 which perpetuates the inflammatory cycle by disruption of cell-to-cell tight junctions8 of the ocular surface epithelium.4

Danger- or damage-associated molecular patterns, also known as alarmins, are released by activated or injured cells to signal the threat of tissue injury.9 We hypothesize that damage-associated molecular patterns may play a vital role in dry eye by perpetuating inflammation once ocular surface homeostasis becomes compromised.10,11 In support of this, many alarmins are increased in systemic inflammatory diseases.12 Elevated blood levels of high-mobility group box 1 have been found in autoimmune diseases such as rheumatoid arthritis,13 lupus erythematosus,13 and Sjögren syndrome, which often present with secondary dry eye.14 Heat shock proteins are increased in response to cellular injury and necrosis15 and are elevated in several inflammatory disorders (e.g., atherosclerosis, rheumatoid arthritis, and multiple sclerosis).16

As typical alarmins, the physiological roles of high-mobility group box 1 and heat shock proteins depend on their intracellular or extracellular localization. Intracellular high-mobility group box 1 is vital to maintain cellular homeostasis owing to its chaperone functions related to DNA repair.17 Extracellular high-mobility group box 1 is known to modulate the inflammatory response of innate immune cells, such as dendritic cells18 and monocytes/macrophages.19 High-mobility group box 1 reaches the extracellular environment from passive release by dead, dying, or injured cells or from active secretion by activated immune cells. Then, depending on its oxidative state, high-mobility group box 1 can bind to different cellular receptors including C-X-C chemokine receptor type 4, receptor of advanced glycation end products, and toll-like receptors 2, 4, and 9 to stimulate cytokine release,20,21 chemotaxis of immune inflammatory cells,20 and/or vascular cell adhesion protein 1 and intercellular adhesion molecule 1 expression.22

Highly conserved heat shock proteins play a critical role in the maintenance of cellular homeostasis. As molecular chaperones, heat shock proteins facilitate the folding and assembly of proteins and also assist in protein refolding and repair after environmental or metabolic stress.23 Although intracellular heat shock proteins are not inflammatory, extracellular heat shock proteins can activate toll-like receptors 2 and 4 to stimulate cytokine mRNA and protein production.24–26

As a key component of the innate immune system, toll-like receptors recognize specific pathogen-associated molecular patterns to induce the release of inflammatory mediators and antimicrobial peptides during infection and inflammation. Toll-like receptors also recognize damage-associated molecular patterns to initiate sterile inflammation to promote tissue repair; however, excessive toll-like receptor activation has been associated with inflammatory and autoimmune diseases.12 Toll-like receptor involvement in dry eye has been studied by others27 and by our group.28–30 At the ocular surface, several cell layers constitute not only a physical barrier to infection but also a key checkpoint of immune surveillance where toll-like receptors 1 to 10 are widely expressed by the corneal and conjunctival epithelia and stroma tissues.30

Dry eye modulates the expression of toll-like receptors on the ocular surface.28,29 Using an ex vivo corneal desiccation model, we observed increased expression of toll-like receptor 429 whose ability to recognize several damage-associated molecular patterns including high-mobility group box 1 has been documented.31–33 In particular, toll-like receptors 2 and 4 have been shown to be activated by damage-associated molecular patterns including heat shock proteins,25,34 high-mobility group box 1,32,33 and calcium-binding protein S100A.35 Elevated levels of S100A have been found in the tear film of dry eye subjects and were found to be positively correlated with dry eye severity.36,37 Therefore, it is plausible that damage-associated molecular patterns released from dying ocular surface cells may activate toll-like receptors to perpetuate the inflammatory response in dry eye (Fig. 1). This study's purpose was to comprehensively examine high-mobility group box 1 or heat shock protein levels in tears and ocular surface cells in normal and dry eye subjects. In addition, the impact of desiccating environmental conditions on damage-associated molecular patterns levels in dry eye subjects was further evaluated.

F1
FIGURE 1:
Damage-associated molecular patterns' potential role in dry eye. In dry eye disease, reduced tear production or increased tear evaporation causes tear film disruption and hyperosmolar stress leading to ocular surface damage and subsequent release of DAMPs (e.g., high-mobility group box 1 and heat shock proteins). Excessive DAMPs in the tear film may activate TLRs to release inflammatory cytokines and MMPs, exacerbating epithelial cell damage and perpetuating the cycle of inflammation in DED. DAMPs = damage-associated molecular patterns; DED = dry eye disease; MMPs = matrix metalloproteinases; TLRs = toll-like receptors.

METHODS

Subjects

This research was reviewed and approved by the University of Houston's Institutional Review Board and conforms with the principles and applicable guidelines for the protection of human subjects in biomedical research. All enrolled subjects reviewed and signed an informed consent form before participation. Thirty normal and dry eye subjects (15 subjects per group) were enrolled in the study. A clinical examination was taken under normal building humidity (74°F and 40 to 45% humidity). Furthermore, 10 of the dry eye subjects were exposed to an enclosed dry environment to evaluate the impact of low-humidity exposure. All subjects recruited were older than 18 years and were currently not using any topical medications other than rewetting drops. Exclusion criteria for both groups included anyone pregnant or nursing, contact lens wear, any ocular surgeries within the previous 6 months, any active eye diseases including ocular allergies but excluding dry eye, anyone currently taking anti-inflammatory medication, and individuals with known allergy or sensitivity to fluorescein, lissamine green, and topical anesthetics. All subjects were instructed not to instill artificial tears 2 hours before starting the study.

Normal and Dry Eye Classification

Dry eye subjects must have reported a symptom score greater or equal to 13 on the Ocular Surface Disease Index questionnaire and have one eye exceed the normal thresholds of three of the four following objective grading measures: tear production, corneal staining, conjunctival staining, and tear film stability. This classification scheme was based on a modified version of the classification schemes previously described.38

Clinical Signs

Normal and dry eye subjects were examined for tear film osmolarity, ocular surface integrity, tear production, and tear film instability. For all clinical signs, a single measurement from each eye was obtained, and the mean value was calculated. Tear film osmolarity was quantitated using the TearLab Osmolarity System (TearLab Corp., Escondido, CA). The system was calibrated following the manufacturer's instructions at the beginning of each day of patient evaluation. For a subject to be classified as dry eye, a cutoff value >308 mOsM39 was applied. The phenol red thread test (ZONE-QUICK; Ayumi Pharmaceutical Corp., Tokyo, Japan) was performed to assess tear production. A sterile phenol red–impregnated cotton thread was inserted between the lower eyelid and eye surface and removed after 15 seconds for measurement of the length of the red portion of the thread. The cutoff value for dry eye was ≤10 mm. Corneal and conjunctival epithelial integrity were evaluated using sodium fluorescein (Soft Glo; HUB Pharmaceuticals, Plymouth, MI) or Lissamine Green staining (Green Glo; HUB Pharmaceuticals, Rancho Cucamonga, CA), respectively. After instillation, grading was performed using a modified National Eye Institute's staining scale of 0 to 3 in each of four bulbar conjunctiva quadrants (nasal, temporal, inferior, and superior) or five corneal areas (central, nasal, temporal, inferior, and superior). Staining ≥4 in the combined five quadrants was considered indicative of dry eye.39 Finally, tear film instability was examined by measuring the tear breakup time using the Dry Eye Test–modified fluorescein strip (Akorn Pharmaceuticals, Chicago, IL), which has been specifically made narrower to deliver a small amount of fluorescein solution to the ocular surface. The cutoff value for dry eye was <10 seconds.

Basal Tears Collection and Analysis

Before clinical examination, basal tears were collected using a 10-μL microcapillary tube (BLAUBRAND intraMARK, Wertheim, Germany), which was placed in the lower temporal fornix, allowing tears from the lower meniscus to fill the tube via capillary action. For each subject, tears from both eyes were pooled into a single sample. All tear samples were immediately stored at −80°C after collection and thawed at the time of analysis. Tear samples were centrifuged at 2000 rpm for 5 minutes before Luminex and ELISA analysis to avoid cellular debris and for optimal sample recovery.

Conjunctival Impression Cytology

A single drop of 0.5% proparacaine hydrochloride anesthetic was instilled onto each eye. A sterile polyether sulfone membrane (Supor Membrane Disc Filters, 0.2-μm pore size, 25 mm, plain; PALL Corp., Port Washington, NY) was cut into eight equal pieces (~60 mm2/piece). Two conjunctival impression cytology samples from each eye were collected from the medial and lateral bulbar conjunctiva and stored at −80°C either in RLT lysis buffer (Qiagen, Germantown, MD) for RNA extraction and quantitative real-time polymerase chain reaction or in phosphate-buffered saline solution containing 0.2% Tween and a protease inhibitors cocktail (Complete Mini; Roche Diagnostics, Nutley, NJ) for protein analysis using Luminex or ELISA assays.

Quantitative Real-time Polymerase Chain Reaction

Total RNA from conjunctival impression cytology membrane samples was extracted using the RNeasy Micro Kit (Qiagen). Quantitative real-time polymerase chain reaction was used to quantitate relative mRNA expression of interleukin 6, interleukin 8, metalloproteinase 9, and high-mobility group box 1. Complementary DNA was generated from 250 ng of total RNA using iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). Samples containing no reverse transcriptase or water in place of RNA (no template control) served as negative controls. Ten nanograms of complementary DNA reactions were analyzed using a CFX96 Real-Time System (Bio-Rad). Real-time polymerase chain reaction amplification of complementary DNA was performed with SsoAdvanced SYBR Green Supermix (Bio-Rad) using PrimePCR SYBR Green primers for human interleukin 6 (unique assay ID: qHsaCID0021314), interleukin 8 (qHsaCED0046633), metalloproteinase 9 (qHsaCID0011597), and high-mobility group box 1 (qHsaCED0048035). For each gene, samples were processed in technical triplicates, and amplified gene products were normalized to housekeeping RPL27 (qHsaCID0023846). The mean relative fold change of interleukin 6, interleukin 8, metalloproteinase 9, and high-mobility group box 1 was calculated then by use of the CFX Manager software (Bio-Rad, Hercules, CA).

High-mobility Group Box 1 ELISA Assay

The levels of high-mobility group box 1 in tear samples and conjunctival impression cytology lysates from dry eye and normal subjects were determined using a commercially available ELISA assay (IBL International, Hamburg, Germany) as per manufacturer's instructions. Each sample was diluted in phosphate-buffered saline and tested in duplicate (10 μg total protein of sample per replicate). Optical density was measured at a wavelength of 450 and 600 nm as reference wavelength with a plate reader (FLUOstar Omega; BMG LABTECH, San Diego, CA). Tear concentration of high-mobility group box 1 was determined using a standard curve and each subject's sample dilution factor.

Heat Shock Proteins Luminex Assay

The relative levels of four heat shock proteins 27, 60, 70, and 90α in tear samples and conjunctival impression cytology lysates were quantitated using a Luminex immunobead-based assay (EMD Millipore, San Diego, CA). Following manufacturer's instructions, each sample was diluted in phosphate-buffered saline and tested in duplicate (1 μg total protein of sample per replicate) using the Luminex MAGPIX system and xPONENT software (EMD Millipore, San Diego, CA). The median fluorescence intensity for each individual analyte per sample was obtained as a readout. Final values were adjusted to account for each subject's sample dilution factor.

Low-humidity Environment

Ten of the 15 dry eye subjects were recruited from the first study and exposed to desiccating conditions in our environmentally controlled room located at The Ocular Surface Institute, College of Optometry, University of Houston. This is a 19′ 8″ × 14′ 7″ enclosed area that is dedicated to dry eye studies requiring environmental control. The room is equipped with built-in humidity sensors and a desiccant unit, which is manually turned on and off by a button placed at one of the room's walls, which allows for switching between the “normal mode” and “dry mode.” Once the system is activated, the relative humidity drops from 40% (normal humidity) to approximately 4% (low humidity) in about 90 minutes and stays with minimal variation indefinitely, even if the door is opened and closed for 5 minutes at a time. Temperature is kept at 75°F, and illumination is motion activated. The room's ventilation and air flow are provided by a wall-mounted fan (airflow range, 2660 to 3190 cubic feet per minute) located about 4 feet above the floor. The room's space is divided into two functional units: the seating area, which mimics a small waiting room where subjects can seat and have access to TV and/or video games, and the clinical examination area, which houses equipment for a comprehensive eye examination as well as a countertop fridge and laboratory materials for sample collection and short-term storage. A layout of the environmentally controlled room is shown in Fig. 2. For the present study, the following workflow was followed: subjects were first examined at normal humidity in a routine clinical examination room at The Ocular Surface Institute, and they were then seated in the environmentally controlled room's waiting area (74.3 to 74.8°F, 4.2 to 4.3% relative humidity) and watched a 2-hour film of their choice on Netflix on a light-emitting diode TV screen. All participants were advised to not use artificial tears or sleep while remaining in the room. After low-humidity exposure, subjects were examined at the environmentally controlled room's clinical examination area. Tear osmolarity, phenol red thread test, tear breakup time, and corneal and conjunctival staining were measured. Before clinical evaluation, basal tears were collected using a microcapillary tube to compare damage-associated molecular patterns levels before and after exposure to a low-humidity environment. Conjunctival impression cytology samples were collected before and after exposure to a low-humidity environment in different eyes to avoid residual inflammation due to previous conjunctival impression cytology collection. Membranes were analyzed for mRNA expression of damage-associated molecular patterns (high-mobility group box 1 and heat shock proteins 27, 60, 70, and 90α) and inflammatory molecules (interleukins 6 and 8, and metalloproteinase 9) using quantitative real-time polymerase chain reaction.

F2
FIGURE 2:
The Ocular Surface Institute environmentally controlled room. The enclosed area is divided into two functional units: the seating area (light gray) where subjects have access to TV and video games, and the clinical examination area that houses all equipment needed for a comprehensive eye examination (dark gray). (1) Entrance, (2) on/off switch, (3) wall-mounted fan, (4) subjects' sofas, (5) TV screen, (6) OCULUS Keratograph (OCULUS Inc., Arlington, WA), (7) TearLab, (8) slit lamp, (9) desk work area, (10) sink, (11) countertop refrigerator, and (12) visual acuity screen.

Statistical Analysis

All data were analyzed using GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). Because the quantity and variability of high-mobility group box 1 and heat shock proteins have not been explored in the tear film, a sample size of 12 was selected based on a formerly published statistical approach.40 An additional three subjects were included in each group (total n = 15) in the event some participants were excluded because of insufficient tear volume for biological analysis. Unpaired t test was used for the subject's age and clinical signs/symptoms. To analyze levels of high-mobility group box 1 and heat shock proteins, normal and dry eye subjects were paired by age, and outliers were removed after being identified by the Grubb's test (α = 0.05). Next, normality of the data was assessed by D'Agostino-Pearson omnibus K2, and statistical significance was determined by applying paired t test or nonparametric Wilcoxon matched-pair signed rank test accordingly. Pre/post–low-humidity environment data were analyzed using a two-way analysis of variance with Dunnett or Bonferroni correction for multiple comparisons.

RESULTS

Normal and Dry Eye Subjects

A total of 30 subjects were included in this study (15 normal subjects and 15 with dry eye disease). Among normal and dry eye subjects, the mean ± standard error of the mean age was 56.5 ± 2.5 (range, 29 to 64) and 56.8 ± 2.7 (range, 35 to 73) years, respectively. In both groups, 60% of the study subjects were female. When comparing clinical scores for normal and dry eye groups, there was a significant difference for Ocular Surface Disease Index, tear breakup time, corneal staining, and conjunctival staining (Table 1). Compared with normal age-matched subjects, the dry eye subjects had significantly higher Ocular Surface Disease Index scores (26.9 ± 3.6 vs. 14.3 ± 2.2; P = .005), corneal staining (5.1 ± 1.2 vs. 1.8 ± 0.6; P = .02), and conjunctival staining (7.6 ± 1.0 vs. 3.6 ± 0.7; P = .003). There was no significant difference in phenol red thread test (25.8 ± 1.3 vs. 23.8 ± 2.1; P = .43) or tears osmolarity (314.8 ± 2.5 vs. 311.2 ± 2.9; P = .36) between the normal and dry eye groups.

TABLE 1 - Clinical signs and symptoms in normal and DED subjects
Normal (n = 15) DED (n = 15) P
Age (y) 56.5 ± 2.5 56.8 ± 2.7 .96
Female (%) 60 60 N/A
OSDI 14.3 ± 2.2 26.9 ± 3.6 .005
PRTT (mm) 25.8 ± 1.3 23.8 ± 2.1 .43
TBUT (s) 6.3 ± 0.9 2.6 ± 0.6 .002
Corneal staining 1.8 ± 0.6 5.1 ± 1.2 .02
Conjunctival staining 3.6 ± 0.7 7.6 ± 1.0 .003
Tear osmolarity (mOsM) 314.8 ± 2.5 311.2 ± 2.9 .36
Data are presented as mean ± SEM and were analyzed using an independent Student t test. DED = dry eye disease; N/A = not available; OSDI = Ocular Surface Disease Index; PRTT = phenol red thread test; SEM = standard error of the mean; TBUT = tear film breakup time.

High-mobility Group Box 1 and Heat Shock Proteins in Normal and Dry Eye Subjects

To assess if damage-associated molecular patterns were increased in the tear film of dry eye compared with normal subjects, the levels of high-mobility group box 1 and heat shock proteins were analyzed using ELISA and Luminex assays, respectively. In addition, to determine whether damage-associated molecular pattern production within conjunctival epithelial cells was upregulated, conjunctival impression cytology samples were taken from the nasal and temporal bulbar conjunctiva of each eye. High-mobility group box 1 was found to have significant higher levels in the tears of dry eye compared with normal subjects (9.5 ± 5.5 vs. 0.5 ± 0.3 ng/mL; P = .03), but no difference was observed in conjunctival cells (103.5 ± 16.6 vs. 121.7 ± 19.9 ng/mL; Table 2). Tear film or conjunctiva levels of heat shock proteins 27, 60, 70, or 90α were not significantly different between dry eye and normal subjects (Table 2).

TABLE 2 - Tear film and conjunctival DAMP levels in normal and DED subjects
DAMP Normal DED P
Tear film HMGB1 (ng/mL) 0.5 ± 0.3 9.5 ± 5.5 .03
Conjunctival HMGB1 (ng/mL) 121.7 ± 19.9 103.5 ± 16.6 .54
Tear film HSP-27 (MFI) 24,056.0 ± 7029.0 47,922.0 ± 17612.0 .22
Conjunctival HSP-27 (MFI) 56,807.0 ± 8138.0 43,204.0 ± 7429.0 .28
Tear film HSP-60 (MFI) 390.0 ± 91.2 391.4 ± 102.8 .99
Conjunctival HSP-60 (MFI) 9793.0 ± 977.9 9146.0 ± 1281 .62
Tear film HSP-70 (MFI) 561.3 ± 65.4 567.6 ± 139.9 .97
Conjunctival HSP-70 (MFI) 8225.0 ± 1059.0 6217.0 ± 1276.0 .22
Tear film HSP-90α (MFI) 13,533.0 ± 3320 17,816 ± 6450 .63
Conjunctival HSP-90α (MFI) 11,600.0 ± 1611 9522.0 ± 1599.0 .38
Tear film and conjunctival impression cytology samples were analyzed to quantitate HMGB1 and determine the MFI for HSPs 27, 60, 70, and 90α. DAMP = damage-associated molecular pattern; DED = dry eye disease; HMGB1 = high-mobility group box 1; HSP = heat shock protein; MFI = median fluorescence intensity.

Effect of Low-humidity Environment on Ocular Surface Integrity and Damage-associated Molecular Pattern Expression

To determine if additional desiccating stress was able to increase the levels of damage-associated molecular patterns at the ocular surface, a subset of 10 subjects from the dry eye group were rerecruited and exposed to a low-humidity environment for 2 hours. The mean ± standard error of the subjects' ages was 61.3 ± 3.0 years. After low-humidity exposure, corneal staining was significantly increased (2.2 ± 1.8 vs. 4.1 ± 2.1; P = .005). There was no significant difference found in phenol red thread test, tear breakup time, conjunctival staining, or tear film osmolarity (Table 3). To assess the magnitude of change in damage-associated molecular patterns levels induced by low-humidity environment, the post/pre–low-humidity environment ratio was calculated for each of the studied damage-associated molecular patterns; only heat shock protein 60 showed an approximately eightfold increase (8.2 ± 3.9; P = .01), whereas the others remained practically unaltered (Fig. 3A). Finally, to evaluate the impact of low-humidity environment on ocular surface inflammation and integrity, mRNA expression of interleukins 6 and 8, and metalloproteinase 9 were assessed at pre– and post–low-humidity environment instances. Environmental desiccating stress significantly upregulated metalloproteinase 9 (2.4 ± 0.4-fold; P = .001) mRNA expression. There was no significant change in interleukins 6 or 8 mRNA expression (Fig. 3B).

TABLE 3 - Effect of LHE on ocular surface signs
Pre-LHE Post-LHE P
PRTT (mm) 20.9 ± 8.2 22.9 ± 6.8 .41
TBUT (s) 4.0 ± 2.6 3.4 ± 1.4 .48
Corneal staining 2.2 ± 1.8 4.1 ± 2.1 .005
Conjunctival staining 3.2 ± 3.3 4.7 ± 4.3 .05
Osmolarity (mOsM) 309.6 ± 5.4 306.9 ± 12.6 .47
Data are presented as mean ± SEM and were analyzed using an independent Student t test. LHE = low-humidity environment; MFI = median fluorescence intensity; OSDI = Ocular Surface Disease Index; PRTT = phenol red thread test; SEM = standard error of the mean; TBUT = tear film breakup time.

F3
FIGURE 3:
Effect of LHE on DAMPs and inflammatory cytokines. Post-LHE/pre-LHE ratio values of HMGB1, HSP-27, HSP-60, HSP-70, and HSP-90α in tear film. (A) mRNA expression of IL-6, IL-8, and MMP-9 in conjunctival impression cytology samples (B). *P < .01; **P < .001. DAMP = damage-associated molecular pattern; HMGB1 = high-mobility group box 1; HSP = heat shock protein; IL = interleukin; LHE = low-humidity environment; MMP = matrix metalloprotein.

DISCUSSION

In this study, high-mobility group box 1 was significantly increased in dry eye subjects compared with normal age-matched subjects. These findings support our previous study where high-mobility group box 1 expression was elevated in the corneal epithelium of mice with experimental dry eye disease and in human corneal epithelial cells treated with dry eye culture conditions (e.g., hyperosmolar stress and tumor necrosis factor α treatment).10 The increase of high-mobility group box 1 was observed only in tears but not in conjunctival impression cytology samples of dry eye subjects. This result is not surprising given that high-mobility group box 1 is a ubiquitous nuclear protein and it is expected that intracellular levels will tend to remain unaltered and lower compared with secreted high-mobility group box 1, which will be modulated by external stimuli.

High levels of proinflammatory high-mobility group box 1 in the tear film of dry eye patients support the evaluation of anti–high-mobility group box 1 agents to reduce inflammation in the ocular surface. In a recent clinical pilot study, the efficacy of glycyrrhizin, a natural high-mobility group box 1 inhibitor isolated from the licorice root, was reported to reduce corneal staining, increase tear breakup time, and increase Schirmer scores in moderate dry eye patients after 28 days of twice daily use of glycyrrhizin 2.5% eye drops.11 However, caution needs to be taken because damage-associated molecular pattern–triggered inflammation is not necessarily detrimental but regenerative, and therefore, low levels of damage-associated molecular patterns are crucial to promote healthy tissue repair and healing through a physiological immune response.12 In agreement, a study found reduced levels of high-mobility group box 1 in the skin of diabetic human and mouse, supporting a beneficial role for high-mobility group box 1 in wound repair.41

Other interesting topic is the potential value of high-mobility group box 1 as a biomarker for dry eye diagnosis or disease severity. Although proinflammatory high-mobility group box 1 has been found in the tears of patients affected by other inflammatory disorders like conjunctivitis and blepharitis,42 its potential value as a potential marker for diagnosis and evaluation of dry eye patients cannot be disregarded given all the challenges that dry eye clinical management implies. Currently, S100A8, S100A9, lipocalin-1, secretory phospholipase A2, and metalloproteinase 943 as well as interleukins 1β and 644 have been proposed as dry eye biomarkers.

Among the objective signs evaluated, only corneal staining showed a minimal correlation when compared with high-mobility group box 1 in tears (R2 ≤ 0.5; data not shown). Although weak, this trend might reflect hyperosmolar stress–mediated corneal epithelial necrosis (which presents clinically as positive staining) as a causative factor for passive release of high-mobility group box 1 extracellularly into the tear film. A large body of publications indicates that high-mobility group box 1 can be actively secreted by immune and nonimmune cells, as well as passively released under injury or stress.45,46 Besides its proinflammatory role, extracellular high-mobility group box 1 has been shown to act as a recruiter for stem cells in bone marrow and cardiac tissue and is necessary for epithelial regeneration.47–49 Thus, areas of epithelium damage, shown by corneal and conjunctival staining, might actively secrete high-mobility group box 1 as a chemoattractant for limbal corneal stem cells to promote healing. Finally, it is also possible that the correlation between high-mobility group box 1 and ocular surface staining results via receptor for advanced glycation end product activation. In fact, S100A proteins and high-mobility group box 1 have been shown to interact with receptor for advanced glycation end products to perpetuate cellular damage and failure of inflammatory resolution.50 In dry eye, high-mobility group box 1 bound to receptor for advanced glycation end products may increase the production of metalloproteinases leading to corneal and conjunctival damage. Further research is needed to determine if the increase in high-mobility group box 1 seen in dry eye is inflammatory, regenerative, or possibly both, in nature.

In this study, there was no significant change in heat shock proteins 27, 60, 70, or 90α in the tear film and conjunctiva levels between dry eye and normal subjects. Interestingly, the levels of all damage-associated molecular patterns in conjunctival impression cytology samples were lower in dry eye subjects, although statistical significance was not reached. Heat shock proteins 27, 60, 70, and 90α are induced by cellular stress and play a key role as cytoprotectants. Among them, intracellular heat shock protein 70 has been shown to reduce inflammatory cytokine production in dendritic cells51 and astrocytes.52 Therefore, low levels of protective intracellular heat shock protein 70 in dry eye may make the ocular surface more susceptible to inflammation. Additional studies are needed given the small sample size of the current study. Using the current data, we calculated the appropriate sample size for these molecules using G*Power 3.1 software53 and standard power conditions (α = 5% two-tailed; power, 80%). Our analysis revealed that our study was underpowered for heat shock protein 60 (tears and conjunctiva), heat shock protein 70 (tears), and heat shock protein 90 (tears), suggesting that a larger sample size is needed for these molecules of interest.

To determine if acute desiccating stress could modulate the expression of damage-associated molecular patterns, dry eye subjects were exposed to acute low-humidity environment. A previous study found that matrix metalloproteinase 9 and inflammatory cytokines are increased after a 2-hour exposure to 5% relative humidity, suggesting a potential increase in damage-associated molecular patterns as well.54 Two hours of low-humidity exposure resulted in increased corneal staining and matrix metalloproteinase 9 mRNA expression in conjunctival samples; however, we did not detect an increase of high-mobility group box 1 in the tear film. Its plausible that additional time would be needed to create additional desiccating stress on the ocular surface. This explanation is based on our results from previous in vitro experiments where a gradient of hyperosmolar stress was cultured with human corneal epithelial cells for 6, 12, and 24 hours. The lowest level of hyperosmolar stress (400 mOsM) did not show a significant increase in extracellular high-mobility group box 1 until after 12 hours of exposure, whereas higher osmolarity levels (450 to 500 mOsM) began showing a significant increase in high-mobility group box 1 as early as 6 hours in our cell culture model.10

To look closer into the low-humidity environment impact on dry eye subjects, the post/pre-exposure ratio was calculated for all the studied damage-associated molecular patterns. Heat shock protein 60 showed an approximately eightfold increase, whereas the others remained unaltered. Heat shock protein 60 is known to have very strong immunogenic properties, and it has been reported to elicit autoimmune chronic conjunctival inflammation and scarring.55 Also, it has been shown to elicit a proinflammatory response in macrophages by acting as an endogenous ligand of toll-like receptor 4.56 Furthermore, heat shock protein 60 can bind to a toll-like receptor 4 ligand, lipopolysaccharide, to enhance their biological activity57,58 and exacerbate inflammation. Therefore, increased levels of heat shock protein 60 after 2 hours of acute low-humidity environment might exacerbate inflammation in dry eye subjects caused by desiccation stress. Interestingly, elevated levels of extracellular (released/secreted) heat shock protein 60 have been reported in saliva and serum from type 2 diabetes patients.59 Diabetic patients are known to be at a higher risk of developing gingivitis and periodontal disease, which could reflect a vicious tissue damage–chronic inflammation circle as it is observed in dry eye.

In conclusion, we demonstrate that damage-associated molecular patterns are elevated on the ocular surface during dry eye. Data revealed elevated levels of high-mobility group box 1 in the tear film of patients with dry eye and increased cellular stress–related heat shock protein 60 after acute environmental desiccating stress. Interestingly, low-humidity environment also increased the levels of damaging metalloproteinase 9, which is measured clinically when assessing the level of dry eye severity. Together, our results suggest damage-associated molecular pattern involvement in the etiology of dry eye disease.

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