Analysis of Cytokine Levels in Meibum and Clinical Correlations with Meibomian Gland Dysfunction

Objectives This study is aimed at investigating the difference of meibum chemokines in MGD subjects with different degrees of MGD and the correlations of meibum chemokines with ocular surface parameters. Methods Twenty MGD subjects (MQ score > 8) and twenty MGD subjects (MQ score ≤ 8) were enrolled to examine ocular surface parameters, including meibomian gland function (MGE, MQ meibograde, and lid margin), tear stability (NIKBUT, FBUT, and LLT), tear secretion (SIT and TMH), OSDI questionnaire, and CFS. These subjects also obtained meibum samples, and then meibum chemokines (MIG, IFN-γ, IL-8, IP-10, and MCP-1) were examined and analyzed the correlations with ocular surface parameters. Results MIG, IP-10, and MCP-1 were found clearly elevated in MGD subjects with higher MQ score than that in MGD subjects with low MQ score (MIG: p = 0.038, IP-10: p = 0.019, MCP-1: p = 0.040). The meibomian function was found mostly positively correlated with level of MIG (MGE: r = 0.600, p < 0.001; MQ: r = 0.579, p < 0.001) and IP-10 (MGE: r = 0.719, p < 0.001; MQ: r = 0.601, p < 0.001). The tear stability was found negatively correlated with the level of MIG (NIKBUT: r = −0.438, p = 0.005; LLT: r = −0.464, p = 0.003) and MCP-1 (NIKBUT: r = −0.425, p = 0.006; LLT: r = −0.761, p < 0.001). The OSDI was positively correlated with IL-8, IFN-γ, and MIG. Conclusion Chemokines in meibum were significantly evaluated in MGD subjects suffering from severe meibomian gland quality. These findings indicate that chemokines play roles in the pathogenesis of MGD, and molecules targeted by chemokines may develop as novel agents for MGD therapy, perhaps through inhibiting inflammation in meibomian glands and microvascular in the eyelid margin.


Introduction
Meibomian gland dysfunction (MGD), a chronic abnormality of the meibomian glands, remarkably affects tear film stability and leads to various ocular surface disease problems [1]. The global prevalence of MGD was reported to range from 10 to 20%, while the prevalence of MGD in the Eastern Asian even achieved more than 50% [2]. In ophthalmology clinics, MGD is one of the most common disorders routinely, so it can be considered as a public health problem [3]. Normal meibomian glands can secrete clear oil meibum, which serve as a mixture of lipids via orifices as the outermost layer of the tear film [4]. The abnormal meibum which MGD patients produce becomes more stagnant than the normal meibum, which can lead to terminal meibomian gland duct obstruction and evaporative dry eye [1]. Although there are different pathogenic mechanisms responsible for DED owing to MGD, a common consensus is that the involvement of inflammation as an integral part of MGD and DED [5]. The malfunction of the meibomian gland leads to exacerbating of the meibum, and abnormal meibum further derangements of the ocular surface and triggers more inflammatory cytokine expression [6]. The vicious circle, named as "dry eye inflammatory vicious cycle", forms between dry eye and inflammation [7], and abundant evidences identified that chronic inflammation on the ocular surface is located at the core pathogenesis of MGD [8].
In the DEWS II report, the etiology of MGD was described to be terminal duct obstruction impairing ocular surface homeostasis and leading to apparent inflammation and tear hyperosmolarity [9,10]. Inflammation is an extremely complex process, and the chemotaxis process is a pivotal step in the promotion and regulation of the inflammatory response. Chemokine family, the most important factors among the chemotaxis process, is a group of low molecular weight proteins (between 8 and12 kDa) which can promote immune cell migration to act on the immune chemotaxis effect [11]. There are two large chemokine families, the C-X-C motif ligand (CXCL) and the C-C motif ligand (CCL) chemokine families, both of which can induce T cell infiltration and regulate autoimmune inflammation [12]. Once these chemokines are bound to the ligands, they can lead to tissue damage and clinical manifestations through polarizing the migration of specific immune cells and amplifying the inflammatory response.
Manuscripts focusing on tear inflammation in MGD are steadily increasing [13,14], but there are rare reports concentrating on meibum chemokine examination in MGD patients. Considering that the core pathogenesis of MGD is located in the meibum secreted by the meibomian gland [1,6], directly study on the inflammation cytokines in meibum has more explicit clinical value than inflammation cytokines in tears.
The aim of the current study was to investigate the levels of different chemokines in meibum in MGD subjects. Furthermore, the correlations between meibum chemokines with ocular surface parameters were analyzed to seek possible meibum biomarkers for potential therapeutic agents in MGD. According to the consensus of The International Workshop on Meibomian Gland Dysfunction, MGD was confirmed by meibomian gland function examination by the same ophthalmology [1,15].

Materials and Methods
The inclusion criteria for MGD based on DEWS II are as follows: OSDI score > 12:5 points, FBUT < 10 s, and the presence of lid margin abnormalities, orifice abnormalities, and meibum abnormalities [1,15]. These MGD patients aged above 18 years old, who voluntarily participated in the experiment. Subjects with immune related dry eye such as Sjogren's syndrome were excluded. Furthermore, subjects with certain ocular diseases (acute ocular inflammation, obvious scar or keratinization in the palpebral margin) or receiving physiotherapy for blepharitis (intense pulsed light, baby shampoo, and demodex blepharitis treatment) in the last 3 months may confound the study results; thus, they were excluded from the study. Subjects were also excluded if they had a related ocular surgery, including cataract surgery, trichiasis surgery, lachrymal duct obstruction, or refractive surgery in the past 3 months. After the procedure and potential consequences of the study were explained elaborately, informed consent was obtained from all subjects before the experiment.
Firstly, OSDI questionnaire was collected to evaluate the symptoms of MGD subjects. Subsequently, ocular parameters including tear meniscus height (TMH), noninvasive tear break-up time (NIKBUT), lipid layer thickness (LLT), incomplete blink rate (%), fluorescein tear film break-up time (FBUT), corneal fluorescein staining (CFS), Schirmer I test (SIT), meibomian gland expressibility (MGE), meibomian gland quality (MQ), lid margin, and meibograde. Finally, the meibum sample was collected and stored for further examination. Based on MQ examination, subjects with mild to severe abnormal meibum quality (ranging from cloudy with granular particulates to toothpaste-like particulates) were included into the MQ > 8 group, while subjects with slight abnormal meibum quality (ranging from clear oil to cloudy oil) were included into MQ ≤ 8 group.

Ocular Surface Parameters and OSDI
2.2.1. OSDI. The OSDI questionnaire, containing a 12-item questionnaire with a scale of 0-100, has been designed to rapidly evaluate different ocular discomfort symptoms (soreness, light sensitiveness, and blurred vision). The OSDI questionnaire provides a rapid assessment of vision-related dyspraxia (difficulty reading, driving, operating a computer, and watching TV). There is a positive correlation between OSDI scores and the severity of ocular discomfort, with higher scores representing greater ocular discomfort [16].
2.2.2. TMH and NIKBUT. TMH and NIKBUT were measured by an OCULUS Keratograph 5M (Wetzlar, Germany) equipped with modified TF-scan software. The procedure was repeated three times following the instructions of OCU-LUS Keratograph 5M by the same ophthalmology in a dark room. TMH was manually gauged at the central point of the lower lid margin on the images. Then, all participants were required to naturally blink twice, and then keep their eyes open as much time as possible until the next blink, the duration is defined as NIKBUT [17].

LLT and Incomplete Blink
Rate. LLT and incomplete blink rate were detected noninvasively by the LipiView® instrument (TearScience, Morrisville, NC, United States). All participants were instructed to blink naturally to record a 15 s video of the tear film interference pattern and analyze the LLT incomplete blink rate (%). The procedure was repeated twice times for each eye.  Each of the eight glands of the lower eyelid was graded on a scale from 0 to 3. The scores of the eight glands were summarized (range: 0-24) [19]. According to the anomalous of the lid margin, lid margin score was graded as 0-4: grade 0 if absent of abnormal, and if present for any of the following parameters is recorded as 1: plugged meibomian gland orifices, vascular congestion, irregularity of the lid margin, and partly expression of the mucocutaneous borderline [20]. Combined with the upper and lower eyelid margins, the total score ranges from 0 to 8.

Demographic Data and Clinical Characteristics.
Twenty MGD subjects whose MQ scores were above 8 with a mean age of 35:30 ± 9:71 years (14 females, 6 males) were enrolled and compared with twenty MGD subjects whose MQ scores were less than 8 (mean age: 34:35 ± 5:76 years; 13 females, 7 males). No significant differences in terms of sex (c2 = 0:109, p = 0:744) and age (p = 0:709) were found between the two groups. The demographic data and ocular surface parameters in MGD subjects with MQ > 8 and MGD with MQ ≤ 8 were listed in Table 1.

Discussion
MGD is a chronic disease with a high level of prevalence among the human population and creates long-term damage to the ocular surface [3]. In consideration of the importance of inflammation in MGD, exploration and identification of         9 Disease Markers the potential inflammatory cytokines that are specifically involved in the pathogenesis of MGD has a large significance. Once a particular cytokine in meibum was found to be tightly related to the severity of the disease, then the inhibitor or agonist targeted to the particular cytokine could be developed as a potential therapeutic agent of MGD. This is the original purpose of our research, and some meaningful data were obtained.
In the current study, the majority of ocular parameters in MQ > 8 group exhibited more serious clinical characteristics, and they were statistically significant in contrast to MQ < 8 group. About meibum inflammation cytokines, the levels of 3 meibum inflammation cytokines (MIG, IP-10, and MCP-1) of the 5 inflammatory cytokines we examined were largely elevated in MGD patients with worse meibum quality. Integrated with these two parts these results, a conclusion can be drawn that the increase of meibum inflammatory cytokines accompanied with more serious disease degree. A previous study [22] showed that increased levels of various inflammatory cytokines in tears (IL-6, IL-8, TNF-α, and IFN-γ) were found in MGD patients than normal people, and these inflammatory cytokines were also associated with meibomian gland function and tear stability. Another report [23] investigated the tear inflammation cytokines between normal subjects and MGD patients, and it was observed that the amount of inflammation cytokines (such as TNF-α, IL-1β, IL-6, IL-8, IL-12p70, and IFN-γ) was significant elevated in the tear of MGD patients, so

10
Disease Markers inflammation may serve as the core characteristic in MGD patients. These reports verified our conclusion that worse meibomian gland function was closely associated with a higher level of tear inflammation cytokines. Thus, an inseparable relationship between the abnormalities of the meibomian glands is in association with ocular surface inflammation [9]. The 3 cytokines found robustly elevated in meibum are all classified to the chemokine family. MIG and IP-10 were included in CXCL family, while MCP-1 was included in CCL family. MIG/CXCL9 and IP-10/CXCL-10, also named "interferon-inducible CXC chemokine receptor 3 ligands", are ELR-negative CXC chemokines induced by IFN-γ or other stimuli during infection or inflammation in several immune cell [24]. Several studies [25,26] have demonstrated that they can effectively activate and recruit T lymphocytes to the target organ in vivo to exert immune chemotaxis function. In the current study, the increases in MIG and IP-10 levels were found positively correlated with worse meibomian gland function, while they were negatively correlated with tear stability. Combined the robust immunomodulatory effect of CXCL family (MIG/CXCL-9 and IP-10/ CXCL-10) [27][28][29] with the highly expression of these two chemokines in MGD patients, we speculated that MIG and IP-10 play a critical role in the pathogenesis of MGD.
According to relevant studies [30], CXC chemokines were pointed out to be associated with neovascularization, and CXC chemokines which lack of ELR (MIG/CXCL-9, IP10/CXCL-10) are potent inhibitors of angiogenesis and microvascular. The antiangiogenesis effect of MIG and IP10 have been applied and certified in the treatment of various diseases [31,32]. Neovascularization and microvascular in lid margin can be found among many MGD patients [6,33], so the treatment targeted on neovascularization and microvascular (IPL) in lid margin has been proved to be effective in MGD patients [34]. Hence, we speculated that the increase of MIG and IP-10 in meibum treatment may potentially achieve the therapeutic effect on MGD by downregulating angiogenesis cytokines to inhibit neovascularization and microvascular in lid margin. Based on the transitional inflammatory and obvious neovascularization responses in MGD, systemic and topical treatment which can combine anti-inflammation with antiangiogenesis should be accepted in regular strategies to maintain the normal function of meibomian glands, and especially novel molecular targeted on MIG and/or IP-10 may have great prospects in clinical.
MCP-1, also named as CCL-2, is an inflammatory cytokine that is specific for monocyte chemotactic proteins and can bind to NF-κB nuclear factor DNA to induce different immune cell migration to the designated tissue and trigger downstream signals to exert immune regulation functions [35,36]. Our study has identified that MCP-1 was closely related with NIKBUT and FBUT, while not correlated with MGE and MQ. Compared to CCL chemokine family, we inferred that CXCL chemokines should be focused as the core pathogenesis of MGD. Based on the transitional expression of CXCL chemokines in meibum in MGD subjects, topical anti-CXCL-inflammation treat-ment have more potential to develop as novel strategies in the treatment of MGD.
Furthermore, there was no significant difference in the levels of IFN-γ and IL-8 between the two groups, but relationships were found between partially ocular parameters with these two cytokines in meibum. IL-8, also named as CXCL-8, is the most well-known molecule in the CXCL chemokine family, which has great attractive chemotactic effects on neutrophils, lymphocytes and basophils [37]. In contrast to normal people, IL-8 was found significantly elevated in tears in all kinds of dry eye patients, not just limited to MGD. Therefore, IL-8 may be positioned as the critical pathogenesis of various types of dry eye, and it strongly affects ocular discomfort symptoms [23,38]. IL-8, as a vital member of the CXCL family, was confirmed to be related with MGE and MQ, thus the key role of CXCL family in maintaining normal meibomian gland function was confirmed once more. IFN-γ, the only member of type II interferon, is mainly secreted by natural killer cells (NK) and natural killer T cells (NKT) cells in the process of innate immunity and is also secreted by CD4 Th1 and CD8 cytotoxic T cells in the process of antigen-specific immunity [39]. Similar to IL-8, IFN-γ has also been proved to be significant elevated in most kinds of dry eye [23,40]. OSDI, the only index to assess the subjective symptoms of dry eye, was found closely related to the levels of IL-8 and IFN-γ. Therefore, a combination therapy including anti-CXCL with anti-IL-8/ anti-IFN-γ agent can simultaneously alleviate the ocular parameters and mitigate the discomfortable symptoms in MGD, to yield twice the result with half the effort.
Since the tear microenvironment is affected by various aspects, including tear quantity secreted by the lacrimal gland, meibum produced by the meibomian gland, and mucins secreted by the corneal and conjunctival epithelium [9], thus, the examination of the tear samples cannot be directly extrapolated to evaluate the function of meibomian gland. Meibum, as a noninvasive and convenient sample to obtain from meibomian glands directly, is the best carrier to reveal the microenvironment of meibomian glands. Therefore, the data obtained from meibum may be more accurate than the tear samples in the study of MGD. It is worth noting that meibum samples from MGD subjects were collected to more directly investigate the inflammation of meibomian glands, and we found that the expression of meibum inflammatory cytokines, especially chemokines (MIG, IP-10, and MCP-1) increased with the aggravation of MGD disease and can be detected as sensitive biomarkers in pre-evaluation the extent of MGD.
Due to the limitation of the small number of subjects, the inadequate number of inflammatory cytokines was also a deficiency in the current study. Furthermore, inflammatory cytokines have only been detected by Luminex chip without rechecked by ELISA assay, which is a shortcoming of the current study. Thus, further studies should be conducted with a larger number of participants examined with more analysis of meibum inflammatory cytokines using both Luminex chip and ELISA assay, to explicit illustrate more novel crucial cytokine biomarkers of MGD patients with strong evidence.

Disease Markers
To the best of our knowledge, this study is the first to identify the chemokines in meibum samples, and we found that they were closely correlated with meibomian function and tear film instability. As the most direct and available carrier of meibomian gland secretion, the meibum sample is clearly more ideal for exploring the microenvironment of the meibomian glands than the tear sample. The current study provides a solid foundation for the technical support of meibum inflammatory cytokines in further meibomian gland inflammation research. Moreover, to prove that chemokines, especially CXCL family, play an important role in the pathogenesis of MGD, it can be well applied in developing novel therapeutic agents for MGD in clinical.

Data Availability
The datasets used and/or analyzed during the present study are available from the corresponding author on a reasonable request.

Ethical Approval
The present study was strictly performed on the basis of the Declaration of Helsinki for clinical and was approved by the Ethics Committee of the EENT Hospital of Fudan University (Shanghai, China). After all details and potential benefits and risks were explicit and explained, written informed consents were obtained from all participants prior to the examination and treatment.