Tear fluid biomarkers in ocular and systemic disease: potential use for predictive, preventive and personalised medicine

Tears are a complex mixture of proteins, lipids, mucins, water and salts, and a recent
study has identified 1526 proteins via proteomics 1], making them less complex (as a body fluid) than serum or plasma. Due to this less
complex nature, and also because at the ocular surface the tears represent the “proximal
fluid”, the final output of the lacrimal functional unit (LFU 2]), the study of their composition has been proposed as an ideal source for discovering
biomarkers associated with the various components of the LFU 3], and there has been increased interest in determining novel tear biomarkers of ocular
diseases, e.g. dry eye disease (DED), vernal conjunctivitis, diabetic retinopathy,
Graves’ ophthalmopathy, ocular tumours and glaucoma, to name a few 1], 4]–8]. Moreover, tears are being investigated for the identification of biomarkers of systemic
disease.

A biomarker, as defined by the National Institute of Health (NIH), is

A characteristic that is objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic responses to therapeutic
intervention 9].

While the US Food and Drug Administration (FDA) describes a biomarker as

Any measurable diagnostic indicator that is used to assess the risk or presence of
disease 10].

In the field of PPPM, biomarkers are playing an increasingly important role in the
discovery and development of new drugs and point-of-care devices design, as diagnostic
tools or for objective monitoring of treatment in clinical trials. As described above,
a specific disease biomarker is defined as a measurable characteristic in a biological
system which changes due to disease, exposure to chemicals or other factors. Both
proteomic and genomic studies are applied to the search for novel and specific biomarkers
of disease processes. Biomarkers are key indicators that can provide vital information
detecting risk of disease, disease progression, disease activity, prediction of response
to therapies or adverse events and drug interactions or establishing baseline risk.
A useful biomarker has to correlate with clinical parameters, such as specific symptoms,
clinical signs and validated diagnostic tests. Also, whenever possible, non-invasive
samples should be used.

Therefore, the utility of biomarkers in personalised medicine for both ocular and
systemic disease is very pertinent to the current interest in tear fluid proteomics.
This review will seek to provide a timely account of the ongoing global search to
identify relevant tear fluid biomarkers, including the proteins of interest and the
technologies employed.

Tear fluid analysis of diseases affecting the ocular surface

Dry eye disease

Dry eye disease (DED) is a multifactorial inflammatory disorder of the LFU that is
characterised by ocular discomfort, visual disturbances, tear film instability, increased
tear osmolarity and inflammation 2], 11]. Research into tear biomarkers has been increasing in DED, mainly due to the fact
that in this multifactorial syndrome there is a lack of concordance between clinical
symptoms and signs 12]–15]. As a consequence, DED diagnosis has been difficult and the development of new pharmacological
therapies is hampered by the lack of objective tests for response outcomes in clinical
trials 16]–18]. For these reasons, there is a growing interest in finding objective biomarkers that
could be used as diagnostic tools for DED, or for objective monitoring of treatment
in clinical trials.

A large number of tear fluid studies in DED have therefore been performed in the last
5 years 19]–22]. These studies comprise research into the different forms of DED, including mild-moderate
DED, evaporative cases (including meibomian gland dysfunction (MGD)) or more severe
hyposecretor forms of DED, e.g. Sjögren’s syndrome (SS) and ocular graft versus host
disease (GVHD). As inflammation is a key component of DED, numerous tear biomarker
studies for this disease have included the analysis of inflammatory molecules, such
as cytokines/chemokines, as well as other molecules, e.g. growth factors, mucins,
neuromediators and lipids. Many of these studies have shown differences in several
tear molecules in DED patients compared to healthy subjects, or among the different
types of DED. A number of reviews on the use of tears as a source of biomarkers have
been published to date, including for non-ocular diseases 23]–25] and in reviews specifically dedicated to DED 26]–29].

Current tear proteins under investigation as DED biomarkers

Various groups have performed tear proteomics analysis in order to determine which
proteins and/or protein profiles are specifically related to DED. For example, a proteomic
approach to detect tear fluid DED biomarkers was used by Aluru et al. 4] by means of 2D electrophoresis and differential gel electrophoresis (DIGE). The authors
found lysozyme proline-rich protein 4 (LPRR4) to be significantly down-regulated in
several types of DED. Based on its differential expression and the correlation of
LPRR4 tear levels with disease severity, this protein has thus been proposed as a
potential biomarker of DED 4]. Other proteomic studies have also reported protein profiles that are specifically
related to DED 30]–35]. For example, in their study, Grus et al. 30] defined a seven-peptide panel for DED. This panel included calgranulin A/100A8, which
was found to be increased in DED patient tears, as well as LPRR3 and LPRR4, nasopharyngeal
carcinoma-associated PRP 4 and ?-1 antitrypsin. The latter four proteins were found
to be decreased in DED 30]. This panel had a 90 % sensitivity and specificity when used together with an artificial
neural network.

By using iTRAQ quantitative proteomics, coupled with 2D nano-LC-nano-ESI-MS/MS alongside
a statistical model, Zhou et al. 31] identified six up-regulated proteins in tears from DED patients versus controls.
These included ?-enolase, ?-1 acid glycoprotein 1, S100 A8/calgranulin A, S100 A9/calgranulin
B, S100 A4 and S100 A11 (calgizzarin), as well as four down-regulated proteins, including
prolactin-inducible protein (PIP), lipocalin-1 (LCN-1), lactoferrin and lysozyme 31]. With a four-protein biomarker panel (including ?-enolase, PIP, LCN-1 and S100 A9/calgranulin
B), this group obtained a diagnostic accuracy for DED of a 96 % (91 % sensitivity
and 90 % specificity). Moreover, using iTRAQ technology Tong et al. 32] found that S100 A8/calgranulin A and S100 A9/calgranulin B correlated to MGD severity
and redness and that LCN-1 correlated with tearing in non-SS-DED. Several of these
tear proteins have been described as being involved in ocular surface defence, with
many of them (such as lysozyme, lactoferrin, LCN-1 and defensins) being essential
components of the innate immune defence system. In fact, due to the fact that many
studies have shown that DED patients present decreased levels, the measure of tear
lysozyme and lactoferrin was one of the first proposals of objective tests for DED
diagnosis 36]–38].

In 2013, Boehm et al. 33] analysed tear protein patterns of dry eye patients by including different clinical
phenotypes, e.g. aqueous-deficient dry eye, (DRYaq), lipid-deficient dry eye (DRYlip)
and a combination of the two (DRYaqlip), in order to examine their influence on tear
film protein composition. By means of a surface-enhanced laser desorption/ionisation-time-of-flight
(SELDI-TOF)/matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF)/TOF
mass spectrometry (MS)-based strategy to detect candidate biomarkers, followed by
a discovery study (study 1) and a validation study (study 2), their results showed
that tear LPRR4 was diminished in both DRYaq and DRYaqlip patients, compared to healthy
subjects. Moreover, mammaglobin B, lipophilin A, S100A8/calgranulin and beta-2 microglobulin
(B2M) precursor were increased in this subgroup. DRYlip patients revealed only slight
tear protein alterations and strongly deviated from the DRYaq or DRYaqlip group 33]. By using this six-protein biomarker set, they achieved a DED patient versus healthy
subjects discrimination sensitivity and specificity of almost 100 % for the DRYaq
and DRYaqlip patients (AUC value?=?1).

Based on tear proteome and protein network analyses for tear film characterisation
in DED and MGD, Soria et al. 34] have presented a pentamarker panel, including S100A6, annexin A1, annexin A11, cystatin
S (CST4) and phospholipase A2-activating protein (PLAA), with an area under the ROC
curve of ?97.7 % (sensitivity ?94.3 %; specificity ?97.6 %) for DED versus controls.
This panel also discriminated between the DED, MGD and control individuals, with a
global correct assignment of 73.2 % between all groups. Versura et al. 35] have also proposed another tear protein panel for its use for DED diagnosis, based
on the simultaneous measurement of tear transferrin, LCN-1 and total protein; this
panel has a sensitivity of 96 % and a specificity of 98 %.

Proteomic studies in tears from dry eye patients, specifically SS, have also been
done. By using SELDI-TOF-MS, Tomosugi et al. 39] found ten protein peaks that could be used to discriminate SS patients from non-SS-DED
patients and controls. However, they did not identify the particular proteins. Li
et al. 40] also examined the tear film proteome of SS-DED patients compared to non-SS patients
with DED symptoms and to normal healthy controls. A total of 435 proteins were identified
by 2D nano-LC-MS/MS, and among them, 56, including defensin-1, clusterin and lactotransferrin,
were found to be unique to SS-DED patients 40]. Cathepsin S activity measurement in tears has also been proposed as a candidate
biomarker for SS 41], as tear activity of this protein was shown to be 4.1-fold higher in SS patients
than that in patients with other autoimmune diseases, 2.1-fold higher than that in
patients with nonspecific DED and 41.1-fold higher than that in healthy subjects 41]. Anti-SS-A and anti-SS-B, as well as anti ?-fodrin antibodies, have also been determined
in tear fluids of patients with SS 42]–44].

Other types of proteins have also been measured in tears of DED patients. For instance,
mucin (MUC)5AC protein tear levels have been shown to be significantly reduced in
tears of patients with SS 45]. Guo et al. 21] have measured tear malate dehydrogenase (MDH) 2 in mild DED patients. They found
that MDH2 activities in the DED group were significantly increased compared to that
in tears from a control (healthy) group. This group also found a significant negative
correlation of tear MDH2 with clinical parameters, such as tear production and tear
quality values, and a positive correlation with soreness symptoms. Recently, a novel
protein of the tear film belonging to the family of surfactant proteins, the palate
lung nasal clone (PLUNC), has been found to be increased in DED patient tears 46].

Neuromediators, such as substance P, calcitonin gene-related peptide (CGRP), neuropeptide
Y (NPY), vasointestinal peptide (VIP) and nerve growth factor (NGF), have also been
determined in tears from DED patients and correlated with clinical findings 47]. Specifically, NGF tear levels were found to be significantly increased in DED patients,
whereas CGRP and NPY were significantly decreased, compared to healthy subjects. Tear
NGF levels correlated directly, and CGRP and NPY inversely, with DED severity. Also,
in a recent study by Chhadva et al. 48], serotonin tear concentration has been correlated to facets of DED and has been found
to be significantly higher in those DED patients that presented with both DED symptoms
and aqueous tear deficiency, compared to those patients with DED symptoms but normal
tear production and those without DED symptoms.

Tear cytokines and chemokines in DED

As mentioned above, DED is associated with immune and inflammatory processes, so there
are a great number of studies particularly focused on the identification in tears
of profiles of cytokines and chemokines in the different clinical subgroups of patients
with DED. The development of multiplex assay technologies, such as cytometric bead
array (CBA), or XMAP technology, developed by the Luminex Corporation (reviewed in
49]), has made it possible to measure multiple molecules in minute volumes of samples,
which has been useful when measuring cytokines in tear samples. Prior to this technological
development, tear protein analysis was limited by the sample amount requirement of
other analysis techniques.

Several inflammatory cytokines/chemokines (such as IL-1, IL-6, TNF-?, metalloproteinase
(MMP)-9, IL-17A, IL-1RA, IL-8/CXCL8, IL-22, INF-?, MIG/CXCL9, IP-10/CXCL10, I-TAC/CXCL11,
macrophage inflammatory protein-1 alpha (MIP-1?/CCL3), MIP-1?/CCL4 and RANTES/CCL5,
among others) have been found to be significantly increased in tears from DED patients
19], 22], 50]–63]. Whereas endothelial growth factor (EGF) has been found to be significantly decreased
51], 55], 64] in the more severe forms of DED. Other studies have also shown differences in cytokine
profiles among the different types of DED forms, e.g. non-SS versus SS-DED, and/or
its severity. For example, Boehm et al. 60] found that while the cytokine profile in patients with aqueous-deficient dry eye
and dry eye patients with a combination of aqueous-deficient and changes in their
lipid layer were quite similar, the cytokine profile of DED patients with only changes
of the lipid layer was similar to that of the healthy controls. Furthermore, Tan et
al. 62] found that IL-17 and IL-22 were significantly increased, not only in tears from patients
with DED (compared to healthy controls), but also in SS patients, compared to non-SS-DED
patients. Another study 65] revealed that, besides IL-17A, tears from SS-DED patients presented with increased
levels of IL-6, IL-10, IL-4, INF-? and TNF-?, compared to non-SS-DED and to controls.
In another study, Lim et al. 66] analysed IL-21 tear concentrations in primary SS patients and found that its level
was significantly increased compared to healthy controls and that it also correlated
significantly with ocular surface stain score and tear production (Schirmer’s I test)
values in SS patients. Moreover, correlations of inflammatory tear molecule levels
such as IL-6, IL-8/CXCL8, TNF-?, IL-1Ra and I-TAC/CXCL11 (among others) with clinical
parameters and/or disease severity have been shown in some of those studies, further
corroborating the utility of tear analysis for this disease 19], 20], 55], 59]–61], 66]–68]. Specifically, MMP-9 measurement in tears has already been proposed as a sensitive
method for DED severity determination 56], 63], and a commercial point-of-care device has already been developed (InflammaDry®,
RPD, USA 69]).

Tear cytokine and chemokine measurement has also been performed in DED patients exposed
to different controlled environmental conditions within an environmental chamber.
For example, the studies carried out by Dr. Calonge’s group 70]–72] have shown that IL-6 and MMP-9 tear levels increase in DED patients, while EGF decreases
after exposure to a controlled environment, simulating an in-flight airplane cabin
(23 °C, 5 % relative humidity, localised air flow and 750 mb of barometric pressure
70]. This research group has also reported 71] that after a 2-h exposure to a controlled desiccating environment (5 % relative humidity),
control non-symptomatic subjects had decreased EGF and increased IL-6 tear levels.
Moreover, MMP-9 tear levels were also increased in both DED patients and non-symptomatic
controls. Besides, under similar desiccating conditions, DED-SS patients showed not
only IL-6 and MMP-9 tear cytokine changes but also IL-1RA and IL-8/CXCL8 tear increased
levels 72].

Tear lipidome in DED

Besides proteomic studies, the tear fluid lipidome in DED patients has been evaluated,
in an attempt to determine the composition and nature of tear lipids (secreted by
the meibomian gland) and to identify any alterations in those patients. Differences
in the meibomian fatty acid composition in patients with MGD and aqueous-deficient
DED were shown by Joffre et al. 73]. Moreover, studies by Lam et al. 74], 75] addressed the meibum lipid composition in DED patients and found several lipid species
that were significantly increased in this demographic, including sphingomyelin and
phosphatidyl species. They reported significant differences in the tear levels of
O-acyl-w-hydroxy-fatty acid (OAHFA) species, depending on DED severity. In MGD patients
undergoing eyelid-warming treatment, Lam et al. 75] found a reduction in tear fluid lysophospholipid and polyunsaturated fatty acid (PUFA)-containing
diacylglyceride species, as well as an increase in some PUFA-containing phospholipids
and OAHFAs, upon treatment. This group suggested that the lipidome changes were related
to reduced rates of ocular evaporation and an improvement in ocular symptoms of patients
75].

Tears have also been used for the evaluation of lipid oxidative stress status in SS
patients. In a study by Wakamatsu et al. 76], tear concentrations of hexanoyl-lysine (HEL) in SS patients were found to correlate
significantly with ocular surface staining scores and inflammatory cell density. More
recently, Choi et al. 77] evaluated the tear concentrations of HEL, 4-hydroxy-2-nonenal (HNE) and malondialdehyde
(MDA) in patients with non-SS-DED and 33 control subjects and found that their levels
significantly correlated with clinical tests for ocular surface health, e.g. TBUT,
Schirmer’s test score, tear clearance rate, keratoepitheliopathy score, conjunctival
goblet cell density and symptom score.

Tear metabolome in DED

As shown earlier, numerous proteins and lipids have been identified by means of proteomics
and lipidomics of tears in DED. By contrast, the number of identified metabolites
is more limited and there are few studies regarding analysis of tear metabolites in
DED. In a recent study by Galbis-Estrada et al. 78], the metabolomic profile of reflex tears from DED patients was analysed by nuclear
magnetic resonance (NMR) spectroscopy of hydrogen-1 nuclei; their results showed that,
when compared to tears from healthy subjects, there were differences in tear composition
of cholesterol, N-acetylglucosamine, glutamate, creatine, amino-n-butyrate, choline, acetylcholine,
arginine, phosphoethanolamine, glucose and phenylalanine levels. In another study
from this group 79], they further studied the metabolomic profile of basal tears from DED patients and
compared to healthy subjects, both before and after oral nutraceutic supplementation
(containing antioxidants and essential PUFAs) after 3 months. Their results showed
that there were significant differences in the tear metabolic profile of both groups
under study, both pre- and post- supplementation.

Tear fluid biomarkers of ocular GVHD-associated DED

Besides SS, another subtype of severe DED is that present in patients that suffer
from ocular GVHD. GVHD is an immune-mediated inflammatory disease that haematological
stem cell-transplanted (HSCT) patients may develop, in which host tissues are attacked
by immunocompetent cells from the donor 80]. Up to 60–90 % of patients have ocular involvement; in particular, chronic GVHD patients
develop a very severe form of dry eye. Signs and symptoms of ocular involvement from
chronic GVHD may mimic typical DED, but in GVHD patients can lead to a serious abnormality
of the ocular surface, affecting patient’s quality of life and eventually leading
to permanent visual loss. GVHD-DED is mainly due to aqueous tear deficiency, Sjögren-like,
and histology shows inflammatory destruction of the conjunctiva and lacrimal gland
with fibrosis, resulting in tear production deficiency 81].

Some studies have already shown that some cytokines have significant different levels
in tears of those ocular GVHD patients when compared to healthy subjects or to GVHD
patients without ocular involvement. One of those studies, Riemens et al. 82], demonstrated that IL-6 and IFN-? were significantly increased in tears from ocular
GVHD patients. While Sakimoto et al. 83] also described that soluble TNF receptor 1 (sTNFR1) expression was significantly
increased in tears from GVHD patients. Recently, Jung et al. 84] also conducted a study in which they studied tear concentration of an 8-cytokine
panel in chronic GVHD patients and compared it to HSCT patients that did not develop
GVHD. Their results showed that IL-2, IL-10, IL-17A, INF-?, IL-6 and TNF-? were elevated
in patients with GVHD compared to transplanted patients without GVHD. Additionally,
they found that IL-10, IL-17A, IL-6 and TNF-? had significant diagnostic abilities,
as based on their calculated odds ratio and AUC values. This group also showed that
those molecules, along with INF-? and IL-2, presented significant correlation with
clinical parameters, particularly with severity 84]. Cocho et al. 85] have also used tear molecule levels to develop a predictive model, based on a panel
of tear cytokines in chronic ocular GVHD. They found that, compared to healthy subjects,
these patients had significantly decreased tear levels of EGF and IP-10/CXCL10 and
increased levels of IL-1RA, IL-8/CXCL8 and IL-10. Significant correlations with clinical
features (including tear production and stability and symptoms, hyperaemia and ocular
surface integrity) were also found for these molecules. A statistically generated
IL-8/CXCL8 and IP-10/CXCL10 tear level-based predictive model was found to have an
AUC value of 0.9004, a sensitivity of 86.36 % and a specificity of 95.24 % 85].

Ocular allergies

Tear molecule analysis has also been addressed in cases of ocular allergy. Several
clinical subtypes, such allergic conjunctivitis, giant papillary conjunctivitis (GPC,
although this remains controversial), vernal keratoconjunctivitis (VKC) and atopic
keratoconjunctivitis (AKC), are considered allergy-related disorders 86]. As for the case of DED, tear analysis in ocular allergy patients has revealed significant
correlations of several molecules with clinical symptoms and signs, with specific
molecule profiles associated to the different clinical subtypes of allergy. Some of
these studies have already been reviewed 26], 27], 87]. Many of the tear molecules analysed in ocular allergy are related to cytokines/chemokines,
as these molecules have been shown to play a key role in allergy. Although, as in
the case of DED, there are also studies of some other proteins and neuromediators.

Tear cytokines and chemokines in ocular allergy

Regarding tear cytokine/chemokine analysis in the different subtypes of ocular allergy,
one of the first studies addressing this was that of Uchio et al. 88]. Using ELISA assays, this group analysed tear levels of Th-1-IFN-? and IL-2 and Th-2-IL-4
and IL-5 in VKC, AKC, allergic conjunctivitis (AC) and normal subjects. They found
that tear IL-4 levels in AKC patients were significantly higher than those in VKC,
AC and controls and that IL-4 tear levels differed significantly in patients with
AKC with proliferative lesions versus VKC patients. Also, tear IL-5 levels in patients
with diseases associated with proliferative lesions were found to be higher than those
in AC and normal controls. Also in this year, Leonardi et al. 89] determined soluble leukocyte activation markers (including neutrophil myeloperoxidase,
eosinophil cationic protein (ECP), eosinophil neurotoxin and soluble IL-2 receptor)
and histamine, in tears from VKC, AKC, seasonal AC and GPC. Later, this group analysed
tear eotaxin-1/CCL11 and eotaxin-2/CCL24 concentrations in VKC and AKC patients (and
normal subjects) and found that eotaxin-2/CCL24 was significantly increased in tears
from allergic patients 90]. Tear levels of both molecules correlated with the percentage of eosinophils in the
tear fluid. Another study by this group showed significantly increased MMP-1 and MMP-9
in VKC patients, compared to controls. Additionally, MMP-9 activity was found to be
correlated with corneal involvement and giant papillae formation 91]. MMP-9 tear levels have also been found to be increased in AC patients in a study
by Acera et al. 54].

Taking advantage of the development of multiplex cytokine analysis (CBA), Cook et
al. 92] analysed tear concentrations of IFN-?, TNF-?, IL-2, IL-4, IL-5 and IL-10, in both
allergic and non-allergic patients. Their study showed that tears from allergic patients
presented with decreased levels of IL-10 and also significant increased ratios of
TNF-?/INF-?, IL-5/INF-? and IL-5/IL-10, versus non-allergic subjects. This was one
of the first studies that established the usefulness of the CBA technique for cytokine
tear analysis. Also by the CBA method, research by Nivenius et al. 93] analysed the concentrations of IFN-?, TNF-?, IL-2, IL-4, IL-5 and IL-10 in tears
from AKC patients and found that they presented significantly increased tear levels
of these molecules. While Leonardi et al. 94] investigated and compared cytokine tear levels among different types of allergic
subtypes, including VKC, AKC, chronic and seasonal AC (SAC), they found specific profiles
of tear molecules that were significantly increased in each one; IL-1?, IL-2, IL-5,
IL-6, IL-12, IL-13 and MCP-1/CCL2 tear levels were found to be increased in all allergic
patients groups (compared to controls); IL-4, IFN-? and IL-10 were elevated in SAC
and VKC, while eotaxin-1/CCL11 and TNF-? were only increased in VKC patients group.
They also noted significant differences in the expression of IL-5, RANTES/CCL5 and
eotaxin-1/CCL11 in VKC patients, compared to that in those with SAC.

By means of a 40-molecule array, Shoji et al. 95] demonstrated that different cytokines were differentially increased or decreased
in tears (compared to controls), depending on whether they were from VKC or GPC patients.
Particularly, in VKC patients, eotaxin-1/CCL11, IL-11, MCP-1/CCL2 and M-CSF increased
to four times the values in the control group, and eotaxin-2/CCL24, IL-4, IL-6, IL-6sR,
IL-7, MIP-1? and TIMP-2 tear levels were increased to eight times the control values.
The increase in tear IL-6sR was statistically significant in both the VKC and GPC
patients compared to that in the controls, while that in eotaxin-2/CCL24 and that
in TIMP-2 were significant only in the VKC group and only in the GPC group, respectively,
compared with that in the controls. Whereas in tears from the GPC patients, IL-6,
M-CSF and MIG/CXCL9 increased to four times than those in the control group, and eotaxin-2/CCL24,
IL-6sR, IL-11, MIP-1? and TIMP-2 increased to eight times the control values. These
same authors also confirmed in another study 96] that in these two groups, tear sIL-6R levels were significantly increased and correlated
to clinical score of allergic inflammation of the ocular surface in VKC patients and
proposed the use of tear sIL-6R as useful biomarker for patients with AC disease.
Another multiplex methodology used for the study of tear molecule concentration is
the use of membrane arrays, a stationary phase protein analysis technique. With an
optimised protocol, Sack et al. 97] used this method to study 16 inflammatory mediators (GM-CSF, IL-1?, IL-1?, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL8, IL-10, IL-12, IL-13, INF-?, MCP-1/CCL2 and
TNF-?) in tears from allergic patients, in both open- and closed-eye environments
and showed detectable levels of most of the protein panel in these patients, particularly
the found enhanced IL-2, IL-4, IL-5 and IFN-? signals in open eye samples and IL1-?
and TNF-? in closed-eye patients’ tear samples, versus controls, in which only IL-8
was detectable.

Other tear biomarkers of ocular allergies

Studies have been undertaken on tear molecules from ocular allergy patients, such
as histaminase, ECP and histamine. In 1995, these molecules were evaluated by ELISA
and RIA in VKC patients 98]. While Montan et al. 99] analysed tear ECP levels of various groups of AC patients (VKC, AKC, SAC and GPC)
versus healthy subjects, they found that subjects with AKC and VKC had significantly
higher tear ECP values than subjects with GPC and SAC. Additionally, they found that
there was a significant correlation between ECP values and disease severity in all
disorders 99].

Other studies have addressed the tear analysis of some other molecules in allergic
tears such as haemopexin, neuromediators and allergen-specific IgE antibodies 8], 100]–103]. Haemopexin tear levels were found to be increased in VKC patients and significantly
associated with disease severity 101]. In agreement with those results by Pong et al. 101], Leonardi et al. 8] —by means of iTRAQ quantitative proteomics analysis of VKC tear samples—found that
levels of haemopexin (and also serum albumin and transferrin) were up to 100 times
higher than the control tear sample levels; those molecule tear levels also correlated
to severity of disease. Additionally, they found that haemopexin, transferrin, mammaglobin
B and secretoglobin 1D were significantly overexpressed in VKC tear samples, compared
to the control ones 8].

Sacchetti et al. 102] showed that after conjunctival allergen provocation test, tear levels of substance
P, CGRP and VIP neuromediators were significantly increased in allergic patients compared
to baseline. More recently, in 2015, the group of Leonardi et al. 103] has determined allergen-specific IgE antibodies in tears from active VKC patients
and age-matched healthy controls, using a multiplex specific microarray technique
for direct measurement of IgE directed against 103 components derived from 47 allergens.

Keratoconus

Keratoconus (KC) is the most common degenerative corneal disease 104], whereby structural changes in the cornea cause it to thin and bulge. KC is characterised
by a distinctive conical shaping of the cornea and results in diminished vision and
reduced quality of life. Despite numerous efforts, no single biomarker for KC has
been discovered that allows early diagnosis of the condition. Recently, however, Priyadarsini
et al. 105] identified a potentially novel tear fluid marker of KC and termed it gross cystic
disease fluid protein-15 (GCDFP-15) or PIP. Another group demonstrated significant
changes in the following molecules: RANTES/CCL5, MMP-13, NGF and IL-6 in tear fluids
of patients with KC 106]. This group also noted age-dependent associations between IL-13, IL-8/CXCL8, RANTES/CCL5
and MMP-13 and the topographical data. A study by You et al. 107], via ELISA analysis of tear fluids from subjects with KC, showed significantly reduced
levels of the glycoprotein secreted frizzled-related protein 1 (SFRP-1). Moreover,
tear fluid inflammatory protein expression has been assessed in subjects with KC.
Most recently, Shetty et al. 108] noted high levels of MMP-9 and IL-6 in tear fluids of KC patients. This group noted
that MMP-9 levels responded (were reduced) to cyclosporine A therapy, thus indicating
this protein as a potential target in arresting KC progression. While Sorkhabi et
al. 109] identified significantly increased IL-6, IL-1? and IFN-? levels in KC tears than
controls, of note was the fact that this study showed significantly lower levels of
the anti-inflammatory mediator IL-10 in tears from KC patients.

Other research indicates roles for metabolites related to the urea cycle, TCA cycle
and oxidative stress in KC patients, as demonstrated by notable tear fluid changes
in proteins associated with these processes 110]. Further evidence for a role in KC of oxidative stress was also shown via lower levels
of tear film prolidase activity (PA) in a study of KC patients and healthy subjects
111].

Keratopathy

Keratopathy is the term used to refer to any disease or dysfunction of the cornea
and can include bullous, band, climatic droplet and neurotrophic keratopathies. Climatic
droplet keratopathy (CDK) is a degenerative disease of the cornea, which is characterised
by progressive opacity of the cornea’s anterior layers. Proteomics, such as iTRAQ,
have been used in numerous studies to define the protein composition of tears from
patients with this disorder. For example, Lei et al. 112] used 2D nano-LC-nano-ESI-MS/MS analysis to quantify N-linked glycoproteins in tears
from patients with CDK, versus controls. This group found that of the 19 novel N-linked
glycoproteins identified in tears, five were found to have significant changes in
N-glycosylation levels in CDK patients, compared to normal controls 112]. As N-linked glycoproteins are found in body fluids, they are of particular interest
in the field of biomarkers and as potential therapeutic targets. Despite this, very
few studies have undertaken tear fluid analysis for N-linked glycoproteins (reviewed
in 113]), indicating these proteins may be difficult to assess. Other potential tear fluid
biomarkers of CDK include cytokines, MMPs and gelatinases 114], 115]. MMPs have also been indicated in the pathology of another form of keratopathy, diabetic
keratopathy. Interest in this particular type of ocular surface disease is on the
increase, due to the global phenomenon of rapidly rising rates of diabetes. For example,
in a tear study of paediatric patients with type 1 diabetes, researchers reported
significantly elevated levels of MMP-9, TIMP-1 and TIMP-2, as well as of MMP-9/TIMP-1
and MMP-9/TIMP-2 ratios versus controls, using ELISA and zymography 116]. Further, they noted a significant correlation between each of MMP-2, MMP-9 and TIMP-2
with Hba1c levels. The authors suggested that the presence of these proteins indicated
local tissue remodelling and of local keratopathy disease progression, which may serve
as early disease markers. Matsumura et al. 117] investigated the tear fluid levels of MMP-2, MMP-9 and MMP-10 in diabetic patients,
pre and post vitrectomy. Using multiplex analysis, they showed significantly higher
levels of MMP-10 in the diabetic patients who subsequently developed keratopathy post-surgery,
indicating a role for this MMP in mediating post-surgical corneal disorders in diabetes.
Tear fluid biomarkers of other diabetes-related ocular disorders, including diabetic
retinopathy, will be discussed later.

Peripheral ulcerative keratitis

Peripheral ulcerative keratitis (PUK) is a chronic, progressive condition characterised
by a crescent-shaped corneal ulcer with epithelial defects adjacent to the limbus
118]. PUK has been linked with various systemic autoimmune conditions, in particular rheumatoid
arthritis, Wegener granulomatosis, systemic lupus erythematosus and polychondritis
119], 120]. Tear analysis has been carried out on patients with PUK, investigating the concentrations
of MMP-2 and MMP-9. These MMPs have been shown to be elevated in those with PUK 121], 122]. Both of these enzymes are involved in the breakdown of collagen, and in PUK, this
relates to the destruction of the cellular structure in the corneal stroma and subsequent
corneal perforation. These studies have also shown that the levels of MMP-2 and MMP-9
are increased during active PUK and are reduced during disease inactivity, indicating
their involvement in the disease process 121], 122].

Trachoma

Trachoma is an infection that is common in developing countries in Africa, the Middle
East and Asia and is the most common infectious cause of blindness worldwide. The
infectious agent is a bacterium, Chlamydia trachomatis, which via trichiasis (ingrowing eyelashes), results in repeated episodes of corneal
and conjunctival scarring. An estimated 8 million people are visually impaired as
a result of trachoma infection, and a further 84 million suffer from active infection
globally (reviewed in 123]). Trachoma is spread by direct contact with eye, nose and throat secretions from
affected individuals, or via contact with contaminated clothing. Therefore, as a major
cause of preventable blindness worldwide, a reliable test for C. trachomatis is necessary in both controlling and eliminating this infection. Numerous programmes
have been undertaken, with the aim of reducing both the infection and the clinical
signs. There is a long history of detecting immune responses to trachoma via immunoglobulin
(Ig) in tears from patients 124]–127]. Immune responses have been measured via tear IgG and IgA (against cHSP60, CT795
and CPAF fusion proteins) using ELISA 128], who reported significantly higher IgG antibody levels against cHSP60, CPAF and CT795
in the inflammatory cases of trachoma, versus controls. This group suggested that
IgG levels to CPAF may serve as a biomarker for patients at risk of inflammatory trachoma.
More recently Mowafy et al. 129] utilised tear ELISA assays to detect IgG and IgM of patients with trachoma.

Finally, in order to improve the understanding of the pathology underlying trachoma
infection, Satici et al. 130] examined tear fluid cytokine expression. They found a significant correlation between
changes in EGF, TGF-?1 and TNF-? levels and conjunctival scar formation of trachoma
patients, indicating a potential role for these inflammatory mediators in scar progression.

Tear fluid analysis for other ocular disorders

Thyroid-associated orbitopathy

Thyroid-associated orbitopathy (TAO) is an autoimmune disease resulting from thyroid
dysfunction. TAO is characterised by enlarged extraocular muscles and increased fatty
and connective tissue, resulting in protruding eyes, restricted ocular motility and,
in severe cases, visual field loss. In recent years, proteomic analysis of tear fluids
from patients with TAO has been undertaken in order to better determine disease activity
and to stratify patients accordingly. These studies have yielded some interesting
data on potential biomarkers of the condition. For example, using CBA, Ujhelyi et
al. 131] reported significantly increased levels of tear fluid IL-1?, IL-6, IL-13, IL-17A,
IL-18, TNF-? and RANTES/CCL5 in TAO patients versus controls. Of interest, IL-6 was
increased 2.5-fold, suggesting this cytokine may serve as a specific marker of disease
activity in TAO patients. In another tear cytokine study, Cai et al. 132] reported differing levels of the cytokine IL-7, depending on disease activity states,
with the highest IL-7 levels observed in patients with inactive TAO. A later study
of patients with active or inactive TAO was performed by Huang et al. 133], and tear fluids were assessed for the cytokines IL-1?, IL-6, IL-7, IL-17A, IFN-?
and TNF-?. They reported that IL-1? was significantly higher in active TAO than inactive
disease and controls, while IL-6 and IL-17A were significantly higher in both active
and inactive TAO than controls. IL-7 levels were highest overall in inactive TAO among
the three groups. Concentration of TNF-? was significantly higher in both active and
inactive TAO than for controls 133].

Taken together, these studies indicate a vital role for inflammatory mediators in
TAO disease progression and may serve as future diagnostic and/or therapeutic targets
for this condition.

Aniridia

Aniridia is a rare congenital condition (linked to the PAX6 gene on chromosome 11),
whereby the iris is missing or incomplete, and it usually affects both eyes. To date,
very few studies have profiled tear fluid proteins in these patients. Recent work
by Ihnatko et al. 134] used 2D electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS)
to compare tear proteins in aniridia and control subjects. The authors noted seven
differentially expressed proteins in aniridia patients and control subjects, including
?-enolase, peroxiredoxin 6, CST4, gelsolin, apolipoprotein A-1, zinc-?2-glycoprotein
and lactoferrin. Of these, the former five proteins were more highly expressed in
healthy subjects, while the latter two proteins were higher in tears of aniridia patients,
and western blot data showed increased tear vascular endothelial growth factor (VEGF)
levels in those with aniridia 134]. Further, a recent study by Peral et al. 135] sought to ascertain the tear levels of diadenosine polyphosphates (Ap4A and Ap5A),
which have been identified previously as potential dry eye biomarkers, in subjects
with aniridia (who also have a propensity to dry eye). In their study of 15 aniridia
patients and 40 controls, the authors observed increased levels in aniridia patients
(than for controls), which correlated with patient age and corneal disorder progression
135]. Although the research into tear fluid proteomics is still at an early stage in this
patient demographic, further biomarker studies are likely to shed light on this condition
and may potentially identify therapeutic targets in treating ocular surface complications
of this disease.

Glaucoma

Glaucoma is an internal ocular condition that usually affects both eyes in which the
aqueous humour builds up, causing an increase in intra-ocular pressure. Around 10 %
of UK blindness registrations are attributable to glaucoma, and its overall prevalence
is approximately around 2 % of people over 40. Moreover, the prevalence of glaucoma
is higher in people of black African or black Caribbean descent and those with a family
history of glaucoma (reviewed in 136]). Globally, it has been estimated that by 2020, at least 53 million people will be
affected by glaucoma 137] and this disease remains the primary cause of irreversible visual impairment.

The main forms of glaucoma include primary open-angle glaucoma (POAG, the most common
form), primary angle-closure glaucoma, secondary glaucoma and developmental (or congenital)
glaucoma. Due to the devastating effects that untreated glaucoma may have on vision
in the working age population, it is imperative that (for improved PPPM) robust biomarkers
are identified for drug development and measuring disease progression in this condition
138]. Using ELISA, Ghaffariyeh et al. 139] assessed brain-derived neurotrophic factor (BDNF) in the tear fluids of normal-tension
glaucoma (NTG) patients. They found this protein to be substantially reduced in tears
of NTG patients versus healthy controls. Pieragostino et al. 140] examined the tears of patients with medically controlled POAG and pseudoexfoliative
(secondary) glaucoma, using SDS-PAGE and MALDI-TOF MS. This group demonstrated differing
levels of Igs, PIP, lysozyme C, LCN-1 and protein S100, between the two disease subtypes,
suggesting different inflammatory pathways underlying the pathologies. This work was
followed by the same group observing tear fluid proteomics in treatment-naïve POAG
subjects, and they reported up-regulation of 25 proteins in POAG subjects (versus
control), 16 of which were inflammatory response mediators 141]. The authors suggested that as a large component of the tear proteins were directly
related to inflammatory pathways, these may serve as future biomarkers and/or therapeutic
targets of POAG.

In addition, Liu et al. 142] assessed tear fluid MUC5AC by ELISA in 25 POAG patients (versus controls) and reported
a reduction following short-term glaucoma medication. By comparison, Roedl et al.
143] found significantly higher levels of tear fluid homocysteine (Hcy, a homologue of
the amino acid cysteine) in 36 POAG patients versus controls. This group noted that
POAG patients with DED had significantly higher tear fluid levels than POAG patients
without DED, indicating that Hcy may serve as a marker for increased risk of both
POAG and dry eye in glaucoma patients.

Table 1 summarises the putative biomarkers in tears for ocular diseases.

Table 1. Current putative biomarkers in tears for ocular diseases