Acta Biomaterialia 71 (2018) 261–270

Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Full length article

Controlled release of an HDAC inhibitor for reduction of inflammation in

dry eye disease
Michelle L. Ratay a, Stephen C. Balmert a, Ethan J. Bassin c, Steven R. Little a,b,c,d,e,⇑
a
Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, United States
b
Department of Chemical Engineering, University of Pittsburgh, Pittsburgh, PA 15216, United States
c
Department of Immunology, University of Pittsburgh, Pittsburgh, PA 15213, United States
d
Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213, United States
e
Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, United States

a r t i c l e i n f o a b s t r a c t

Article history: Dry eye disease (DED), also known as keratoconjunctivitis sicca, is an ocular surface disease characterized
Received 27 November 2017 by T-cell-mediated inflammation. Current therapeutics, such as immunosuppressive agents, act to sup-
Received in revised form 24 February 2018 press the clinical signs and inflammation. However, long-term usage of these treatments can cause severe
Accepted 1 March 2018
side effects. In this study, we present an alternative therapeutic approach that utilizes a histone deacety-
Available online 9 March 2018
lase inhibitor (HDACi) to regulate transcription of a variety of immunomodulatory genes. Specifically,
HDACi have emerged as a potential anti-inflammatory agent, which can modulate the functions of a sub-
Keywords:
set of suppressive T lymphocytes known as regulatory T cells (Tregs), enhancing FoxP3 acetylation and
HDACi
SAHA
subsequently guarding the transcription factor from proteasomal degradation. Here, a specific HDACi
Microspheres known as SAHA (suberoylanilide hydroxamic acid) was formulated to controllably release in the lacrimal
PLGA gland. Intralacrimal gland injection of PLGA-based SAHA microspheres prevented clinical signs of DED in
Dry eye disease mice with Concanavalin A-induced DED, reduced expression of pro-inflammatory cytokines, and
increased expression of FoxP3 in the lacrimal glands. Murine T cell culture experiments also revealed that
SAHA decreased effector T cell proliferation and enhanced suppressive function of Tregs in co-cultures of
Tregs and effector T cells.

Statement of Significance

In this study, we demonstrate a therapeutic approach that utilizes a histone deactylase inhibitor (HDACi)
to regulate transcription of a variety of immunomodulatory genes. HDACi have emerged as a potential
anti-inflammatory agent, which can modulate the functions of a subset of suppressive T lymphocytes
known as regulatory T cells (Tregs). Here, HDACi microspheres composed of a biocompatible and
biodegradable polymer (poly(lactic-co-glycolic acid) (PLGA)), were able to locally release the HDACi
and prevent clinical signs of DED. This work is timely given the recent shift in treatments of DED towards
immunological based therapies to reduce ocular inflammation. However, notably, many of these treat-
ments require large amounts of drug, and non-specifically suppress the immune system, leading to sev-
eral systemic side effects. Instead of merely suppressing or blocking inflammation, the formulation
described herein intends to balance the microenvironment promoting immunological homeostasis.
This particular drug delivery system may also have broad implications in the field of inflammatory medi-
ated ocular disorders such as uveitis, Sjögren’s syndrome, allergic conjunctivitis.
Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction of the immunological homeostasis of the lacrimal functional unit

Dry eye disease (DED) affects more than 10 million individuals
in the United States [1–5]. The disease originates from disruption
⇑ Corresponding author at: Department of Chemical and Petroleum Engineering,
University of Pittsburgh, Pittsburgh, PA 15261, United States.
E-mail address: srlittle@pitt.edu (S.R. Little).
(LFU) [6–9]. This disruption stems from persistent infiltration of
pro-inflammatory effector CD4+ T lymphocytes (Teff) into the
LFU, and reduced numbers and/or functionally less suppressive
anti-inflammatory regulatory T cells (Tregs) [10,11]. Current ther-
apies are aimed at blocking pro-inflammatory signaling by sup-
pressing T-cell proliferation, or preventing T-cell infiltration

https://doi.org/10.1016/j.actbio.2018.03.002
1742-7061/Ó 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
262 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270
[12,13]. However, in a healthy state, the body utilizes sophisticated
regulatory mechanisms that include Tregs, which play an integral
role in the immune system by suppressing the proliferation of
pathogenic effector T cells and promoting the maintenance of the
immunological microenvironment [14–16]. Due to the vital role
Tregs play in suppressing aberrant inflammation, our group previ-
ously developed a method to bolster the overall pool of Tregs, uti-
lizing a sustained release of a combination of Treg-inducing factors
(two cytokines and a small molecule drug) [17–19]. This therapeu-
tic approach yielded promising results in an inflammatory murine
model of DED, and ultimately shifted the immunological microen-
vironment in the lacrimal gland tissue. Although, clinical signs of
DED were prevented by the combined delivery of these three fac-
tors, treatment with a single small molecule drug, instead of a
combination of biologics and small molecule drug, would drasti-
cally reduce the regulatory hurdles and translational potential of
such a therapy.
Histone deacetylase inhibitors (HDACi) are a family of small
molecule drugs with the ability to shift Treg-Teff balance [20].
HDACi are best known for their ability induce differentiation and
cell cycle arrest in cancer cells [21]. A specific HDACi, known as
Vorinostat, or SAHA (N-hydroxy-N0 -phenyl-octanediamide,
suberoylanilide hydroxamic acid), has been previously approved
by the FDA for treatment of cutaneous T-cell lymphoma (marketed
by Merck as ZolinzaTM) [21]. In addition to use of SAHA as an anti-
cancer therapeutic, this small molecule drug has recently attracted
interest as a potential anti-inflammatory therapeutic [22]. HDACi,
such as SAHA, have also recently been shown to enrich or enhance
local populations of Tregs through several mechanisms [20]. HDACi
are capable of directly acting on Tregs to increase their suppressive
function by promoting FoxP3 acetylation and binding to DNA
[23,24], or by increasing expression of regulatory molecules like
CTLA-4 [22]. SAHA can also increase the number of Tregs through
multiple routes. For instance, HDACi, including SAHA, can cause
conventional T cells to express FoxP3, thereby converting them
into induced Tregs [25]. Additionally, HDACi can also promote
the generation of tolerogenic dendritic cells (DCs), which can then
induce Tregs [22,26]. This effect of HDACi is mediated by increased
acetylation of STAT3, which leads to reduced expression of costim-
ulatory molecules and production of pro-inflammatory cytokines,
as well as increased expression of the immunosuppressive enzyme
indoleamine 2,3-dioxygenase (IDO) [26,27]. Finally, it has also
been suggested that SAHA can inhibit T effector (Teff) proliferation,
independent of Tregs [28].
Given that HDACi can both expand Tregs and enhance their sup-
pressive function, we hypothesized that the controlled release of
SAHA from degradable, polymer-based microspheres injected into
the lacrimal gland would cause local induction of Tregs and prevent
clinical symptoms associated with DED. In order to test this hypoth-
esis, SAHA was formulated into degradable microspheres made
from a polymer with an excellent track record of prior FDA approval
(poly (lactic-co-glycolic) acid). These microspheres are capable of
sustainably releasing SAHA for several days. Local release of SAHA
from these formulations in the lacrimal gland of mice prevent dam-
age to the ocular tissue, enhance FoxP3 mRNA expression in the
lacrimal gland, and reduce the pro-inflammatory microenviron-
ment observed in a model of murine DED.
2. Experimental methods
2.1. Materials and reagents
Suberoylanilide Hydroxamic Acid (SAHA) was acquired from
Selleck Chem (Houston, TX). Poly(lactic-co-glycolic acid) (PLGA;
65:35 lactide:glycolide, acid terminated, MW 7–17 kDa, viscosity
0.16–0.24 dL/g at 0.1% w/v), Concanavalin A (Con A), and Tween80
were purchased from Sigma Aldrich (St. Louis, MO). Poly(vinyl
alcohol) (PVA; MW 25 kDa, 98% hydrolyzed) was purchased from
PolySciences (Warrington, PA). Dichloromethane, methanol, and
neutral buffered formalin were obtained from Fisher Scientific
(Hampton, NH). Phosphate-buffered saline (PBS or ‘‘saline”) with-
out calcium and magnesium from Corning (Corning, NY) was used
for injections and release assays. RPMI-1640 media (Gibco via
Thermo Fisher Scientific, Waltham, MA) and the following supple-
ments were used for cell culture experiments: fetal bovine serum
(FBS; Atlanta Biologicals, Atlanta, GA), non-essential amino acids
(NEAA; Lonza, Walkersville, MD), HEPES buffer (Lonza), L-
glutamine (Gibco), 2-mercaptoethanol (Gibco), sodium pyruvate
(Sigma), and antibiotic-antimycotic solution (Sigma). Fluorescein,
CellTrace CFSE, CellTrace Far Red, Dynabeads Mouse T-Activator
anti-CD3/CD28, and eBioscience Fixable Viability Dye eFluor 780
were obtained from Thermo Fisher. The following fluorescently
labeled antibodies were obtained from BD Biosciences (San Jose,
CA) and used for FACS and flow cytometry: CD4 (RM4-5), CD25
(PC61.5), and CD45RB (16A). Finally, qRT-PCR reagents included
TRI-reagent (Molecular Research Center, Cincinnati, OH), Quanti-
Tect Reverse Transcription Kit (Qiagen, Valencia, CA), VeriQuest
Probe qPCR Mastermix (Affymetrix, Santa Clara, CA), and TaqMan
primer/probes (Thermo Fisher).
2.2. Fabrication of SAHA microspheres
HDACi microspheres were fabricated using a single-emulsion
evaporation technique due to the hydrophobic nature of SAHA.
Specifically, 200 mg of PLGA and 40 mg of SAHA were added to a
mixture of 2.68 ml dichloromethane and 1.32 ml methanol, and
sonicated for 1 h. Subsequently, this emulsion was then mixed
with 60 ml of 2% PVA and homogenized (L4RT-A, Silverson, pro-
cured through Fisher Scientific) at 3000 rpm for 1 min. Subse-
quently, the homogenized mixtures were added to 80 ml of 1%
PVA on a stir plate and left for approximately 1.5 h in order for
the organic solvent to evaporate. After 1.5 h, the microspheres
were centrifuged (200g, 5 min, 4 °C), washed 4 times with deion-
ized water, and lyophilized for 48 h (Virtis Benchtop K freeze dryer,
Gardiner, NY). Microspheres without drug, referred to as ‘‘Blank
MS,” were fabricated similarly, except without the addition of
SAHA.
2.3. Characterization of SAHA microspheres
The morphology of the microspheres were characterized using
scanning electron microscopy (SEM) (JEOL, JSM-6330F, Peabody,
MA) and volume impedance measurements were performed on a
Beckman Coulter Counter (Multisizer-3, Beckman Coulter, Fuller-
ton, CA). In order to determine the release kinetics of the SAHA
microspheres, 10 mg of SAHA MS or Blank MS (unloaded control)
were added to 1 ml of 0.2% Tween 80 in PBS, which was placed
onto a rotator at 37 °C. The supernant was sampled daily and the
release profile was determined using a NanoDrop 2000 Spec-
trophotometer (ThermoFisher Scientific). The wavelength utilized
for this test was 242 nm. A standard curve was completed on the
NanoDrop to determine the corresponding absorbance values and
the range of concentrations detected in this study, which were
approximately 2 ng/ml to 0.375 mg/ml. The samples were diluted
as necessary to be within the detectable range of the standard
curve.
2.4. Mice
Female Balb/c mice aged 6–8 weeks from Charles Rivers Labora-
tories (Wilmington, MA) were utilized for this experimental study.
 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270
All murine experiments were approved by the Institutional Animal
Care and Use Committee, University of Pittsburgh, Pittsburgh, Pa.
2.5. Murine model
To induce inflammation-mediated DED, 20 ll of a 10 mg/ml
solution of ConA in PBS was injected into the left and right lacrimal
glands of mice with the aid of a dissecting microscope (Olympus
SZX10, Waltham, MA) [29]. For microsphere treatments, 10 mg/
ml of Blank MS or SAHA MS were administered together with ConA
in the same total volume of 20 ll. For non-diseased saline controls,
20 ll of PBS was injected into the lacrimal gland, instead of ConA
and/or microspheres. Prior to lacrimal gland injections, mice were
anesthetized by intraperitoneal injection of ketamine (80–100 mg/
kg) and xylazine (5–10 mg/kg) [29].
2.6. Tear production
In order to measure tear production, phenol red cotton threads
(Oasis Medical, San Dimas, CA) were utilized. Specifically, 7 days
after lacrimal gland injections, mice were physically restrained
by hand, and the phenol red thread was placed in the lateral can-
thus of the eye for a period of 60 s. The amount of tears absorbed
onto the thread (when tears are absorbed onto the thread a color
change occurs from yellow to red) was measured using a dissecting
microscope (Olympus SZX10, Waltham, MA) [30,31]. To convert
linear thread wetting measurements (mm) to tear volumes (lL),
a standard curve was generated using known volumes of basic sal-
ine solution (0.9% w/v NaCl + 16.7 mM NaOH, pH  12), as
described in [32]. The following linear regression equation was
obtained from thread wetting measurements of standard volumes
of basic saline solution using Prism GraphPad: Volume (lL) = 0.0
5106 * [thread wetting length (mm)] + 0.04596; R2 = 0.9741, n = 3
independent measurements per volume.
2.7. Corneal fluorescein staining
Seven days after lacrimal gland injections, and after phenol red
thread testing, mice were physically immobilized by hand, and
approximately 1 ml of fluorescein (1% solution in PBS) was applied
to the conjunctival sac. Subsequently, the ocular surface was
imaged using a dissecting microscope with camera, and a masked
ophthalmologist used these images to score staining. Corneal fluo-
rescein was scored 0 for no staining, score 1 for a quarter of stain-
ing, score of 2 for less than a half, score of 3 for half, and 4 for more
than half of the cornea.
2.8. Ocular histology
At the end of the one-week study, mice were euthanized by car-
bon dioxide asphyxiation and secondary cervical dislocation, and
murine eyes were exenterated and immediately fixed in 10% neu-
tral buffered formalin solution for a period of 48 h. Ocular tissue
samples were embedded at the same depth and orientation to
ensure comparable regions of tissue were used for histology. Then,
the paraffin embedded tissue was cut into approximately 5 mm
thick sections and stained with Periodic Acid Schiff (PAS). These
histological sections were scanned and the goblet cell density
was quantified using a Zeiss Axio Scan. Z1 (Thornwood, NY) and
Pannoramic Viewer software (3D HISTECH Ltd.).
2.9. qRT-PCR
Total RNA was extracted from excised lacrimal glands using TRI-
reagent, and quantified using a NanoDrop 2000 (Thermo Scientific).
For the reverse transcriptase assay, 2 lg RNA was converted to cDNA
263
using a QuantiTect Reverse Transcription Kit. Quantitative real-time
PCR was then performed using VeriQuest Probe qPCR Mastermix
and TaqMan primer/probes specific for IFN-c (Mm01168134_m1),
IL-12 (Mm01288989_m1), IL-6 (Mm00446190_ml), FoxP3
(Mm00475162_m1), and Gusb (Mm01197698_m1). Primer/probes
for target genes were FAM-MGB labeled, and that for the endoge-
nous control (GUSB) was VIC-MGB_PL labeled. Duplex reactions
(target gene + GUSB) were run and analyzed on a StepOnePlus
Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Relative
fold changes of IFN-c, IL-12, IL-6, and FoxP3 expression were calcu-
lated and normalized based upon the 2DDCt method [33], using the
Saline group as the untreated control.
2.10. Suppression assay
Lymphocytes were isolated from Balb/c spleens and stained
with antibodies against CD4, CD25, and CD45RB. Flow activated
cell sorting (FACS) was performed (FACSAria, BD Bioscience) to
obtain Teff (CD4+CD25CD45RBhi) and Treg (CD4+CD25+CD45RBlo).
Teff and Treg were labeled with CellTrace CFSE or CellTrace Far
Red, respectively. Teff were cultured in round-bottom 96 well
plates (5  104 cells/well) with Dynabeads Mouse T-Activator
CD3/CD28 stimulation (1 Dynabead: 4 Teff) and 0:1, 1:1, 1:2, or
1:4 ratios of Treg: Teff. T cells were cultured for 72 h in RPMI media
supplemented with 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 1
mM sodium pyruvate, 1x antibiotic–antimycotic, 1x NEAA, and
55 lM 2-mercaptoethanol. Culture media also contained 0, 50, or
200 nM SAHA. T cells were stained with Fixable Viability Dye,
and CFSE dilution was analyzed by flow cytometry.
2.11. Statistical analysis
Data expressed as mean ± S.D. Comparisons between multiple
treatment groups were performed using one-way ANOVA, followed
by Bonferroni correction for multiple comparisons, and p < 0.05 was
considered statistically significant. For PCR data, a Grubb’s test was
performed to identify any significant outliers. If a significant outlier
was found (p < 0.05) it was excluded from the statistical analysis. If
assumptions of ANOVA were not met, a non-parametric Kruskal-
Wallis test, followed by Dunn’s post-hoc analysis, was performed.
All animal experiments in this study were repeated twice. The
microsphere fabrication and characterization was repeated three
times for this study. Statistical tests were performed using GraphPad
Prism Software 6.0 (GraphPad Prism, San Diego, CA).
3. Results
3.1. Characterization of SAHA microspheres
Scanning electron micrographs (SEM) (Fig. 1) illustrate that
individual, polymer particles are spherical with average size distri-
bution average of 17 mm (Blank MS) and an average of 17 mm
(SAHA MS), which was confirmed by utilizing a Coulter Counter
(representative plots of volume impedance measurements shown
below in Fig. 1). Additionally, the release kinetics were character-
ized using a NanoDrop (UV–vis spectrophotometer) through detec-
tion of the absorbance of SAHA, which demonstrates a cumulative
release of approximately 50 ng/mg of microspheres over the course
of 5–6 days (Fig. 2).
3.2. Aqueous tear production with the administration of SAHA
microspheres
To determine whether SAHA MS were capable of preventing
loss of tear production in our model of DED, a standard phenol
264 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270

Fig. 1. Characterization of SAHA Microspheres. (A) Representative scanning electron microscopy (SEM) images (1000X). (B) Volume Impedance Measurements (Coulter
Counter) of Blank Microspheres and SAHA Microspheres.

red threads test was utilized at the experimental endpoint [29].
Compared to non-diseased mice (injected with saline), mice that
received intralacrimal gland injections of ConA, with or without
Blank MS, experienced less tear production, as shown in Fig. 3. This
loss of tear production caused by ConA was prevented by the
administration of SAHA MS (Fig. 3).
3.4. Impact of SAHA microspheres on corneal fluorescein staining
Representative corneal fluorescein images were captured using
a fluorescent dissecting microscope and scored by a masked oph-
thalmologist (Fig. 5A). Compared to the ConA alone and ConA +
Blank MS group, the uptake of fluorescein to the cornea was signif-

3.3. Impact of SAHA microsphere administration on goblet cell density

To determine whether SAHA MS could reduce loss of goblet

cells caused by ConA-induced inflammation, Periodic Acid Schiff
(PAS staining) of the ocular tissue was used to identify mucin-
producing goblet cells. SAHA MS treatment of ConA-induced DED
reduced the loss of goblet cells compared to ConA alone (Fig. 4A),
thereby potentially preserving the goblet cells (pink/purple cells)
located in the conjunctiva. Notably, there was a significant preser-
vation of goblet cell density with the administration of SAHA MS as
compared to the ConA alone group shown in Fig. 4B.

Fig. 3. Tear production in mice with ConA-induced DED is restored by adminis-

Fig. 2. Release kinetics of SAHA from microspheres (n = 3).
tration of SAHA MS. Tear production was measured by phenol red thread test and
converted to tear volumes using a standard curve. Data represent mean ± SD for
saline (non-diseased) (n = 5), ConA (diseased) (n = 5), Blank MS (w/ ConA) (n = 5),
and SAHA MS (w/ ConA) (n = 4) groups. A significant difference between mice with
ConA-induced DED that were treated with Blank MS (vehicle control) and SAHA MS
was identified by one-way ANOVA, followed by Bonferroni post-hoc test (*p < 0.05).
 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270 265

Fig. 4. SAHA MS Preserve Goblet Cell Density in the Conjunctival Epithelial Layer. (A) Quantification of the Number of Goblet Cells per field shown as mean ± S.D. (n = 5 per
group). (B) Representative Histological Images of the goblet cells in the conjunctiva (10X) of Saline (non-diseased), ConA (diseased), Blank MS (diseased + unloaded
microspheres), SAHA MS (diseased + SAHA microspheres). Significant differences were determined by one-way ANOVA, followed by Bonferroni post-hoc tests, and are
indicated by *p < 0.05, ****p < 0.0001.

Fig. 5. Corneal Fluorescein Staining is Reduced with the Administration of SAHA MS. (A) Corneal Fluorescein Staining scored on a scale of (0–4) shown as mean ± S.D. (n = 6).
(B) Representative Corneal Fluorescein Images of Saline (non-diseased), ConA (diseased), Blank MS (w/ ConA), and SAHA MS (w/ ConA). Significant differences were
determined by one-way ANOVA, followed by Bonferroni post-hoc tests, and are indicated by *p < 0.05, **p < 0.01, ***p < 0.001.
266 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270

icantly reduced in the Saline and SAHA MS groups shown in Fig. 5B.
Ultimately, the local administration of SAHA MS to lacrimal gland
demonstrated that the preventative therapy was able to reduce the
corneal fluorescein scores by approximately 4-fold compared to
the ConA alone group.
3.5. mRNA expression altered in the lacrimal gland with SAHA
microspheres
To determine if SAHA MS could reduce production of inflamma-
tory cytokines, mRNA expression levels were examined in the
lacrimal gland tissue after ConA-induced inflammation. Quantita-
tive RT-PCR data suggested that IL-12, IFN-c, and IL-6 pro-
inflammatory cytokines were significantly reduced in the lacrimal
gland of the SAHA MS treated as compared to the ConA (diseased)
group as shown in Fig. 6. In contrast, the level of FoxP3 (transcrip-
tion factor and marker of regulatory T cells) mRNA expression was
significantly higher in the SAHA MS group as compared to the ConA
(diseased) group [34]. Though mice treated with Blank MS had
intermediate levels of FoxP3 expression (greater than ConA alone,
but less than SAHA MS treatment), these differences were not sig-
nificant. Together, this data suggests that the SAHA MS was able to
reduce the pro-inflammatory microenvironment initiated by ConA
and enhance the expression levels of a Treg-associated transcrip-
tion factor.
3.6. Lymphocyte suppression assay
Various mechanisms by which SAHA can influence the Treg-Teff
balance have been proposed including direct suppression of Teff
proliferation and enhancement of Treg function [20,22,28,35,36].
In order to gain insight into the mechanism of inflammation reduc-
tion in our model of murine DED, CFSE labeled Teff alone or CFSE
labeled Teff co-cultured with Tregs were treated with SAHA and
proliferation was assessed by CFSE dilution. It was observed that
Teff alone, treated with 200 nM SAHA, exhibited significantly
reduced proliferation compared to Teff alone that were not treated
with SAHA (Fig. 7A). Teff co-cultured with Tregs exhibited less pro-
liferation, and this was further reduced in a dose-dependent man-
ner by the addition of SAHA as shown in Fig. 7B. Although 200 nM
SAHA inhibited the proliferation of Teff alone by 18% and a 1:1
Teff: Treg ratio (SAHA not treated) inhibited proliferation by 27%,
the combination of 200 nM SAHA and a 1:1 Treg: Teff ratio inhib-
ited T cell proliferation by 66%. Overall, the impact of SAHA alone
on solely T effector cells appears to be smaller compared to the
co-culture of Tregs and Teff cells.
4. Discussion
Recently, immunosuppressive agents such as corticosteroids
have been investigated as a therapeutic to reduce inflammation
associated with DED [37,38]. However, long-term topical use of
corticosteroids has been implicated in conditions such as glaucoma
and retinopathy [39]. As an alternative to non-specific corticos-
teroids and associated side effects, a histone deactylase inhibitor
(HDACi) was utilized to alter the adaptive immune response to
restore immunological homeostasis. Specifically, the HDACi, SAHA,
was selected due to its ability to cause epigenetic modifications
that regulate gene expression and protein function to modulate
the function of immune cells such as T lymphocytes [21,22]. In par-
ticular, SAHA and other HDACi can stimulate thymic production of
anti-inflammatory Tregs, enhance Treg suppressive function, and
promote the peripheral conversion of CD4+ naïve T cells into Tregs
[22,36]. Although, HDACi have shown promising immunomodula-
tory effects on T lymphocytes in vitro and in pre-clinical inflamma-
tory models at a dose of 0.1 mg/kg/d [20], this class of small

Fig. 6. mRNA Expression is altered in the lacrimal gland tissue with the administration of SAHA MS, shown as mean ± S.D. (n  5 mice per group). Relative fold changes of
IFN-c, IL-12, IL-6, and FoxP3 mRNA expression were calculated and normalized based upon the 2DDCt method, with the Saline group as the untreated control. Significant
differences were determined by one-way ANOVA, followed by Bonferroni post-hoc test, or Kruskal-Wallis test, followed by Dunn’s post-hoc test (for IL-6), and are indicated
by *p < 0.05; **p < 0.01.
 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270 267

Fig. 7. Lymphocyte Suppression Assay. (A) Representative Flow Cytometry Histograms of T effector cells only untreated (no SAHA) and cells treated with 50 nM and 200 nM
SAHA and bar graphs shown as mean ± S.D. (B) Representative Flow Cytometry Histograms of 1:1 Treg: T effector cells untreated (no SAHA) and cells treated with 50 nM and
200 nM SAHA and bar graphs shown as mean ± S.D. Significant differences were determined by one-way ANOVA, followed by Bonferroni post-hoc test, and are indicated by
*
p < 0.05; **p < 0.01; ****p < 0.0001.

molecules possesses a narrow therapeutic window (both efficacy
and toxicity) [40], which limit their broader applicability. The abil-
ity to locally and sustainably deliver the HDACi would in theory
permit a much lower drug dosage, reducing toxicity concerns, as
well as permit reduced frequency of injections. Therefore, in this
study a controlled release system was fabricated to provide local
administration of SAHA to the lacrimal gland in an inflammatory
murine model of DED.
SAHA releasing microspheres (MS) were fabricated using the
biodegradable and biocompatible polymer poly (lactic-co-
glycolic) acid (PLGA) (Fig. 1), and showed the ability to release
SAHA in vitro over the course of approximately five days (as shown
in Fig. 2) with the intention of altering the local microenvironment
in the ocular tissue [17,41]. Notably, the data suggest that local
administration of SAHA MS in the ocular tissue prevented several
clinical signs of DED. For instance, a common clinical sign of DED
is a reduction of overall tear production, which subsequently can
lead to ocular dryness and irritation [42]. While aqueous tear
secretion was significantly reduced in diseased (ConA ± Blank
MS), as expected [29], SAHA MS treatment prevented ConA-
induced loss of aqueous tear secretion (Fig. 3). In addition to pre-
serving aqueous tear production, effective therapeutics should also
maintain the composition of tears by protecting gel-forming,
mucin-producing goblet cells [2]. These goblet cells are located
on the apical surface of the conjunctiva, within the stratified
columnar conjunctival epithelial cells, and produce important
non-aqueous components of tears, such as mucin [43,44,45].
Mucin 5AC (MUC5AC), a glycoprotein produced by goblet cells, acts
as a gel layer to trap pollen and allergens [44,46]. Depletion of gob-
let cells due to chronic inflammation in conditions, such as DED,
can ultimately lead to conjunctival epithelial squamous metaplasia
and abnormal tear film [46]. Interestingly, loss of goblet cells in
ocular tissue in DED is reportedly due to an increase in the pro-
inflammatory milieu [44]. Thus, in this study, histological sections
of the conjunctiva were examined to determine whether SAHA MS
treatment preserved mucin-producing goblet cells. Indeed, there
was a significantly greater goblet cell density in SAHA MS-
treated mice, compared to diseased (ConA) mice (Fig. 4). Although,
the preservation of goblet cell density was not as profound as other
measurements (ex: corneal fluorescein staining) there was a statis-
tically significant preservation of goblet cell numbers in the SAHA
MS as compared to the ConA (diseased) group as can be seen in
Fig. 4. These results are in agreement with a previous report by
DeZoeten et al., which demonstrated that administration of an
HDACi from the same class as SAHA, led to preservation of intesti-
nal goblet cells in an inflammatory colitis model [47].
Since loss of aqueous tear production and/or mucin-producing
goblet cells in DED ultimately leads to damage and increased per-
meability of the ocular surface [48,49–51], we investigated
whether SAHA MS, which prevented both of the former patholog-
ical features, also maintained integrity of the corneal epithelium.
Corneal integrity was assessed with fluorescein [12,48], a dye that
stains dead epithelial cells and can diffuse into areas where cellular
tight junctions are compromised [52]. Corneal fluorescein staining
is a standard diagnostic indicator of ocular surface damage and
measurement for DED severity [48]. Fluorescein staining was
observed in mice with DED induced by ConA (with or without
Blank MS), as shown in Fig. 5. Notably, there was a 4-fold reduction
in the average fluorescein staining score in the SAHA MS group,
compared to ConA alone, suggesting that the HDACi was able to
prevent damage to the ocular tissue initiated by ConA.
In order to demonstrate that the prevention of clinical signs of
DED was attributed to changes in the underlying T cell mediated
immune response, the lacrimal gland microenvironment was eval-
268 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270
uated using PCR analysis. The underlying pathogenesis of DED
involves effector T cells that infiltrate the ocular tissue and secrete
pro-inflammatory cytokines, which can directly affect the health of
the ocular microenvironment [9,53–55]. Since increases in pro-
inflammatory cytokines have been found in inflamed lacrimal
gland tissues of mice, the microenvironment of the tissue was
investigated to determine whether the SAHA MS-mediated preven-
tion of clinical signs of DED was due to a reduced pro-
inflammatory milieu in the ocular tissue [56–58]. Specifically,
mRNA expression levels of pro-inflammatory cytokines (IL-12,
IFN-c, and IL-6) in lacrimal gland tissue were evaluated by qRT-
PCR. Expression of IL-12 and IFN-c was induced by ConA injection,
relative to saline controls, as shown in Fig. 6. This is likely attribu-
ted to ConA acting as a T-cell mitogen, ultimately leading to the
production of Th1 cytokines [59,60]. Both of these findings in the
ConA-induced murine model of DED are consistent with clinical
reports that IL-12 and IFN-c are upregulated in tears of patients
with DED [42,43,56]. In addition to increases in IL-12 and IFN-c
in the lacrimal gland, IL-6 was also significantly elevated by ConA,
which is consistent with other reports of increased IL-6 production
in lacrimal glands of DED murine models [61]. Importantly, treat-
ment with SAHA MS significantly inhibited ConA-induced expres-
sion of IL-12, IFN-c, and IL-6 (Fig. 6). These reductions in the
expression levels of pro-inflammatory cytokines prompted the
question of whether SAHA MS were altering the immunological
microenvironment by increasing anti-inflammatory Tregs. Previ-
ous studies have demonstrated that HDACi can expand FoxP3+
Tregs in vitro and in vivo [20,28]. Interestingly, we observed a sig-
nificant increase in FoxP3 mRNA expression in the lacrimal gland
tissue of SAHA MS-treated mice (Fig. 6), suggesting that the reduc-
tion of signs of DED and pro-inflammatory microenvironment in
the lacrimal gland may be due to Tregs suppressing ocular inflam-
mation. Future studies will be needed to determine whether
increased FoxP3 expression in lacrimal glands of SAHA MS-
treated mice is due to an increase in the number of Tregs in the tis-
sue, or enhanced expression of FoxP3 by Tregs, which is associated
with greater suppressive function [62].
In vitro experiments suggest, however, that SAHA may enhance
Treg function in addition to directly inhibiting Teff (Fig. 7). Notably,
neither Treg induction, nor increased expression of FoxP3 by Tregs
was observed with SAHA treatment in vitro in our hands (data not
shown). It is possible that this discrepancy between in vitro and
in vivo results regarding FoxP3 expression is due to the fact that
increased FoxP3 mRNA in the lacrimal gland may not necessarily
correspond to increases in FoxP3 protein, as was measured
in vitro experiments. Alternatively, enhanced FoxP3 expression
in vivo could be due to the effects of dendritic cells (DCs) and other
antigen-presenting cells on T cells in vivo [22]. In addition to direct
effects on T cells, SAHA has also been shown to cause DCs to reduce
costimulatory molecule expression and increase IDO production,
both of which can promote Treg induction [26]. Due to the ubiqui-
tous nature of histone acetylation and the dynamic nature of epi-
genetic regulation, it is not surprising that HDACi shift the Teff-
Treg balance through multiple direct and indirect mechanisms.
The results presented here are consistent with several previous
reports of SAHA directly inhibiting Teff proliferation and enhancing
the suppressive function of Tregs [22,25,28,36].
In summary, administration of SAHA MS preserved aqueous
tear production (Fig. 3), reduced inflammation-induced loss of
mucin-producing goblet cells (Fig. 4), and prevented damage to
ocular tissue (Fig. 5), likely by reducing the pro-inflammatory
milieu in the lacrimal gland (Fig. 6) and potentially by enhancing
numbers and/or suppressive function of FoxP3+ Tregs (Figs. 6–7).
While the ConA-induced DED model in these studies is frequently
used as an acute model of inflammation-mediated DED for pre-
clinical assessment of preventative therapies [18,29,30,63–65], it
may fail to capture some of the multifactorial pathophysiology of
chronic and/or autoimmune forms of DED. Therefore, future stud-
ies in chronic DED models (e.g. induction by chronic desiccating
stress) will be needed to determine whether restoring tear and
mucin production (e.g. by resolving lacrimal gland inflammation
with SAHA MS) could also help to reduce ocular inflammation
mediated by immune cells in the conjunctiva [9,66,67]. In such
chronic models, use of SAHA MS with longer release kinetics, or
novel delivery systems that could provide sustained topical deliv-
ery of SAHA to the conjunctiva (as in [68]), rather than lacrimal
gland, could be investigated. Chronic DED models would also allow
us to evaluate treatment of ongoing DED and to study the kinetics
of disease progression and resolution following treatment with
SAHA MS (or soluble SAHA). Additional clinical tests for DED (e.g.
tear osmolarity and evaporation rate) could also provide useful
information regarding the status of the ocular surface, and could
be performed throughout the course of disease progression. In
the future, large animal pre-clinical rabbit models would also be
needed to move toward potential clinical translation and to better
test local toxicity of SAHA MS and/or topical SAHA sustained deliv-
ery formulations in various compartments of the eye. Additionally,
by ameliorating inflammation in the lacrimal glands, SAHA MS
could also potentially be used to treat Sjögren’s syndrome, an
autoimmune disease targeting the lacrimal and salivary glands
that causes a severe form of DED and dry mouth. Accordingly,
future studies using SAHA MS in murine models of Sjögren’s syn-
drome would be of interest [69,70]. Such studies would also pro-
vide additional insight into the various types of DED (e.g.
evaporative, aqueous deficient, Sjögren’s, non-Sjögren’s) for which
this therapeutic approach may be more or less effective. Ulti-
mately, the controlled delivery of SAHA may also have pharmaco-
logical benefits for other inflammation-mediated conditions (based
on previous literature reports) [23,25,28,71] by locally inducing
FoxP3+ Tregs and suppressing the pro-inflammatory milieu.
Acknowledgements
This work was supported in part by the National Institutes of
Health (NIH) CORE Grant P30 EY008098, the University of Pitts-
burgh, Pittsburgh, PA; an unrestricted grant from Research to Pre-
vent Blindness, NY. Research reported in this publication was
supported by the National Center for Advancing Translational
Sciences of the National Institutes of Health under Award Number
TL1R001858. The content is solely the responsibility of the authors
and does not necessarily represent the official view of the National
Institutes of Health. We would also like to acknowledge Dr. Julia K.
Polat for scoring the corneal fluorescein images and Katherine A.
Davoil for assisting with ocular histology. Finally, thanks to the
Department of Dermatology at the University of Pittsburgh for
use of their facilities and equipment for RNA isolation and qRT-
PCR.
References
[1] R.D. William Stevenson, Sunil K. Chauhan, Dry Eye Disease, Arch. Ophthamol.
130 (2012) 90–100..
[2] M. Rolando, M. Zierhut, The ocular surface and tear film and their dysfunction
in dry eye disease, Surv. Ophthalmol. 45 (Suppl 2) (2001) S203–S210.
[3] F. Brewitt, H. Sistani, Dry eye disease: the scale of the problem, Surv.
Ophthalmol. 45 (2001) 199–202.
[4] J.L. Gayton, Etiology, prevalence, and treatment of dry eye disease, Clin.
Ophthalmol. 3 (2009) 405–412.
[5] J. Yu, C.V. Asche, C.J. Fairchild, The economic burden of dry eye disease in the
United States: a decision tree analysis, Cornea. 30 (2011) 379–387.
[6] M.E. Stern, J. Gao, K.F. Siemasko, R.W. Beuerman, S.C. Pflugfelder, The role of
the lacrimal functional unit in the pathophysiology of dry eye, Exp. Eye Res. 78
(2004) 409–416.
 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270
[7] S.K. Chauhan, J. El Annan, T. Ecoiffier, S. Goyal, Q. Zhang, D.R. Saban, R. Dana,
Autoimmunity in dry eye is due to resistance of Th17 to Treg suppression, J.
Immunol. 182 (2009) 1247–1252.
[8] J.Y. Niederkorn, M.E. Stern, S.C. Pflugfelder, C.S. De Paiva, R.M. Corrales, J. Gao,
K. Siemasko, Desiccating stress induces T cell-mediated Sjogren ’ s Syndrome-
like lacrimal keratoconjunctivitis, J. Immunol. 176 (2006) 3950–3957.
[9] Y. Chen, S.K. Chauhan, H.S. Lee, D.R. Saban, R. Dana, Chronic dry eye disease is
principally mediated by effector memory Th17 cells, Mucosal Immunol. 7
(2014) 38–45.
[10] M. Miyara, S. Sakaguchi, Natural regulatory T cells: mechanisms of
suppression, Trends Mol. Med. 13 (2007) 108–116.
[11] J. Stein-Streilein, A.W. Taylor, An eye’s view of T regulatory cells, J. Leukoc. Biol.
81 (2007) 593–598.
[12] S. Goyal, S.K. Chauhan, Q. Zhang, R. Dana, Amelioration of murine dry eye
disease by topical antagonist to chemokine receptor 2, Arch. Ophthalmol. 127
(2009) 882–887.
[13] C.A. Dinarello, J.W.M. van der Meer, Treating inflammation by blocking
interleukin-1 in humans, Semin. Immunol. 25 (2013) 469–484.
[14] M. Yadav, S. Stephan, J.A. Bluestone, Peripherally induced Tregs-role in
immune homeostasis and autoimmunity, Front. Immunol. 4 (2013) 1–12.
[15] A. Schmidt, N. Oberle, P.H. Krammer, Molecular mechanisms of Treg-mediated
cell suppression, Front. Immunol. 3 (2012) 1–20.
[16] B.D. Singer, L.S. King, F.R. D’Alessio, Regulatory T cells as immunotherapy,
Front. Immunol. 5 (2014) 1–10.
[17] S. Jhunjhunwala, S.C. Balmert, G. Raimondi, E. Dons, E.E. Nichols, A.W. Thomson,
S.R. Little, Controlled release formulations of IL-2, TGF-b1 and rapamycin for the
induction of regulatory T cells, J. Control. Release. 159 (2012) 78–84.
[18] M.L. Ratay, S.C. Balmert, A.P. Acharya, A.C. Greene, T. Meyyappan, S.R. Little,
TRI Microspheres prevent key signs of dry eye disease in a murine,
inflammatory model, Sci. Rep. 7 (2017) 17527.
[19] S.C. Balmert, C. Donahue, J.R. Vu, G. Erdos, L.D. Falo, S.R. Little, In vivo induction
of regulatory T cells promotes allergen tolerance and suppresses allergic
contact dermatitis, J. Control. Release. 261 (2017) 223–233.
[20] R. Tao, E.F. De Zoeten, E. Ozkaynak, C. Chen, L. Wang, P.M. Porrett, B. Li, L.A.
Turka, E.N. Olson, M.I. Greene, A.D. Wells, W.W. Hancock, Deacetylase
inhibition promotes the generation and function of regulatory T cells, Nat.
Med. 13 (2007) 1299–1307.
[21] K. Ververis, A. Hiong, T.C. Karagiannis, P.V. Licciardi, Histone deacetylase
inhibitors (HDACIS): Multitargeted anticancer agents, Biol. Targets Ther. 7
(2013) 47–60.
[22] T. Akimova, U.H. Beier, Y. Liu, L. Wang, W.W. Hancock, Histone/protein
deacetylases and T-cell immune responses, Blood 119 (2012) 2443–2451.
[23] C. Chen, L. Wang, P.M. Porrett, B. Li, R. Tao, E.F. De Zoeten, O. Engin, L.A. Turka,
E.N. Olson, M.I. Greene, A.D. Wells, W.W. Hancock, Deacetylase inhibition
promotes the generation and function of regulatory T cells, Nat. Med. 13
(2007) 1299–1307.
[24] B. Li, A. Samanta, X. Song, K.T. Iacono, K. Bembas, R. Tao, S. Basu, J.L. Riley, W.W.
Hancock, Y. Shen, S.J. Saouaf, M.I. Greene, H.T. N-terminal, FOXP3 interactions
with histone acetyltransferase and class II histone deacetylases are required
for repression, Proc. Natl. Acad. Sci. USA 104 (2007) 4571–4576.
[25] J.L. Lucas, P. Mirshahpanah, E. Haas-Stapleton, K. Asadullah, T.M. Zollner, R.P.
Numerof, Induction of Foxp3+ regulatory T cells with histone deacetylase
inhibitors, Cell. Immunol. 257 (2009) 97–104.
[26] J. Frikeche, Z. Peric, E. Brissot, M. Grégoire, B. Gaugler, M. Mohty, Impact of
HDAC inhibitors on dendritic cell functions, Exp. Hematol. 40 (2012) 783–791.
[27] Y. Sun, Y.E. Chin, E. Weisiger, C. Malter, I. Tawara, T. Toubai, E. Gatza, P.
Mascagni, C.A. Dinarello, P. Reddy, Cutting edge: negative regulation of
dendritic cells through acetylation of the nonhistone protein STAT-3 1, J.
Immunol. 182 (2009) 5899–5903.
[28] J. Johnson, A. Pahuja, M. Graham, B. Hering, W.W. Hancock, P. Bansal-Pakala,
Effects of histone deacetylase inhibitor SAHA on effector and FOXP3
+Regulatory T cells in Rhesus macaques, Transplant. Proc. 40 (2008) 459–461.
[29] M.L. Ratay, A.J. Glowacki, S.C. Balmert, A.P. Acharya, J. Polat, L.P. Andrews, M.V.
Fedorchak, J.S. Schuman, D.A.A. Vignali, S.R. Little, Treg-recruiting
microspheres prevent inflammation in a murine model of dry eye disease, J.
Control. Release. 258 (2017) 208–217.
[30] M.J. Lee, D.H. Kim, J.S. Ryu, A.Y. Ko, J.H. Ko, M.K. Kim, W.R. Wee, S.I. Khwarg, J.Y.
Oh, Topical TSG-6 administration protects the ocular surface in two mouse
models of inflammation-related dry eye, Investig. Ophthalmol. Vis. Sci. 56
(2015) 5175–5181, https://doi.org/10.1167/iovs.14-16307.
[31] D. Dursun, M. Wang, D. Monroy, D.-Q. Li, B.L. Lokeshwar, M.E. Stern, S.C.
Pflugfelder, A mouse model of keratoconjunctivitis sicca, Invest. Ophthalmol.
Vis. Sci. 43 (2002) 632–638.
[32] P. Stewart, Z. Chen, W. Farley, L. Olmos, S.C. Pflugfelder, Effect of experimental
dry eye on tear sodium concentration in the mouse, Eye Contact Lens. 31
(2005) 175–178.
[33] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using real-
time quantitative PCR and the 2-DDCT method, Methods 25 (2001) 402–408.
[34] Q. Tang, J.A. Bluestone, The Foxp3+ regulatory T cell: a jack of all trades, master
of regulation, Nat. Immunol. 9 (2008) 239–244.
[35] F. Blanchard, C. Chipoy, Histone deacetylase inhibitors: new drugs for the
treatment of inflammatory diseases?, Drug Discov Today. 10 (2005) 197–204.
Pii s1359-.
[36] T. Akimova, G. Ge, T. Golovina, T. Mikheeva, L. Wang, J.L. Riley, W.W. Hancock,
Histone/protein deacetylase inhibitors increase suppressive functions of
human FOXP3+ Tregs, Clin. Immunol. 136 (2010) 348–363.
269
[37] C.S. De Paiva, R.M. Corrales, A.L. Villarreal, W.J. Farley, D.Q. Li, M.E. Stern, S.C.
Pflugfelder, Corticosteroid and doxycycline suppress MMP-9 and
inflammatory cytokine expression, MAPK activation in the corneal
epithelium in experimental dry eye, Exp. Eye Res. 83 (2006) 526–535,
https://doi.org/10.1016/j.exer.2006.02.004.
[38] H.D. Perry, Evaluation of topical cyclosporine for the treatment of dry eye
disease, Arch. Ophthalmol. 126 (2008) 1046.
[39] J.P. Kersey, D.C. Broadway, Corticosteroid-induced glaucoma: a review of the
literature, Eye 20 (2006) 407–416.
[40] J.R. Whittle, J. Desai, Histone deacetylase inhibitors in cancer: What have we
learned?, Cancer 121 (2015) 1164–1167
[41] X. Rong, W. Yuan, Y. Lu, X. Mo, Safety evaluation of poly(lactic-co-glycolic
acid)/poly(lactic-acid) microspheres through intravitreal injection in rabbits,
Int. J. Nanomed. 9 (2014) 3057–3068.
[42] J.M. Tiffany, Tears in health and disease, Eye (Lond). 17 (2003) 923–926.
[43] T.G. Coursey, J.T. Henriksson, F.L. Barbosa, C.S. de Paiva, S.C. Pflugfelder,
Interferon-c–induced unfolded protein response in conjunctival goblet cells as
a cause of mucin deficiency in Sjögren Syndrome, Am. J. Pathol. 186 (2016) 1–
12.
[44] Z. Zhang, W.Z. Yang, Z.Z. Zhu, Q.Q. Hu, Y.F. Chen, H. He, Y.X. Chen, Z.G. Liu,
Therapeutic effects of topical doxycycline in a benzalkonium chloride-induced
mouse dry eye model, Investig. Ophthalmol. Vis. Sci. 55 (2014) 2963–2974.
[45] C. Baudouin, P. Aragona, E.M. Messmer, A. Tomlinson, M. Calonge, K.G.
Boboridis, Y.A. Akova, G. Geerling, M. Labetoulle, M. Rolando, Role of
hyperosmolarity in the pathogenesis and management of dry eye disease:
Proceedings of the ocean group meeting, Ocul. Surf. 11 (2013) 246–258.
[46] Vamsi K. Gangaraju, Lin Haifan, Conjunctival epithelial and goblet cell function
in chronic inflammation and ocular allergic inflammation, Curr. Opin. Clin.
Immunol. 14 (2014) 467–470.
[47] Edwin F. De Zoeten, Liqing Wang, Hong Sai, Wolfgang H. Dillmann, W.W.
Hancock, Inhibition of HDAC9 increases T regulatory cell function and
prevents colitis in mice, Gastroenterology 138 (2010) 583–594.
[48] Z. Lin, X. Liu, T. Zhou, Y. Wang, L. Bai, H. He, Z. Liu, A mouse dry eye model
induced by topical administration of benzalkonium chloride, Mol. Vis. 17
(2011) 257–264.
[49] E. Badaro, E.A. Novais, F.M. Penha, M. Maia, M.E. Farah, E.B. Rodrigues, S. Paulo,
Vital dyes in ophthalmology: a chemical perspective, Curr. Eye Res. (2014) 1–
10.
[50] D. Zoukhri, A. Fix, J. Alroy, C.L. Kublin, Mechanisms of murine lacrimal gland
repair after experimentally induced inflammation, Investig. Ophthalmol. Vis.
Sci. 49 (2008) 4399–4406.
[51] J.D. Nelson, H. Helms, R. Fiscella, Y. Southwell, J.D. Hirsch, A new look at dry
eye disease and its treatment, Adv. Ther. 17 (2000) 84–93.
[52] A.J. Bron, P. Argueso, M. Irkec, F.V. Bright, Clinical staining of the ocular surface:
Mechanisms and interpretations, Prog. Retin. Eye Res. 44 (2015) 36–61.
[53] J. El Annan, S.K. Chauhan, T. Ecoiffier, Q. Zhang, D.R. Saban, R. Dana,
Characterization of effector T cells in dry eye disease, Invest. Ophthalmol.
Vis. Sci. 50 (2009) 3802–3807.
[54] T.G. Coursey, N.B. Gandhi, E.A. Volpe, S.C. Pflugfelder, C.S. De Paiva, Chemokine
receptors CCR6 and CXCR3 are necessary for CD4+ T cell mediated ocular
surface disease in experimental dry eye disease, PLoS One 8 (2013).
[55] M.E. Stern, C.S. Schaumburg, S.C. Pflugfelder, Dry eye as a mucosal
autoimmune disease, Int. Rev. Immunol. 32 (2013) 19–41.
[56] M.L. Massingale, X. Li, M. Vallabhajosyula, D. Chen, Y. Wei, P.A. Asbell, Analysis
of inflammatory cytokines in the tears of dry eye patients, Cornea 28 (2009)
1023–1027.
[57] K.S. Na, J.W. Mok, J.Y. Kim, C.R. Rho, C.K. Joo, Correlations between tear
cytokines, chemokines, and soluble receptors and clinical severity of dry eye
disease, Investig. Ophthalmol. Vis. Sci. 53 (2012) 5443–5450.
[58] S.C. Pflugfelder, R.M. Corrales, C.S. de Paiva, T helper cytokines in dry eye
disease, Exp. Eye Res. 117 (2013) 118–125.
[59] R. Palacios, Concanavalin A triggers T lymphocytes by directly interacting with
their receptors for activation, J. Immunol. 128 (1982) 337–342.
[60] F. Gantner, M. Leist, A.W. Lohse, P.G. Germann, G. Tiegs, Concanavalin A-
induced T-cell-mediated hepatic injury in mice: the role of tumor necrosis
factor, Hepatology 21 (1995) 190–198.
[61] X. Xiao, X. Shi, Y. Fan, X. Zhang, M. Wu, P. Lan, L. Minze, Y.-X. Fu, R.M. Ghobrial,
W. Liu, X.C. Li, GITR subverts Foxp3(+) Tregs to boost Th9 immunity through
regulation of histone acetylation, Nat. Commun. 6 (2015) 8266.
[62] S.K. Chauhan, D.R. Saban, H.K. Lee, R. Dana, Levels of Foxp3 in regulatory T cells
reflect their functional status in transplantation, J. Immunol. 182 (2009) 148–
153.
[63] M.J. Seo, J.M. Kim, M.J. Lee, Y.S. Sohn, K.K. Kang, M. Yoo, The therapeutic effect
of DA-6034 on ocular inflammation via suppression of MMP-9 and
inflammatory cytokines and activation of the MAPK signaling pathway in an
experimental dry eye model, Curr. Eye Res. 35 (2010) 165–175.
[64] M.J. Lee, A.Y. Ko, J.H. Ko, H.J. Lee, M.K. Kim, W.R. Wee, S.I. Khwarg, J.Y. Oh,
Mesenchymal stem/stromal cells protect the ocular surface by suppressing
inflammation in an experimental dry eye, Mol. Ther. (2014) 1–8.
[65] M.T.T.J. Nagelhout, D.A. Gamache, L. Roberts, J.M. Yanni Brady, Preservation of
Tear Film Integrity and Inhibition of Corneal Injury by Dexamethasone in a
Rabbity Model of Lacrimal Gland Inflammation -Induced Dry Eye, J. Ocul.
Pharmacol. Ther. 21 (2005).
[66] C.S.D.P. Zhang, E.Al. Xiaobo, Topical interferon-gamma neutralization prevents
conjunctival goblet cell loss in experimental murine dry eye, Exp. Eye Res. 118
(2014) 117–124.
270 M.L. Ratay et al. / Acta Biomaterialia 71 (2018) 261–270

[67] V.L. Perez, S.C. Pflugfelder, S. Zhang, A. Shojaei, R. Haque, Lifitegrast, a novel
integrin antagonist for treatment of dry eye disease, Ocul. Surf. 14 (2016) 207–
215.
[68] M.V. Fedorchak, I.P. Conner, J.S. Schuman, A. Cugini, S.R. Little, Long term
glaucoma drug delivery using a topically retained gel/microsphere eye drop,
Sci. Rep. 7 (2017) 8639.
[69] Y. Park, A.E. Gauna, S. Cha, Y. Park, Mouse models of primary Sjogren ’ s
Syndrome, Curr. Pharm. 21 (2015) 2350–2364.
[70] T.N. Lavoie, B.H. Lee, C.Q. Nguyen, Current concepts: mouse models of
Sjögren’s syndrome, J. Biomed. Biotechnol. 2011 (2011) 1–14.
[71] L. Wang, E.F. de Zoeten, M.I. Greene, W.W. Hancock, Immunomodulatory
effects of deacetylase inhibitors: therapeutic targeting of FOXP3+ regulatory T
cells, Nat. Rev. Drug Discov. 8 (2009) 969–981.