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\begin{document}

\title{Altered Sphingolipid metabolism in Vernal Keratoconjunctivitis: Newer
Insights}



\author[1]{Menta Vignesh}%
\author[2]{Shweta Agarwal}%
\author[3]{Ujjwalkumar Das }%
\author[3]{Lakshmi Moksha}%
\author[1]{Gurumurthy Srividya}%
\author[2]{Amrutha Mahalakshmi Anandan}%
\author[2]{Bhaskar Srinivasan}%
\author[2]{Geetha Iyer}%
\author[3]{Thirumurthy Velpandian}%
\author[1]{Narayanasamy Angayarkanni}%
\affil[1]{Vision Research Foundation}%
\affil[2]{Medical Research Foundation}%
\affil[3]{All India Institute of Medical Sciences}%


\vspace{-1em}



  
  \date{May 28, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: The etiopathogenesis of vernal keratoconjunctivitis (VKC),
is incompletely understood. Bioactive lipids play a key role in the
allergic disorders and there are no studies exploring it in the ocular
surface allergic disorder such as VKC. This study focused on the
sphingolipid metabolism in VKC and explored the association of ocular
surface sphingolipids with the refractory nature of the disease.
Methods: Active VKC cases and age-matched healthy controls were
recruited as part of a one-year prospective study at our tertiary eye
care centre in South India. Imprint cytology was used to assess gene
expression of enzymes of sphingolipids metabolism in conjunctival cells.
Sphingolipids levels were estimated in the tears by LC-MS/MS analysis.
Systemic levels of Sphingosine-1-phosphate (S1P) and
Ceramide-1-Phosphate and IgE were estimated by ELISA. IgE induced
regulation of S1P receptor gene expression was assessed by western blot
analysis of histone deacetylases in cultured mast cells. Results:
Significantly altered gene expression of the sphingolipids enzymes was
noted in VKC in conjunctival cells. Pooled tears showed significantly
lowered levels of S1P (d17:1), S1P (d17:0) and S1P (d20:1) in VKC.
Additionally, Cer (d18:/17:0) was significantly lowered in R-VKC
compared to NR-VKC. Lowered C1P and raised IgE were observed at serum
level in VKC. Increased S1P receptors and histone deacetylases
expression indicate regulation by histone modification as evaluated in
IgE induced mast cells. Conclusion: Altered sphingolipid metabolism is
associated with VKC pathogenesis and is differential in refractory cases
that showed lower ceramide and specific sphingosines compared to
non-refractory VKC.%
\end{abstract}%



\sloppy


\textbf{Introduction:}

Vernal keratoconjunctivitis (VKC) is a chronic, bilateral allergic
inflammation of the ocular surface, seasonal or perennial affecting
children in the first decade of their life. It can present as limbal,
palpebral or as a mixed disease with varying grades of severity, with a
small subset bordering on refractory disease not amenable to currently
available treatment modalities(1). Being a tropical country, the
incidence is high in India and almost 36\% children have the perennial
form of VKC(2).

The exact etiopathogenesis of allergic disorders is still not completely
understood. Pathways involving IgE antibodies or T-cells and
inflammatory cytokines(3,4) are well known. However, the ones involving
bioactive lipid mediators are still being explored. Of the four classes
of immune modulating lipids; platelet activating factor, leukotrienes,
prostanoids and sphingolipids, immense focus has been on the
sphingolipid group in influencing the pathogenesis of allergic disease
as seen in the last decade(5). Sphingolipids (SL) comprise a large
family of bioactive lipids containing a sphingoid base (aliphatic amino
alcohol) as backbone that is acylated with various fatty acids to form
several SL species. Ceramide is the central molecule which is generated
by \emph{de novo} synthesis or formed by degradation from sphingomyelin
at plasma membrane level, while sphingosine is formed from ceramide by
ceramidase(6). Cytosolic conversion to their respective phosphorylated
forms, ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) and
further to other sphingolipids including glycosphingolipids constitute
the bioactives that are regulated by the various kinases, phosphatases,
hydrolases and lyases \textbf{(Figure 1)} . Activation of the pathway
occurs in response to a variety of cellular stresses due to UV
radiation, pathogen exposure, allergens and chemotherapeutic agents,
leading to the production of the bioactive lipid ceramide(7). Ceramide
and sphingosine are interconvertible components of the ``sphingolipid
rheostat'' that regulates immune function(8). These immune modulating
sphingolipids accumulate in cell membrane and compartmentalize
intracellularly to play a major role in immune cell trafficking,
inflammation, barrier cell integrity and cell survival(6,7,9).

In light of the above recent understanding from reports of systemic
allergic disorders, we decided to explore such bioactive lipids in the
ocular allergic disorder, specifically VKC. The etiopathogenesis of VKC
is still being explored. In addition, with the available reports in
literature, we are presently unable to explain the etiopathology of
refractoriness of this condition, barring reports of higher levels of
inflammatory cytokines in these severely affected eyes compared to the
less severe ones(10). We therefore specifically studied the sphingolipid
metabolism in patients with vernal keratoconjunctivitis.

\textbf{Method:}

As part of a two-year observational prospective study in the year 2017,
VKC cases and controls were recruited for one year in our tertiary eye
care center, as approved by the Institutional review board. The study
was conducted in strict adherence to the tenets of Declaration of
Helsinki after written informed consent from all study participants.

VKC was diagnosed and graded based on the classification by
Bonini\emph{et al} (11). Patients with ocular itching, photophobia,
presence of active tarsal/limbal papillae, bulbar congestion, Horner
Tranta's dots or superficial punctate keratitis were diagnosed as active
VKC. The quiescent stage was diagnosed based on inactive or flat-topped
papillae, a non -inflamed ocular surface with previous history of
chronic itching. All cases were included after a complete ophthalmic
examination and ruling out any other ocular or systemic morbidity or
history of ocular surgery. Tear specimen and conjunctival imprints were
collected in cases and controls, while blood sample was collected in a
sub-set based on consent of the study participant. All cases with active
VKC and age \textgreater{}6 years were included in the study. Patients
with history of previous ocular surgery, ocular co-morbidity other than
VKC, any systemic disease other than allergic disorders; age \textless{}
6 years and quiet eye at presentation were excluded from the study.

VKC cases were further graded based on their severity as non-refractory
(NR-VKC) and refractory (R-VKC). On basis of Bonini's classification,
patients with grade 1 and 2 (mild to moderate form of seasonal VKC) were
considered as non-refractory and those with grade 3 and 4 (severe to
very severe form of perennial VKC) were grouped as refractory. Patients
with refractory VKC were further sub-grouped based on the duration and/
or use of topical steroid or immunomodulator, (C) those who were on
topical steroid/immunomodulator \textless{}8 weeks; (D) those without
steroid or immunomodulator, who presented as fresh case with a repeat
episode; (E) those who were on steroid/immunomodulator \textgreater{} 8
weeks. A duration of 8 weeks was considered so that the maximum effect
of topical immunomodulator could be assessed. All cases had active VKC
at recruitment and were advised to defer use of any topical on the day
of collection. Non refractory and fresh refractory cases (group D) were
only on lubricants at presentation whereas refractory (groups C and E)
were on dual acting mast cell stabilizer (Olopatadine 0.2\% w/v, Alcon
Laboratories, India) in addition to lubricants and topical steroid /
immunomodulator (Fluoromethalone 0.1\% w/v, Allergan India Private
Limited/ Tacrolimus ointment 0.03\% w/w, Aurolab, India).

\textbf{Tear sample collection}

Tear was collected using sterile Schirmer strip by placing it at the
lower cul-de-sac for 5 min time to obtain tear fluid as reported in our
previous study(12); transferred using forceps to a vial and stored at
-80\textsuperscript{o} C until analysis.

\textbf{Conjunctival impression cytology}

Conjunctival imprints using sterile Whatman paper were obtained after
local anaesthesia with proparacaine HCL (Ophthalmic solution of 0.5\%)
from superior, inferior, temporal, nasal regions of the conjunctiva as
published earlier(13)\textsuperscript{(13)}. The samples were stored at
-80\textsuperscript{o} C until analysis.

\textbf{Blood Collection:}

Blood sample (plain) was collected in a sub-set of controls and cases
who gave consent, the serum was separated and stored at -80 degree C
until analysis of IgE, S1P and C1P by ELISA.

\textbf{Estimation of IgE, Ceramide 1 phosphate (C1P) and Sphingosine 1
phosphate (S1P) by ELISA~}

The reagents, working standards and samples were prepared according to
manufacture protocol for the estimation of serum IgE, (Human IgE ELISA
Kit (ab195216); MA, USA), serum C1P (Human Ceramide-1-phosphate
-MBS2601367 ELISA kit, MyBio Source, CA, USA) and~serum S1P (Human
S1P-MB5S2516132 ELISA kit, MyBio Source, CA, USA). Detection was based
on TMB substrate and the absorbance at 450nm was measured (Spinco
spectramax-M2e multimode reader, Molecular Devices, CA, USA).

\textbf{Total RNA isolation from conjunctival imprints}

The imprint samples stored were extracted for RNA using 300-400\selectlanguage{greek}µ\selectlanguage{english}l of
trizol as published by us earlier(13). The concentration and purity of
the RNA were estimated using Nano drop method and then converted to cDNA
using Bio-Rad iScript cDNA conversion kit.

\textbf{Sphingolipid Enzyme gene expression by qPCR}

The gene expression studies were carried out using the 50 ng/\selectlanguage{greek}µ\selectlanguage{english}L
concentration of cDNA for assessing the sphingolipid metabolism enzymes
mentioned in \textbf{Supplementary Table 1.0.} One \selectlanguage{greek}µ\selectlanguage{english}L (50 ng total RNA
equivalent) in 10 \selectlanguage{greek}µ\selectlanguage{english}L reaction mixture was used for 50 cycles reaction in
the qPCR and the fold change were analysed by \selectlanguage{greek}Δ\selectlanguage{english}Cq method according to
Thomas(14).

\textbf{Tear Lipid extraction:}

For tear lipid analysis, one tear specimen each from 3 patients was
pooled (tear collected using Schirmer's strip) in both control and VKC
groups. The analysis was done in 7 pooled sets in controls and 22 in VKC
(NR-VKC: 6 sets; and R-VKC: 16 sets). Briefly, the Schirmer strips were
cut and measured the volume of the extraction solvent,
CHCl\textsubscript{3}: Methanol Reagent (1:1) was added, vortexed and
sonicated for 5 minutes. Samples were then transferred to a fresh vial
and nitrogen purged. Extraction solvent was further added to the sample
followed by vortexing for 1 minute. Sample was centrifuged for 5 min at
10000 rpm. Supernatant (20 \selectlanguage{greek}µ\selectlanguage{english}l) was loaded for analysis in LC-MS/MS.

\textbf{LC-MS/MS analysis of sphingolipids in tear}

The sphingolipid standards were procured from Avanti Polar Lipids
(Alabama, USA). Based on calibration of the LC column and linearity
check, the following standards were finalized for the assay in pooled
specimens of tear in cases and controls \textbf{(Supplementary Table 2)}
. LC-ESI-MS/MS experiments were performed using a triple quadrupole
tandem mass spectrometer (4000 Q-Trap, AB Sciex, Foster City, CA, USA)
coupled with high performance liquid chromatography system (HPLC,
Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA) that
consisted of quaternary pump (G1311C), multi-sampler (G7167A), HIBAR (30
x 2.1mm, 2\selectlanguage{greek}µ\selectlanguage{english}m) column compartment (G1316A) with variable wavelength UV
detector (G1314F) and online degasser. All the parameters of tandem mass
spectrometer and HPLC were controlled by Analyst software, version 1.5.2
(AB Sciex, Foster City, CA, USA) and OpenLAB control panel software
(Agilent Technologies, 1260 Infinity, Santa Clara, CA, USA),
respectively.

\textbf{Mast cell culture~and~sphingolipid receptor expression on IgE
exposure:}

Mast cell lines were purchased from~(Kerafast-LUVA-Human Mast Cell Line,
(EG1701-FP, MA, USA). Suspended cells~were grown to confluence in six
well culture plate~and were exposed to 10ng/ml \& 20ng/ml concentration
of~ IgE (Abcam-Native human IgE protein~ (ab65866, MA, USA) for 16
hours~ in serum free medium (STEM PRO-34 nutrient supplement (Gibco
\#10641-025, MA, USA); Pen Strep (Gibco \#15140, MA, USA);
L-glutamine-200 mM~ (Gibco \# 25030-081). At the end of 16 hours, cells
were centrifuged at 3000g, washed~in 1X-PBS.~ RNA was isolated from
these cells using Trizol (FavorPrep Tri-RNA Reagent, Merck, St.Louis,
USA) followed by cDNA~conversion (Biorad-iScript 1708841, CA, USA).~The
gene expressions were analysed~by~RT-PCR (Biorad-Touch real time PCR,
CA, USA) for the enzymes.

\textbf{Mast cell expression and HDAC expression on IgE exposure:}

Mast cell were grown to confluence in six well cell culture
plates~exposed to 10ng/ml \& 20 ng/ml concentration of~IgE for 16
hours~in serum free medium. Cells were then collected and centrifuged at
3000g, washed~in 1X-PBS, lysed using RIPA buffer.~ The lysate after
protein estimation~(Pierce BCA Protein Assay Kit Cat No:~ 23225, MA,
USA)~was used for western blot analysis (30 \selectlanguage{greek}μ\selectlanguage{english}g/\selectlanguage{greek}μ\selectlanguage{english}l) to assess endogenous
Histone Deacetylase (HDAC) expression using Antibody Sampler Kit
(\#9928, Cell signalling Technology, MA, USA) using the corresponding
antibodies namely, HDAC1, HDAC2, HDAC4, HDAC6 and 7 (representing Zinc
dependent class I, class IIa and IIb ) as well as the Beta actin
(\selectlanguage{greek}β-\selectlanguage{english}Actin Antibody (AC-15) sc-69879, Santa Cruz Biotechnology, CA, USA)
used as the loading control. Chemidoc-XRS+ (Biorad, CA, USA) was
used~for gel documentation and image lab tool (Biorad, CA, USA) was used
for~estimating the pixel density of the bands.

\textbf{Statistical analysis}

Statistical analysis was performed in all patient samples using Analysis
of variance one-way ANOVA student-t test with Microsoft Excel and
GrapPad Prism was used for graphical representation. A p-value
\textless{}0.05 was considered statistically significant.

\textbf{Results:}

The study included 87 cases of vernal keratoconjunctivitis (NR-VKC-28,
R-VKC-59), with a gender distribution of 74 male; 13 female and a mean
age of 12.5 \selectlanguage{ngerman}± 5.3 years. Controls (n=60) were age matched healthy
individuals with a mean age of 13.7±5.2 years and the gender
distribution of male and female as 37 and 23 respectively.

\textbf{3. 1 Conjunctival gene Expression of Enzymes of Sphingolipids
Metabolism:}

Gene expression of enzymes of the sphingolipid metabolism was evaluated
in the conjunctival cells of VKC cases compared to age matched control.
A significant increase in the expression of the enzymes involved in the
removal of ceramide to form sphingosine namely, Alkaline ceramidase
(ACER) (p\textless{}0.05); Sphingosine kinase (SPHK1) that forms S1P
from sphingosine (p\textless{}0.001); Sphingosine 1- phosphate lyase
(SGPL) that acts on S1P to breakdown to phosphoethanolamine
(p\textless{}0.001) were seen. CERK which converts ceramide to C1P was
raised though not statistically significant. But UDP- Glucose ceramide
glucosyl transferase (UGCG) which forms glycosphingolipid from ceramide
was increased significantly (p\textless{}0.05) in VKC
patients\textbf{(Figure 2A)} . However, the enzyme, acid
sphingomyelinase (ASMA) involved in ceramide synthesis from
sphingomyelin was significantly reduced (p\textless{}0.01). No
significant difference between R-VKC and NR-VKC were observed in the
gene expressions studied (data not shown).

\textbf{3.2 Sphingolipid receptor expression in the conjunctival cells:}

Alteration in the sphingolipid metabolism was further supported by the
increased S1P receptor 3 expressions (S1P3R) in conjunctival cells of
VKC cases compared to control. S1P1R did not show significant
change\textbf{(Figure 2B)} showing that the cellular uptake of S1P is
S1P3R mediated. The receptor expression however did not show significant
variation between NR-VKC and R-VKC (data not shown).

\textbf{3.3 Gene expression of fibrotic, apoptotic and inflammation
marker in the conjunctival cells}

The mRNA expression of IL6, vimentin and Bcl2/Bax were evaluated. The
conjunctival IL6 expression was increased by 2-fold though not
statistically significant indicating a pro-inflammatory status in VKC.
Bcl2/Bax ratio was down regulated, however; vimentin was found to be
significantly upregulated in VKC cases compared to
control\textbf{(Figure 2C)} .

\textbf{3.4 Tear Sphingolipid concentration assessed by LC-MS/MS}

Pooled tear specimen of the normal and VKC group were analysed by mass
spectrometry to quantify the sphingolipids on the ocular surface.Based
on standards available and sensitivity of detection, 11 different
sphingolipids were estimated by LC-MS/MS analysis. Amongst these, S1P
(d17:1), S1P (d17:0) (both p\textless{}0.01) and S1P (d20:1) (p
\textless{}0.001) sphingolipids were significantly decreased in the tear
of the total VKC patients when compared to the control. Further, S1P
(d20: 0) and S1P (D17:1 DMA adduct) were raised more than two-fold in
VKC though not statistically significant \textbf{(Figure 3A)} .

The difference in the levels of these sphingolipids was also noted
within the VKC subgroups when classified as NR-VKC and
R-VKC\textbf{(Figure 3B)} . Looking at characteristic changes in R-VKC,
specific sphingolipids were found to be significantly low as compared to
control namely, S1P (d17:1) by 4-fold, (p\textless{}0.001),S1P (d17:0)
by 3 folds (p\textless{}0.001), S1P (d20:1) by nearly 2.5-fold
(p\textless{}0.01) and C1P (d18:1/ 2:0) by nearly 2-fold
(p\textless{}0.05). All these SL were also relatively lower in R-VKC in
comparison to NR-VKC of which S1P (d17:0) and S1P (d20:1) were
significantly lower (p\textless{}0.001). In addition, significant
lowering of Cer(d18:1/17:0) and C1P(d18:1/18:0) (p\textless{}0.001 and
p\textless{}0.01 respectively) was observed. Both Cer(d18:1/17:0) and
C1P(d18:1/1/8:0) showed an increase by nearly 1.5 fold in NR-VKC
compared to control but were not statistically significant.

\textbf{3.5 Altered serum Sphingosine 1-phosphate (S1P);
ceramide-1-phosphate (CIP) and serum IgE in VKC}

In order to check, changes in the systemic level, the metabolites namely
S1P and C1P were estimated by ELISA. The serum C1P level was lowered in
the total VKC group compared to the control (p\textless{}0.05) and this
was seen in both R-VKC and NR-VKC \textbf{(Figure 4A)} . Serum SIP was
significantly higher in the non-refractory VKC compared to control
(p\textless{}0.05), while R-VKC had levels similar to
control\textbf{(Figure 4B)} .

IgE levels were significantly increased in VKC (P=0.0007) and this was
observed both in NR-VKC (p=0.013) and in R-VKC (p=0.004) with no
difference amongst the two \textbf{(Figure 4C)} . The quantitative
estimation revealed a mean of 1433 ± 2061 ng/mL in control (median = 504
ng/mL), while VKC group revealed a mean of 3801±3719 ng/mL (median =
3719 ng/mL) with \textgreater{} 95 \% case showing IgE levels more than
the median of the control.



\textbf{3.6 IgE induced expression of the sphingolipid receptors in mast
cells is mediated by HDAC: \emph{In vitro studies}}

In order to evaluate the altered sphingolipid metabolism in mast cells,
the mast cells treated with IgE were evaluated for the S1P receptor
expression. A significant increase in the expression of both receptors
S1P1R and S1P3R (p\textless{}0.05) was observed in response to IgE
treatment \textbf{(Figure 5A)} .

Histone deacetylation was evaluated by HDAC protein expression to see if
IgE mediated sphingolipid metabolism is regulated through histone
deacetylation. HDAC1 (p\textless{}0.05), HDAC2, HDAC4 and HDAC 6
(p\textless{}0.05) were found to be increased in response to IgE
treatment in the mast cells cultured \emph{in vitro} \textbf{(Figure 5B,
C)} .

\textbf{Discussion:}

In the last few decades sphingolipids have emerged as vital structural
and signaling mediators that regulate varied cellular activities like
cell death, proliferation and inflammation(15,16). Ceramide,
sphingosine, C1P, S1P are all bioactive lipids and are key modulators in
various inflammatory cascades(16,17). Next to cholesteryl esters and
waxes, sphingolipids are the major lipid components in the tear and
along with phospholipids contribute to the amphiphilic lipids in tear as
shown by mass spectrometry analysis which not only comes from the meibum
but also from the cellular ocular surface(18). The first link between
altered sphingolipids and eye originated from lysosomal storage
disease(19). However, recently they have been implicated in dry eye
disease, corneal infections, diabetic cornea and age-related macular
degeneration(20,21). But their role in conjunctival disorders including
VKC needs to be explored.

Our present study revealed altered markers of sphingolipid metabolism in
the ocular surface in VKC patients along with differences in the
refractory cases. The conjunctival epithelial cells in VKC had
significantly reduced levels of ASMA which facilitates ceramide
synthesis. However, the ceramide that was estimated in tear namely
Cer(d18:1/17:0) showed differential levels in NR and R-VKC. Increased
expression of SPHK1 (5-fold) along with increase in SGPL (20-fold) as
compared to 4 folds in CERK was noted indicating accumulation of S1P
over that of C1P. These findings corroborated with the tear levels of
sphingolipids too. Concomitantly, we also found S1PR3 receptor in the
conjunctival imprints to be significantly upregulated indicating
cellular accumulation of S1P. An altered sphingolipid metabolism was
observed at systemic level too with raised S1P and lower C1P in VKC both
in NR-VKC and in R-VKC. Both S1P and C1P promote chemotaxis, cell
survival and inflammation. A critical balance between these
interconvertible metabolites is crucial for normal cell function and any
derangement can be detrimental to cells(22,23).

SIP plays an important role in the egress of lymphocytes, while C1P and
S1P are involved in eicosanoid synthesis; both prostaglandins and
leukotrienes. Besides, they also regulate the cellular arm of
inflammatory cascade by promoting neutrophil migration, extravasation,
macrophage survival and cytokine production. S1P receptors are present
on dendritic cells, eosinophils, Natural Killer cells in addition to
mast cells(24). Mast cells exposed to IgE in vitro showed increase in
expression of S1PR1 in addition to SIPR3. Activated mast cells release
S1P which acts in an autocrine fashion via S1P receptors to enhance mast
cell function in addition to causing chemotaxis and degranulation(25).

\textbf{Refractory VKC shows low levels of tear SL}

Interestingly, tear sphingolipids showed differential levels based on
the refractory nature of the VKC disease. Specific S1Ps and C1P (4 of
them) were found to be characteristically lowered in R-VKC compared to
control as well as NR-VKC. While Cer(d18:1/17:0) was significantly
increased (by 30\%) in NR- VKC than control, this response was not
observed in R-VKC. Looking at the cumulative levels of tear
sphingolipids a relatively lower level of the total sphingolipids, S1P,
C1P and ceramide were seen in R-VKC compared to NR-VKC, while NR-VKC
showed an increase compared to the control (\textbf{Supplementary Figure
1} ). Of the refractory cases classified based on intervention, those
who were on topical immunomodulators \textgreater{} 8 weeks and still
active, showed maximal lowering of tear ceramide and raised S1P/ceramide
ratio thus correlating with the more severe/refractory grade of VKC
(Supplementary Figure 1). Ceramide is an important component of the
lipid raft in cell membranes and plays a pivotal role in signal
transduction via FceRI receptors in mast cells. Among the multiple roles
of ceramide, apoptosis is considered as the key function which it
induces by both intrinsic and extrinsic pathways. The effect depends on
the chain length of the fatty acids in the ceramide as short (C16; C18)
or long (C24)(26,27). Apoptosis is necessary to resolve inflammation and
delayed apoptosis has been observed in autoimmune diseases(28). A higher
or raised ceramide probably induces apoptosis and the inflammation
eventually subsides in the non-refractory group whereas reduced levels
of ceramide in the refractory group with a raised S1P/ceramide ratio
inhibits apoptosis and the inflammation persists contributing to the
refractory status. Epithelial barrier permeability is influenced by
ceramide and its deficiency reportedly contributes to the pathogenesis
of atopic dermatitis(29). Similarly, a lower level of ceramide in the
conjunctival epithelium probably alters the permeability making them
more susceptible to allergens and reduced apoptosis further contributes
to the refractory behavior in a select group of VKC patients. The
reduced apoptosis in VKC and persistence of inflammation was further
validated by the lower expression levels of Bcl2 and Bax and a 2-fold
increase in the gene expression of IL6 expression in the conjunctival
cells of VKC cases noted in our study(30). Moreover, there was an
increase in the epithelial mesenchymal transition (EMT) marker namely
vimentin, indicating pro-survival and EMT changes in VKC. EMT and
sphingolipid metabolism has been explored in cancer and each inducing
the changes in other is reported(31).

Since altered sphingolipid metabolism has been reported in allergic
asthma, atopic dermatitis and many other autoimmune diseases, they have
also been considered as therapeutic targets(23). Topical ceramide cream
has been found to improve the transepidermal barrier permeability in
patients with atopic dermatitis(32). Topical liposomal C6-ceramide was
found to inhibit corneal inflammation in murine models(33). SphK
inhibitors like SK1-I and DMS have been found to reduce airway
inflammation in mice(16,34). S1P receptor modulators like FTY 720 are
being used in allergic rhinitis, asthma, allergic skin diseases such as
contact hypersensitivity or atopic dermatitis(35). Owing to its lesser
side effects than immunomodulators, topical FTY720 has been attempted to
reduce rejection in corneal allograft as it was found to decrease
infiltration of CD4 T cells in corneal grafts in animal experiments(36).
A similar approach based can be explored in VKC.

\textbf{HDAC mediated regulation of the sphingolipids}

In addition, recently role of epigenetic regulation in allergic
disorders has gained interest. The most common mechanisms are DNA
methylation, histone modification and noncoding RNA's. There are 4
subclasses of HDAC enzymes of which classI/II have been most widely
studied.Evidence suggests contribution of HDAC enzymes in allergic
disorders primarily in two areas; one is regulation of cells
contributing to allergy (Tcells, macrophages) and the other is with
various allergic phenotypes(37). Grausenburger et al showed in
conditional HDAC1 knockout mice that HDAC was important for development
of Th2 cytokine production and allergic airway inflammation(38). We
found significantly raised levels of HDAC1 and 6 when mast cells were
exposed to IgE along with increased levels of S1PR1and 3 receptors,
similar to published reports(39). Increased S1PR1 and 3 expressions that
internalize the S1P seem to target HDAC for transcriptional modulation
in mast cells. However, further studies are required to study the role
of HDAC enzymes in pathogenesis of VKC.

One of the drawbacks of our study was that all samples were analyzed
only in the active stage and not compared with the quiet stage of the
disease to further delineate the cause and effect of the sphingolipids.
Besides this was the first time that role of sphingolipid metabolism was
being evaluated in VKC and not all the species of the sphingolipids
including various ceramides in the tear were estimated. However, altered
enzymes expression, receptor levels and corresponding variation in the
levels of specific sphingolipids confirmed an altered metabolism in VKC
which is differential in R-VKC in terms of relatively lower levels of
ceramide and specific S1P compared to NR-VKC.

To summarize, this study brings forth for the very first time an
association between altered sphingolipid metabolism and pathogenesis of
VKC which was observed both at ocular and systemic levels. In addition
it unveils a possible explanation for the severe or refractory cases of
VKC as compared to the seasonal ones and thus opens up fresh avenues in
therapeutic intervention.

\textbf{Acknowledgement} : We acknowledge High Precision Bioanalytical
Facility (HPBAF) with Mass spectrometry (LC-MS/MS) at Ocular
Pharmacology and Pharmacy Division, RP Center, AIIMS, New Delhi, India.

\textbf{Author Contributions:}

Menta Vignesh: Experimentation and data analysis, First draft of
manuscript

Gurumurthy Srividya: Experimentation, analysis, manuscript drafting and
discussion

Narayanasamy Angayarkanni: Concept and study design, data analysis,
discussion,~drafting, and funding

Agarwal Shweta: Clinical specimen and data, study design, discussion and
drafting paper

Amrutha Anandan, Srinivasan Bhaskar, Iyer Geetha: ~ Clinical data and
discussion

Das S Ujjwalkumar, Lakshmi Moksha, Thirumurthy Velpandian:~ ~Analysis
(LC-MS/MS) and data analysis

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\textbf{Figure Legends}

\textbf{Figure 1:} Sphingolipid Metabolism

\textbf{Figure 2A:} mRNA expression of the key enzymes of sphingolipid
metabolism in the conjunctival cells of VKC.

UGCG: UDP-Glucose Ceramide Glucosyltransferase; CERK: Ceramide Kinase;
SPHK1: Sphingosine Kinase 1; ACER: Alkaline ceramidase; SGMS2:
Sphingomyelin Synthase; ASMA: Acid sphingomyelinase; ASAH1: Acid
ceramidase; SGPL: Sphingosine-1-phosphate lyase

*p\textless{}0.05; **p\textless{}0.01 is the significance based on
comparison of control versus VKC for the corresponding enzyme.

Control: n=60 and VKC: n=87

\textbf{Figure 2B:} S1P1 and S1P3 receptor expressions in the
conjunctival cells of VKC

*p\textless{}0.01 is a comparison of control vs VKC;

S1P1R: Control, n=13 and VKC, n= 13; S1P3R: Control, n=13 and VKC, n=9

\textbf{Figure 2C:} Expression of fibrosis marker \selectlanguage{greek}α-\selectlanguage{english}Smooth Muscle Actin,
Vimentin and apoptosis markers BCl\textsubscript{2} and Bax

p\textless{}0.05; ** P\textless{}0.01: are the comparison between
control and VKC

Control, n=22 ; VKC, n= 19

\textbf{Figure 3:} Tear sphingolipids in VKC patients analyzed using
LC-MS/MS.

\textbf{Figure 3A} : Tear Sphingolipids in control and Total VKC

\textbf{Figure 3B:} Tear sphingolipids in Refractory VKC (R-VKC) and
Non-Refractory VKC (NR-VKC). Details of pooled sample sizes in each
group is given in the methods section.

1: C17 Ceramide (d18:1/17:0); 2: Sphingosine-1-Phosphate (d18:1); 3:
Sphingosine-1-Phosphate (d20:1); 4: Sphingosine-1-Phosphate (d17:1); 5:
Sphinganine-1-Phosphate (d20:0); 6: sphinganine-1-phosphate (d17:0); 7:
Sphinganine-1-Phosphate (d18:0); 8: C8 Ceramide-1-Phosphate (d18:1/8:0);
9: C8 Ceramide-1-Phosphate (d17:1/8:0); 10: C2 Ceramide-1-Phosphate
(d18:1/2:0); 11: Sphingosine-1-Phosphate (d17:1) (DMA Adduct). X-axis
represents the Mean concentration in (ng/ml) in the tear samples Y axis
represents the different groups. * p\textless{}0.05; **
P\textless{}0.01:***p\textless{}0.001 is the comparison between control
vs VKC in Fig 3a. and R-VKC vs NR-VKC in Fig 3b.\# p\textless{}0.05;
\#\# P\textless{}0.01:\#\#\#p\textless{}0.001 is the comparison between
control vs R-VKC

Tear specimen were analyzed after making pooled sets of each group.

Control: 7 pooled sets; VKC: 22 sets (NR-VKC: 6 sets; and R-VKC: 16).
One set is a pool of 3 tear specimen in the group

\textbf{Figure 4:} Serum S1P, S1P and IgE in VKC estimated by ELISA

\textbf{Figure 4A} : Serum C1P in VKC, NR-VKC and R-VKC (Control: n= 14;
VKC: 29; (NR-VKC: n= 6; R-VKC: n=23)

\textbf{Figure 4B} : Serum S1P in VKC, NR-VKC and R-VKC (Control: n= 9;
VKC: 29; (NR-VKC: n= 6; R-VKC: n=23)

\textbf{Figure 4C} : Serum IgEin VKC, NR-VKC and R-VKC (Control: n= 12;
VKC: 29; (NR-VKC: n= 6; R-VKC: n=23)

\textbf{Figure 5:} Effect of IgE on gene expression of the S1P receptors
in cultured Mast cells

\textbf{Figure 5A:} S1P Receptors expression in Mast cells exposed to
IgE

\textbf{Figure 5B} : Representative western blot showing protein
expressions of the Histone Deacetylases, HDAC1, HDAC2, HDAC5 and HDAC6

Histone Deacetylase (HDAC) protein expression in mast cells exposed to
IgE (10 and 20 ng/mL) for 16 hours compared to control \emph{in vitro}as
detailed in method section.

Lanes L1, 3, 5, 7- treated with 10ng/ml of IgE; L2,4,6,8- treated with
20ng/ml of IgE)

\textbf{Figure 5C} : Densitogram

Densitometry of the bands of the western blot was done using ImageJ
software,

Y-axis: fold variation normalized to control

Student's--t test was performed to obtain the statistical significance
between control and treated *p\textless{}0.05; ** P\textless{}0.01.

The data is expressed as mean \selectlanguage{ngerman}± SD of three independent experiments\selectlanguage{english}
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