Distinct ocular surface soluble factor profile in human corneal dystrophies

Journal Pre-proof Distinct ocular surface soluble factor profile in human corneal dystrophies Rohit Shetty, Jagadeesh R. Naidu, Archana Padmanabhan Nair, Tanuja Arun Vaidya, Sharon D'Souza, Himanshu Matalia, Vrushali Deshpande, Swaminathan Sethu, Arkasubhra Ghosh, Koushik Chakrabarty PII:

S1542-0124(19)30233-2

DOI:

https://doi.org/10.1016/j.jtos.2019.11.007

Reference:

JTOS 454

To appear in:

Ocular Surface

Received Date: 13 July 2019 Revised Date:

30 September 2019

Accepted Date: 18 November 2019

Please cite this article as: Shetty R, Naidu JR, Nair AP, Vaidya TA, D'Souza S, Matalia H, Deshpande V, Sethu S, Ghosh A, Chakrabarty K, Distinct ocular surface soluble factor profile in human corneal dystrophies, Ocular Surface (2019), doi: https://doi.org/10.1016/j.jtos.2019.11.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

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Distinct ocular surface soluble factor profile in human corneal dystrophies

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Rohit Shetty, FRCS, PhD1*; Jagadeesh R Naidu, MSc2*; Archana Padmanabhan Nair

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MSc2; Tanuja Arun Vaidya, MSc2; Sharon D'Souza, MS1; Himanshu Matalia, MS1;

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Vrushali Deshpande, PhD2; Swaminathan Sethu, PhD2; Arkasubhra Ghosh, PhD2,3,+;

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Koushik Chakrabarty, PhD2, #

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1

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India; 2GROW Research Laboratory, Narayana Nethralaya Foundation, Bengaluru,

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India;

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Department of Cornea and Refractive Surgery, Narayana Nethralaya, Bengaluru,

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Singapore

Eye

Research

Institute,

Singapore;* Equal

contributors;

#

Corresponding author; + Co-corresponding author

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Corresponding authors address

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Koushik Chakrabarty, PhD

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GROW Research Laboratory, Narayana Nethralaya Foundation,

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Narayana Nethralaya, Narayana Health City,

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# 258/A, Bommasandra, Hosur Road, Bangalore - 560 099, India.

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Email: [email protected] ; Phone: +91-7411613923

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Arkasubhra Ghosh, PhD

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GROW Research Laboratory, Narayana Nethralaya Foundation,

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Narayana Nethralaya, Narayana Health City,

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# 258/A, Bommasandra, Hosur Road, Bangalore - 560 099, India.

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Email: [email protected]; Phone: +91-08066660712

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Short title: Tear cytokines in corneal dystrophies

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Declaration of interest: None

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Abstract

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Purpose: Corneal dystrophies (CD) are classified as rare eye diseases that results in

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visual impairment and requires corneal transplant in advanced stages. Ocular surface

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inflammatory status in different types of CD remains underexplored. Hence, we

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studied the levels of tear soluble factors in the tears of patients with various types of

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corneal dystrophies.

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Methods: 17 healthy subjects and 30 CD subjects (including epithelial, stromal and

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endothelial CD) were included in the study. Schirmer’s strips were used to collect the

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tear fluid in all subjects. 27 soluble factors including cytokines, chemokines, soluble

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cell adhesion molecules and growth factors were measured in the eluted tears by

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multiplex ELISA or single analyte sandwich ELISA.

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Results: Percentages of subjects with detectable levels of tear soluble factors were

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significantly higher in CD compared to controls. Significant higher level of IL-2 was

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observed in both epithelial and stromal CD. IL-4, TGFβ1 and IgE were significantly

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higher in stromal CD. VCAM, IL-13 and Fractalkine were significantly elevated in

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epithelial and macular CD. IL-1α, IL-8, IL-12, ANG, Eotaxin, MCP1, RANTES,

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ICAM1, L-selectin and P-selectin were significantly higher in epithelial CD. TGFBIp

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was significantly elevated in lattice CD and endothelial CD.

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Conclusion: Distinct set of the tear soluble factors were dysregulated in various

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types of CD. Increase in tear inflammatory factors was observed in majority of the CD

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subjects depending on their sub-types. This suggests a plausible role of aberrant

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inflammation in CD pathobiology. Hence, modulating inflammation could be a

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potential strategy in improving the prognosis of CD.

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Keywords: Corneal dystrophy; Tear fluid; TGFBIp; Inflammation; ELISA; Epithelial

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Corneal dystrophy; Granular Corneal dystrophy; Lattice corneal dystrophy; Macular

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Corneal dystrophy; Fuch’s endothelial corneal dystrophy.

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Abbreviations: Angiogenin (ANG); Corneal dystrophies (CD); Corneal stromal

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dystrophy (SCD); Epithelial basement membrane dystrophy (EBMD); Extracellular

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matrix (ECM); Fuch’s endothelial corneal dystrophy (FECD); Gelsolin (GSN);

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Granular corneal dystrophy (GCD),

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Intercellular Adhesion Molecule 1(ICAM1); Interferon gamma-induced protein 10

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(IP10); interferon-inducible T-cell alpha chemoattractant (ITAC); Lattice corneal

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dystrophy (LCD); Macular corneal dystrophy (MCD); Map dot fingerprint dystrophy

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(MDFPD); Monokine induced by gamma interferon (MIG); Monocyte Chemoattractant

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Protein (MCP)1; Regulated on Activation; Regulated on activation normal T cell

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expressed and secreted (RANTES); Transforming Growth Factor β1 (TGFβ1); TGFβ

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induced protein (TGFBIp); Vascular cell adhesion protein (VCAM).

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1. Introduction

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Corneal dystrophies (CD) are a heterogeneous group of rare hereditary disorders

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characterized by bilateral abnormal deposition of insoluble material in the cornea

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leading to visual impairment [1]. The disease is often progressive and clinically

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categorized based on the anatomical locations of the abnormal deposits or opacities

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and their gross phenotypic appearance in the cornea [1, 2]. The major types include

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epithelial, stromal and endothelial corneal dystrophies, where specific gene mutations

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are known to underlie their pathogenesis (Supplementary table 1). Epithelial

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basement membrane dystrophy (EBMD) and map dot fingerprint dystrophy (MDFPD)

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are some of the common corneal epithelial dystrophies [3-4]. The common stromal

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CD includes granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD) and

Immunoglobulin (Ig) E; Interleukin (IL);

3

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macular corneal dystrophy (MCD) [3-4]. Fuch’s endothelial corneal dystrophy (FECD)

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is one of the more prevalent and studied endothelial CD [3-4]. The key pathological

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characteristic feature common among CD is extracellular protein aggregate formation

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that presents as corneal opacities [5]. The distribution and intensity of these opacities

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or aggregates contribute to vision impairment. Gene mutations have been implicated

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in the formation of protein aggregates that results in different types of CD [6-7].

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The progression of CD to an advanced stage that requires corneal grafts happens

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over years [8]. The current strategies to manage CD are quite broad from no

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definitive treatment to corneal transplant based on the phenotype [8]. Although the

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genetic underpinnings of many CD are widely examined, therapeutic options for CD

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are limited due to the little understanding of the disease pathophysiology and factors

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affecting its progression. CD is traditionally viewed as non-inflammatory disease with

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no cellular infiltration and/or apparent vascularization of the affected cornea [1].

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Nevertheless, there have been reports where inflammation modulation has improved

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the prognosis of CD. Treatment with tropical steroids, autologous serum and artificial

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tears have been attempted to manage the stromal edema, guttae formation, epithelial

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erosion and endothelial pump functions under the assumption that an inflammatory

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milieu may be responsible for the exacerbation of the condition [9, 10,11]. Tear fluid

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is a well-known noninvasive sample source to determine the ocular surface

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inflammatory status [12-13]. It has proven to be useful in treatment planning and

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disease monitoring [14-15]. Despite, the knowledge of altered tear film dynamics in

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CD [16-18], a detailed analysis of the tear film elucidating inflammatory factors which

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are deregulated is not yet available, with only a report of decreased levels of tear

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cystatins in CD [19]. Therefore, we hypothesized that there may be specific subsets

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of molecular factors influencing the inflammatory and pro-fibrotic pathways that cause 4

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disease progression in addition to the underlying genetic trigger. This study was

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designed to analyze a set of tear soluble factors to determine their relative

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association with different types of CD as it can provide insights into new diagnostic

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and management modalities.

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2. Materials and methods

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This cross-sectional observational study was approved by the Narayana Nethralaya

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Ethics Committee and was conducted in adherence to the Indian Council for Medical

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Research (ICMR) guidelines and the tenets of the Declaration of Helsinki. Written

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informed consent was obtained from all participants prior to recruitment and tear fluid

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collection. All study subjects were recruited from patients visiting Department of

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Cornea and Refractive Surgery, Narayana Nethralaya, Bangalore, India. Clinical

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examination included slit lamp examination with topographic and pachymetric

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evaluation on the Pentacam HR (Oculus, Germany) and Orbscan (Orbtek, Bausch &

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Lomb). In some cases, Anterior Segment Optical Coherence Tomography (Topcon

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DRI Triton OCT, Japan) was carried out for clinical diagnosis.

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2.1 Clinical cohort: A total of 30 CD patients diagnosed based on the criteria laid by

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International Committee for Classification of Corneal Dystrophies (IC3D) [2] and 17

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healthy volunteers were included for the study. GCD and LCD diagnosis were based

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on the observation of granular and lattice opacities that are bilateral and often

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asymmetric [5]. Subjects diagnosed with EBMD and MDFPD were taken together as

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epithelial corneal dystrophies (ECD). As a primary endpoint, we have determined the

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levels of a set of soluble inflammatory factors in the tear fluid of patients with different

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types of CD and compared them with healthy controls. Inclusion criteria were patients

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with clinically confirmed corneal dystrophy. Exclusion criteria were the following: (i)

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CD patients who underwent prior refractive surgery or other ocular surgery; (ii) recent 5

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use of topical medications in the last month; (iii) current ocular infection or clinical

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signs of inflammation; (iv) subjects with other ocular surface or ocular co-morbidities;

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(v) subjects with systemic disease. Tear fluid from a total of 48 eyes from 30 CD

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patients and 18 eyes from 17 healthy volunteers were included in the study. ECD

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(n=4) subjects include EBMD (2 eyes, n=2) and MDFPD (2 eyes, n=2). SCD subjects

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(38 eyes, n=23) include MCD (13 eyes, n=8), GCD (16 eyes, n=9) and LCD (9 eyes,

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n=6). Endothelial corneal dystrophy included FECD (6 eyes, n=3). Table 1, shows the

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demography of the subjects included in this study. There were no significant

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differences in age and sex between the control and patient groups.

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2.2 Tear collection: Schirmer's strips (Whatman filter paper, 5 × 35-mm2, ContaCare

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Ophthalmics and Diagnostics, India) were used to collect the tear fluid from the study

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subjects by following Schirmer's test I protocol as previous described [20]. The

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collected strips were stored at -80°C until further use. Tear analytes were extracted

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from Schirmer's strips by incubating the cut /shredded pieces of the strips in 300 µL

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of ice-cold sterile 1xPBS for 1.5 hours at 4°C with agitation. The tear protein

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containing eluate was separated from the pieces of the Schirmer’s strip by

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centrifugation.

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2.3 Tear total protein measurements – Bicinchoninic acid assay (BCA assay):

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Total protein concentration in the tear fluid was estimated by BCA assay (G-

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Biosciences, USA) as per manufacturer’s protocol. Briefly, 25 µL of standard (Bovine

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serum albumin, BSA) and samples (1:3 diluted) were added into respective wells.

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200 µL of BCA working solution was added to each well, mixed and incubated at

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37°C for 30 min. Plate was brought to room temperature and absorbance measured

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at 562 nm, using a multimodal plate reader (Spark 10M, Tecan, Austria). The

6

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absolute protein concentration in the tear samples were determined using a standard

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curve.

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2.4 Measurement of tear soluble / secreted factors: Cytokines, chemokines,

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secreted cell adhesion molecules and growth factors were quantified in control and

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patient tears by multiplex ELISA using cytometric bead array (BDTM CBA Human

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Soluble Protein Flex Set System, BD Biosciences, USA). A total of 26 analytes (IL-1α

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IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12/IL23p40, IL-13, IL-17A, IL-17F, IFNα, monokine

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induced by gamma interferon (MIG), interferon gamma-induced protein 10 (IP10),

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interferon-inducible T-cell alpha chemoattractant (ITAC), Fractalkine, monocyte

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chemoattractant protein 1 (MCP1), regulated on activation normal T cell expressed

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and secreted (RANTES), Eotaxin, Angiogenin, VEGF, TGFβ1, sVCAM, sICAM1, sP-

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Selectin, sL-selectin and IgE were measured by bead based multiplex ELISA using a

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cocktail of respective capture beads and detection antibodies. The assay was

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performed as per manufacturer's instructions using BD Human Soluble Protein

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Master buffer kit. The fluorescent signal intensity for each analyte was determined on

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flow cytometer (BD FACS Canto II, BD Biosciences, USA) using BD FACS DIVA

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software (BD Biosciences, USA). The absolute concentration (pg/ml) for the various

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analytes were determined using standard curve for the respective analytes using

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FCAP array V3.0 software (BD Biosciences, USA). Analytes that were not detected in

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more than fifty percent of the study samples were excluded from the study. The

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concentration of the analytes was normalized to the total protein concentration for

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each tear sample and the concentration of each specific analyte is represented as

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pg/µg of total protein.

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2.5 ELISA based estimation of Tear Human Transforming Growth Factor beta

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induced protein (TGFBIp): TGFBIp was measured in tears of control and dystrophy 7

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cases using sandwich ELISA (Human TGFBIp/ (BIGH3) ELISA Kit, Thermo Scientific,

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USA) as per manufacturer’s instructions. Absorbance was measured at 450 nm using

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a multimodal plate reader (Spark 10M, Tecan, Austria). The absolute concentration

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was determined based on a standard curve. Further, the concentration of TGFBIp

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was normalized to the total protein concentration for each tear sample and is

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represented as pg/µg of total protein.

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2.6 Statistical analysis: Distribution of data was determined using Shapiro-Wilk

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normality test. Statistical tests such as Mann-Whitney test and Kruskal–Wallis test

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were performed to determine the difference between the study groups. Spearman

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Rank correlation test was performed to study the relationship between the analytes in

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the study. P value < 0.05 was considered to statistically significant. Software:

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GraphPad Prism Version 6 (GraphPad Software, Inc, USA) and MedCalC version

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18.5 (MedCalc Software bvba, Belgium) was used for statistical analysis. The open-

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source

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(https://orange.biolab.si/; Slovenia) was used for generating the heatmaps.

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3. Result

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3.1 Tear soluble factors level in corneal dystrophies

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Concentration of total protein in the tear fluid of CD patients was observed to be

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significantly higher compared with controls (Figure 1). Increase in total protein in

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tears of inflammatory and corneal allergic conditions has been reported [22, 23]. To

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compensate for the variability in tear collection or extraction of protein from the

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Schirmer’s strips, the measured levels of soluble factors in the tears were normalized

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to total protein concentration in each sample [24,25]. The percentage of tear samples

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with detectable levels of soluble factors was observed to be significantly higher in CD

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compared to controls (Figure 2). A total of 17/27 soluble factors were detected in

data

visualization

Heatmapper

software

[21]

and

Orange

8

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higher percentage in the tear samples of CD subjects compared to controls are

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shown in and Table 2. The levels of IL-2, IL-4, IL-8, IL-13, Angiogenin, Eotaxin,

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Fractalkine, IFNα, RANTES, ICAM-1, VCAM, P-selectin, TGFBIp, TGFβ1 and IgE

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were significantly higher in CD patients (Table 3). The median concentration of IL-1α,

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IL-1β, IL-2, IL-6, IL-8, IL-12, IL-13, ANG, Eotaxin, fractalkine, IFN-α, RANTES and

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VCAM was higher in ECD compared to other types of CD (Figure 3). The median

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concentration of IL-1α, IL-2, IL-4, IL-8, IL-12, ANG, Fractalkine, IFN-α, RANTES,

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VCAM and IgE was significantly higher in MCD compared to FECD (Figure 3, Table

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5b and 6). In GCD, the median concentration of IL-1α, IL-4, IL-8, ANG and

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Fractalkine was found significantly higher compared to FECD (Table 5b and 6). In

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LCD, median concentration of IL-1α, IL-2, ANG, RANTES and VCAM was

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significantly higher compared to FECD (Table 5b and 6).

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3.2 Tear soluble factors level in epithelial corneal dystrophies

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In epithelial corneal dystrophies (ECD), a significant increase in levels of IL-1α, IL-1β,

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IL-2, IL-8, IL-12/ IL23p40, IL-13, ANG, Eotaxin, Fractalkine, IFN-α, MCP-1, RANTES,

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ICAM-1 VCAM, L-selectin, P-selectin, was observed compared to controls (Figure 3

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& Table 4). However, IL-4, IL-17A, TGFβ1 and IgE were observed to be below the

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detection limit in the tears of ECD subjects, albeit detectable and measured in the

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tears of controls and other corneal dystrophy subjects (Table 4 – 6).

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3.3 Tear soluble factors level in stromal corneal dystrophies

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We observed that subjects with stromal corneal dystrophies (SCD) exhibited

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significantly higher level of tear IL-2, IL-4, IL-8, IL-13, ANG, Eotaxin, Fractalkine, IFN-

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α, RANTES, ICAM-1, VCAM, TGFβ1 and IgE compared to the control (Table 5a).

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Furthermore, distinctly varying levels of tear soluble factors were seen within SCD

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(Granular corneal dystrophy – GCD, Lattice corneal dystrophy – LCD and Macular 9

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corneal dystrophy – MCD) as shown in Table 5b. Level of IL-2, IL-4, IL-17F,

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Fractalkine was significantly elevated in all the SCD types compared to controls.

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Levels of IFN-α was significantly higher in tear fluid of MCD and GCD (Figure 3m,

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Table 5b). Although IFN-α level was high in LCD compared to controls, the difference

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was not statistically significant. VCAM in MCD and LCD was found was significantly

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higher compared to GCD (Figure 3o, Table 5b). Compared to control, significant

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increase in TGFβ1 was found in GCD tear fluid with MCD tear fluid having the

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highest fold change (Figure 3q, Table 5b).

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3.4 Tear soluble factors level in EnCD

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The levels of the analytes measured in the tear fluid of EnCD patients were mostly

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lower than the controls (Table 6). Levels of IL-1β, IL-17F, IP-10, MIG were found to

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be significantly lower in EnCD compared to the controls except for TGFBIp, which

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was significantly elevated in EnCD compared with the controls (Table 6).

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3.5 Correlations and signature of tear fluid soluble factors in control and corneal

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dystrophies.

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The analytes correlated more significantly in CD (147 total correlations of which 3

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correlated negatively) compared with the control (57 total correlations of which 3

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correlated negatively). Statistically significant Spearman’s rank correlation coefficient

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(r) was seen between TGFB1 and VEGF in CD. In CD, negative correlation existed

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between ANG and IL-6 (r = -0.408). In addition, IgE negatively correlated with ICAM-

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1 and IL-1β (r = - 0.3) in the CD group. In the control, negative correlation was found

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between RANTES and IL-8; MCP and MIG; and MIG and VCAM1 (Figure 4). Heat

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map representation as depicted in figure 5 indicates the distinct tear soluble factor

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signature present in various human corneal dystrophies. 10

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4. Discussion

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The key pathological hallmarks of corneal dystrophies are corneal erosion,

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aggregation of misfolded proteins, guttae and edema formation depending on the

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type of CD, which eventually cause visual compromise [26]. These pathologic

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features are exacerbated due to ensuing inflammatory processes ultimately leading

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to disease progression. Thus, unraveling the underlying inflammatory milieu in

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various CD cases can expand our understanding of the disease pathobiology.

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This study was designed to test if dysregulated soluble inflammatory and signaling

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factors such as cytokines, chemokines, cell adhesion factors, growth factors and

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others [27-29] are associated with CD pathobiology. To this end, we used tear fluid to

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evaluate the inflammatory mediators [30-33] which could be present in the ocular

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surface. Our study revealed significant and distinct levels of pro-inflammatory

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cytokines, IgE and growth factors in the tear fluid of CD patients compared to control

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subjects. IL-4 and TGFβ1 levels were found to be unique across the different types of

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CD with significantly high level in the SCD group which included GCD, LCD and

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MCD. Here, our observation of significant increase of IL-4, considered to be an anti-

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inflammatory cytokine in SCD group suggests a possible regulatory role for the

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individual corneal cell types on cytokine production which can potentiate a local

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expression of protective immune reactions in that specific corneal region

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Indeed, the quantity of the bioactive tear soluble factors and their spatio-temporal

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action has been shown to greatly influence their properties [35]. In this context, IL-4 is

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also known to play a critical role in allergy by inducing immunoglobulin (Ig) isotype

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switching in B cells and sustain IgE production [36]. An elevated level of IgE in tear

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fluid is indicative of an allergic state [37]. We observed a marked elevation of IgE

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levels in the CD group compared to the controls. Studies have shown IL- 4 mediating

[34].

11

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many of the critical functions in epithelial cells and fibroblasts [38, 39]. IL-4

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possesses the potential to partner with IL-13 towards inducing IgE production [40]

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and inducing expression of other adhesion factors such as ICAM-1 [41]. TGFβ1 has

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been shown to be essential in initiating corneal wound healing response [42]. Our

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observation of increased TGFβ1 expression across the different SCDs could indicate

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the scarring process in the diseased cornea which can be attributed to the context

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specific capabilities of TGFβ1 [42].

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We found significant increase in levels of IL-2, a critical cytokine involved in activating

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T cells [43] in CD tears compared with the control. The importance of IL-2 in

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promoting immune responses and its pivotal role in autoimmune diseases makes it a

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prime addition as a diagnostic candidate for CD. Lowering IL-2 levels in CD can be

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considered to be one of the strategies to decrease the inflamed or inhospitable

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vascularized recipient corneal bed for enhancing the survival of corneal allografts

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[44]. We report significant increase in the levels of IL-8, IL13, IFN-α, ANG,

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Fractalkine, Eotaxin, RANTES, VCAM, P-selectin and ICAM, in the tear fluid of CD

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patients providing further evidence of the critical inflammatory processes involved.

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Both IL-1α and IL-8 levels was distinctively high in ECD tears suggesting

289

inflammation [45, 46] to be a major aspect of the disease process. Expression of

290

CXCR1 (3) (receptor for fractalkine) has been reported in the mammalian corneal

291

epithelia [47].

292

indicates its role in the possible modulation of the macrophages and dendritic cells

293

(DCs) in the diseased cornea. We also found other chemotactic factors such as IL-8

294

and RANTES to be dysregulated in ECD tears, which could be crucial participants in

295

prolonging the inflammation in ECD [48]. Differential expression pattern of cell

296

adhesion factors in inflamed human cornea has been previously demonstrated [49].

Our observation of increased fractalkine in ECD and MCD tears

12

297

Nameth et al [50] reported selective and increased expression of ICAM1 in stromal

298

corneal dystrophies (SCD) which corroborates with our observation of significantly

299

elevated levels of ICAM1 in the tear fluid of SCD and ECD. Furthermore, we found

300

the levels of another soluble cell adhesion molecule VCAM to be significantly

301

elevated in tears of ECDs and all the SCD types (GCD, LCD and MCD). Interestingly,

302

up- regulation of ICAM1 and VCAM expression in corneal fibroblasts has been

303

implicated on the pathogenesis of allergic keratopathy [51] suggesting their possible

304

role in CD pathogenesis. Li et al [52] reported that p-selectin is a significant

305

determinant in the events after corneal epithelial abrasions that contribute to wound

306

healing of the cornea. Our observation of significant elevation of p-selectin levels in

307

ECD tears hints to the possibility of its role in dysregulated healing response of the

308

corneal epithelia in ECD pathology. The levels of the various soluble factors

309

measured in the healthy subjects were comparable to those reported earlier [53, 54].

310

The variation in the concentration range for some of the factors could be attributed to

311

the platform used in the measurement of these factors, in addition to ethnic and age

312

variations. Further, it is important to note that the presence of these factors in normal

313

healthy subjects suggests their essential role in the maintenance of ocular surface

314

homeostasis [24]

315

The correlation within the analytes was relatively lesser in control subjects compared

316

to CD cases. The correlation between the analytes were predominantly positive in

317

both the cohorts, with the high to moderate correlation size in the control relative to

318

broader size range spanning from high to low in CD [55]. We found the heterodimeric

319

pro-inflammatory cytokine, IL-12 to have the highest correlation size in both the

320

cohorts. IL-12 correlated with IL-17F in the control (r 0.92) and with IL-13 in CD (r

321

0.83). Angiogenin (ANG) correlated negatively with IL- 6 and IgE in CD cohort. While 13

322

IgE negatively correlated with ICAM-1 and IL-1β in the CD cases. These correlations

323

are interesting in view of our observed significantly reduced levels of ANG in FECD

324

tears compared with the control. Although not statistically significant, the decrease in

325

ANG levels in FECD cases was quite distinct compared to the elevated levels of ANG

326

in ECD and SCD. This observation is important since reports indicate ANG to

327

facilitate corneal endothelial wound healing as a novel target for therapeutic

328

exploitation [56]. Incidentally, an ANG based pharmaceutical composition has been

329

patented to treat FECD [57]. Formation of guttae in FECD has been shown to affect

330

the migration of corneal endothelial cells (CEnC) [58] where IL- 17F has been shown

331

to play a crucial role in endothelial cell migration [59]. Interestingly, we found

332

significant reduction in IL-17F levels in FECD tear fluid which may reflect the effects

333

of the aberrant production of extra cellular matrix (ECM) proteins and guttae

334

formation observed in the FECD. Along with IL-17F we found IL-1β, IP-10, MIG levels

335

to be significantly reduced in FECD while the levels of IL-6, IL-8 and MCP-1

336

remained unchanged compared with the control. De Roo et al [60] reported

337

unchanged IL-6, IL-8 and MCP-1 levels in the aqueous humor of FECD patients

338

which corroborates with our observations. We found the levels of TGFβ1 to be

339

significantly increased in CD cases compared with the controls. TGFβ signaling has

340

been shown to be a major player in the extracellular matrix (ECM) deposit formation

341

[42] which in turn triggers the intrinsic apoptotic pathway leading to cell death in

342

FECD [61]. Reducing TGFβ levels have been demonstrated to effectively diminish

343

edema, reduce opacification and inhibit inflammatory cell infiltration [62]. Moreover,

344

TGFβ signaling has been shown to modulate TGFBIp expression [63] which is one of

345

the key proteins in the cornea [64]. We found TGFBIp levels significantly higher in

346

the tears of CD cohort compared with the control. In LCD (which arise from mutations

347

in the TGFBIp gene [65] ) tears, the expression of TGFBIp was significantly higher 14

348

compared with the control and other SCD, thereby indicating a distinct pattern of

349

TGFBIp levels in the tear fluid of different types of SCD. TGFBIp apart from being the

350

most abundant in the CEnC layer has been shown to be co-localized in the guttae,

351

which is a key pathological hallmark of FECD [66]. The pathogenic role of TGFBIp in

352

the formation of guttae [67] suggests that the analysis of FECD patient tear fluid for

353

TGFBIp expression might represent as an additional diagnostic tool reflecting the

354

status of the CEnC layer. Our observation of the pronounced and significant increase

355

in the inflammatory cytokines and fibrogenic factors in CD compared to the control

356

suggests modulating these factors may have therapeutic potential for CD.

357

5. Conclusion:

358

Observations from the current study demonstrate an altered, disease specific ocular

359

surface inflammatory profile in the tears of CD subjects. The aberrant inflammatory

360

factors could be associated with the progression of CD. Therefore, dampening ocular

361

surface inflammation may be used as a prophylactic strategy in retarding the CD

362

progression and/ or improving graft outcomes. In addition, our study highlights the

363

potential of tear fluid as an additional diagnostic tool for evaluating CD and its

364

objective monitoring.

365

6. Acknowledgements

366

This study is supported by the Narayana Nethralaya Foundation (NNF). KC is funded

367

by BT/PR26190/GET/119/118/2017 grant from the Department of Biotechnology,

368

Government of India, and Narayana Nethralaya Foundation (NNF). AG is funded by

369

EMR/2016/003624 grant from Science and Education Research Board (SERB),

370

Department of Science and Technology, Govt. of India. The authors thank Harsha

15

371

Nagaraj (Cornea consultant, Narayana Nethralaya, Bangalore) for his assistance in

372

collecting the tear samples.

373

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Figure Legends Figure 1: Total protein concentration in the tear fluid of controls and corneal dystrophy patients. The graph indicate the total protein concentration (µg/ml) in tear fluid of control subjects (18 eyes; n=17) and CD patients (48 eyes; n=30). Bar graph depicts Mean±SEM; ****P<0.0001, Mann-Whitney test; SEM - Standard error of mean. Ctrl - Controls; CD - Corneal Dystrophy. Figure 2: Percentage of tear samples with detectable levels of soluble factors in controls and corneal dystrophy patients. Bar graph depicts the percentage of tear samples with detectable levels of analytes in control subjects (18 eyes; n=17) and CD patients (48 eyes; n=30). Mean ±SEM; **P<0.001, Mann-Whitney test; SEMStandard error of mean (b) Graph depicts the percentage of tear samples with detectable levels of analytes in controls and corneal dystrophy. Figure 3: Significantly altered tear soluble factors in different types of corneal dystrophies. Box-and-whisker plots depict levels of analytes in the tear fluid of control subjects (18 eyes, n=17), epithelial corneal dystrophy (4 eyes, n=4), macular corneal dystrophy (13 eyes, n=8), granular corneal dystrophy (16 eyes, n=9), lattice corneal dystrophy (9 eyes, n=6) and Fuch’s endothelial corneal dystrophy (6 eyes, n=3). The boxes represent the 25th to 75th percentiles. The whiskers represent the lowest and highest value (pg/µg) and horizontal line within the box represent median values. Mean±SEM; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Kruskal-Wallis test. C - Controls; E - Epithelial Corneal Dystrophy; M - Macular Corneal Dystrophy; G - Granular Corneal Dystrophy; L- Lattice Corneal Dystrophy; F- Fuch’s Endothelial Corneal Dystrophy; ND - Not Detected; SEM - Standard error mean. Figure 4: Correlations patterns between the tear soluble factors in controls and CD. Heatmap depicts the correlation patterns among the analytes within the study 1

groups. Spearman’s rank coefficients (P<0.05) was used to generate the heatmap to determine the differences in the relationship within the analytes in controls and CD samples. The color green denotes positive correlation coefficient and the color red denotes negative correlation coefficient. Correlation coefficient color scale -1.0 in color green denote positive correlation coefficient and +1.0 in red color denote negative correlation coefficient. Figure 5: Tear soluble factors signature in different types of corneal dystrophies. The rows of the heatmap denote the different analytes measured in the tear fluid. The columns depict different samples of epithelial, granular, lattice, macular and Fuch’s corneal dystrophy. The color scale corresponds to the levels of analytes; with Min=0 and Max=214 arbitrary units. The grey color cell represents sample not run (NR). Figure 6: The schema indicates the sites of different types of corneal dystrophies and associated dysregulated tear factors. The upper panel depicts the anatomical location of the various corneal dystrophies. The lower panel lists the significantly altered tear factors in CD compared with the controls. This suggests plausible dysregulation of the ocular surface inflammatory status in CD and the potential to use anti-inflammatory agents in its management. Red up-ward arrow indicates analytes significantly increased levels compared with the controls. Blue down-ward arrow indicates analytes significantly decreased levels compared with the controls. Corneal epithelial cells (CECs); Epithelial corneal dystrophy (ECD); Granular corneal dystrophy (GCD); Lattice corneal dystrophy (LCD); Macular corneal dystrophy (MCD) are the Stromal corneal dystrophies (SCD). Corneal endothelial cells (CEnCs) and the Descemet’s membrane (DM) is affected in Fuch’s endothelial corneal dystrophy (FECD). 2

Table 1: Cohort characteristics Corneal dystrophy Control

N Age (Mean±SD) Sex (M/F) Total

17 30 ± 4 11/6 17

Epithelial

Stromal

Endothelial

EBMD/MDFPD 4

MCD 8

GCD 9

LCD 6

FECD 3

23 ± 8

31± 16

30 ± 11

21±22

43±10

1/3

3/5

4/2

1/2

3/6 30

Epithelial basement membrane dystrophy – EBMD; Map-dot-fingerprint dystrophy – MDFPD, Macular corneal dystrophy – MCD, Granular corneal Dystrophy – GCD, Lattice corneal Dystrophy – LCD, Fuch’s endothelial corneal dystrophy – FECD.

Table 2: Percentage of tear samples with detectable levels of the various analytes measured in controls and corneal dystrophy patients. Class

Cytokines & Chemokines

Soluble cell adhesion molecules

Growth factors and others

Analytes IL-1α IL-1β IL-2 IL-4 IL-6 IL-8 IL-12/IL-23p40 IL-13 IL-17A IL-17F ANG Eotaxin Fractalkine IFNα IP-10 ITAC MCP-1 MIG RANTES ICAM-1 VCAM L-selectin P-selectin TGFBIp TGFβ1

Ctrl 10/18 11/18 7/18 8/18 11/18 18/18 17/18 13/18 10/18 10/18 18/18 12/18 13/18 11/18 18/18 16/18 16/18 17/18 14/18 12/18 12/18 17/18 17/18 18/18 7/18

Detected % 56 61 39 44 61 100 94 72 56 56 100 67 72 61 100 89 89 94 78 67 67 94 94 100 39

CD 42/48 41/48 48/48 36/48 41/48 48/48 44/48 45/48 33/48 47/48 42/48 40/40 44/48 45/48 47/48 48/48 45/48 48/48 48/48 47/48 46/48 44/48 39/48 48/48 26/48

Detected% 88 85 100 75 85 100 92 94 69 98 88 83 92 94 98 100 94 100 100 98 96 92 81 100 54

VEGF IgE

18/18 9/18

100 50

47/48 34/48

98 71

Table shows the percentage of the analytes detected in the controls (18 eyes) and corneal dystrophy (48 eyes). Ctrl – Controls; CD – Corneal Dystrophy

Table 3: Concentration of tear fluid soluble factors in controls and corneal dystrophy patients Ctrl (pg/µg) Class

Cytokines & Chemokines

Soluble cell adhesion molecules

Growth factors & others

P value

FC (Ctrls vs CD)

0.040

ns

2

0.004

0.01

ns

1

1.29

0.15

0.94

****

16

0.001

0.07

0.02

0.03

****

35

0.001

0.007

0.09

0.04

0.014

ns

15

0.54

0.11

0.52

4.05

1.38

1.04

**

7

IL-12/IL-23p40

3.21

0.89

2.61

5.27

0.76

3.36

ns

2

IL-13

0.02

0.01

0.005

0.09

0.01

0.059

****

5

IL-17A

0.002

0.0004

0.002

0.01

0.003

0.002

ns

5

IL-17F

3.45

0.38

2.94

2.65

0.55

0.73

*

-1

ANG

311

48

259

8019

1982

1836

*

26

Eotaxin

0.04

0.02

0.006

0.14

0.02

0.091

**

3

Fractalkine

1.56

0.48

0.75

7.35

1.43

3.93

**

5

IFNα

0.02

0.01

0.003

0.1

0.02

0.06

***

5

Analytes

CD (pg/µg)

Mean

SEM

Median

Mean

SEM

Median

IL-1α

0.03

0.01

0.01

0.07

0.01

IL-1β

0.02

0.001

0.01

0.02

IL-2

0.08

0.04

0.005

IL-4

0.002

0.001

IL-6

0.006

IL-8

IP-10

71

21

54.37

699

344

28.25

ns

10

ITAC

5.12

1.19

5.15

4.64

0.58

3.11

ns

-1

MCP-1

0.49

0.18

0.08

0.59

0.09

0.34

ns

1

MIG

1.94

0.64

1.36

2.11

0.45

0.57

ns

1

RANTES

0.07

0.02

0.016

0.2

0.03

0.14

**

3

ICAM-1

6.58

2.59

0.61

20.32

3.9

11.76

**

3

VCAM

0.79

0.27

0.1

5.24

1.04

1.95

**

7

L-selectin

6.96

1.68

7.32

11.62

2.52

4.98

ns

2

P-selectin

0.78

0.31

0.15

1.09

0.24

0.5

*

1

TGFBIp

2.7

0.05

2.26

8.13

1.4

4.65

**

3

TGFβ1

0.18

0.05

0.11

12.63

2.59

6.94

****

69

VEGF

3.42

0.85

2.8

3.83

0.76

1.87

ns

1

IgE

0.09

0.06

0.01

53.36

8.7

37.23

****

589

Table showing the mean and median concentration of analytes (pg/µg) in tear fluid of control subjects (18 eyes, n= 17) and corneal dystrophy (48 eyes, n=30) patients. Ctrl – Controls; CD – Corneal Dystrophy; SEM –Standard error mean; FC –Fold change; ns –not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, MannWhitney Test (versus controls). Picogram and microgram denoted as pg and µg, respectively.

Table 4: Concentration of tear fluid soluble factors in epithelial corneal dystrophy patients

Mean

SEM

Median

Mean

SEM

Median

P value

IL-1α

0.03

0.01

0.01

0.18

0.01

0.19

**

FC (Ctrls vs ECD) 6

IL-1β

0.02

0.001

0.01

0.09

0.01

0.088

**

5

IL-2

0.08

0.04

0.005

1.66

0.07

1.72

***

22

IL-4

0.002

0.001

0.001

ND

ND

ND

na

na

IL-6

0.006

0.001

0.007

0.43

0.17

0.312

ns

71

IL-8

0.54

0.11

0.52

3.59

0.2

3.77

**

7

IL12/IL23p40

3.21

0.89

2.61

11.19

0.57

11.19

*

3

IL-13

0.02

0.01

0.005

0.16

0.01

0.159

***

9

IL-17A

0.002

0.0004

0.002

ND

ND

ND

na

na

IL-17F

3.45

0.38

2.94

5.57

0.27

5.61

ns

2

ANG

311

48

259

38677

4577

42462

**

124

Eotaxin

0.04

0.02

0.006

0.51

0.12

0.446

***

12

Fractalkine

1.56

0.48

0.75

15.87

1.17

15.5

**

10

IFN-α

0.02

0.01

0.003

0.17

0

0.17

***

8

IP-10

71

21

54.37

465

115

414.6

ns

7

ITAC

5.12

1.19

5.15

6.91

0.22

6.75

ns

1

MCP-1

0.49

0.18

0.08

1.17

0.07

1.2

*

2

MIG

1.94

0.64

1.36

3.51

0.26

3.43

ns

2

RANTES

0.07

0.02

0.016

0.25

0.01

0.24

**

4

ICAM-1

6.58

2.59

0.61

31.8

2.44

32.31

**

5

VCAM

0.79

0.27

0.1

10.14

0.42

10.41

**

13

L-selectin

6.96

1.68

7.32

23.4

2.2

23.33

*

3

P-selectin

0.78

0.31

0.15

5.27

1.23

3.98

**

7

TGFBIp

2.7

0.05

2.26

7.3

4.5

3.95

ns

3

TGFβ1

0.18

0.05

0.11

ND

ND

ND

na

na

VEGF

3.42

0.85

2.8

4.97

0.07

5.01

ns

1

IgE

0.09

0.06

0.01

ND

ND

ND

na

na

Ctrl (pg/µg) Class

Cytokines & Chemokines

Soluble cell adhesion molecules

Growth factors & others

Analytes

ECD (pg/µg)

Table shows the mean and median concentration of analytes (pg/µg) in tear fluid of control subjects (18 eyes, n= 17) and epithelial corneal dystrophy (4 eyes, n=4) patients. Ctrl – Controls; ECD – Epithelial Corneal Dystrophy; SEM – Standard error mean; FC – Fold change; ND – not detected; na – not applicable; ns – not significant; *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney Test (versus controls). Picogram and microgram denoted as pg and µg, respectively.

Table 5a: Concentration of tear fluid soluble factors in stromal corneal dystrophy patients

Mean

SEM

Median

Mean

SEM

Median

IL-1α

0.03

0.01

0.01

0.07

0.01

0.047

ns

FC (Ctrls vs SCD) 2

IL-1β

0.02

0.001

0.01

0.02

0

0.012

ns

1

IL-2

0.08

0.04

0.005

1.37

0.19

0.96

***

18

IL-4

0.002

0.001

0.001

0.08

0.02

0.04

***

40

IL-6

0.006

0.001

0.007

0.06

0.04

0.017

ns

10

IL-8

0.54

0.11

0.52

4.72

1.72

1.13

**

9

IL-12/IL-23p40

3.21

0.89

2.61

5.43

0.85

3.45

ns

2

IL-13

0.02

0.01

0.005

0.1

0.02

0.06

***

6

IL-17A

0.002

0.0004

0.002

0.01

0.0034

0.003

ns

6

IL-17F

3.45

0.38

2.94

2.75

0.66

0.88

ns

-1

Ctrl (pg/µg) Class

Cytokines & Chemokines

Soluble cell adhesion molecules

Growth factors & others

Analytes

SCD (pg/µg) P value

ANG

311

48

259

9235

2866

2209

*

30

Eotaxin

0.04

0.02

0.006

0.11

0.01

0.09

**

3

Fractalkine

1.56

0.48

0.75

7.56

1.68

4.6

**

5

IFNα

0.02

0.01

0.003

0.12

0.03

0.07

**

6

IP-10

71

21

54.37

837

434

31.55

ns

12

ITAC

5.12

1.19

5.15

4.99

0.68

3.64

ns

-1

MCP-1

0.49

0.18

0.08

0.61

0.11

0.34

ns

1

MIG

1.94

0.64

1.36

2.28

0.54

0.68

ns

1

RANTES

0.07

0.02

0.016

0.22

0.03

0.15

**

3

ICAM-1

6.58

2.59

0.61

22

4.81

13.39

*

3

VCAM

0.79

0.27

0.1

5.51

1.26

2.39

**

7

L-selectin

6.96

1.68

7.32

12.1

3.09

5.44

ns

2

P-selectin

0.78

0.31

0.15

0.86

0.13

0.56

ns

1

TGFBIp

2.7

0.05

2.26

7

1.3

4.32

ns

3

TGFβ1

0.18

0.05

0.11

16

2.96

12.93

***

87

VEGF

3.42

0.85

2.8

4.29

0.94

2.12

ns

1

IgE

0.09

0.06

0.01

63.4

9.55

54.7

***

700

Table shows the mean and median concentration of analytes (pg/µg) in tear fluid of control subjects (18 eyes, n= 17) and stromal corneal dystrophy (38 eyes, n=23) patients. Ctrl – Controls; SCD – Stromal Corneal Dystrophy; SEM – Standard error mean; FC – Fold change; ND – not detected; na – not applicable; ns – not significant; *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney Test (versus controls). Picogram and microgram denoted as pg and µg, respectively.

Table 5b: Concentration of tear fluid soluble factors in different types of stromal corneal dystrophy patients

Mean

SEM

Median

Mean

SEM

Median

P value

IL-1α IL-1β IL-2 IL-4 IL-6 IL-8 IL12/IL23p40 IL-13 IL-17A

0.03 0.02 0.08 0.002 0.006 0.54 3.21 0.02 0.002

0.01 0.01 0.005 0.001 0.007 0.52 2.61 0.005 0.002

0.07 0.03 1.26 0.12 0.11 2.86 4.83 0.07 0.01

0.02 0.01 0.26 0.04 0.08 1.59 1 0.01 0.01

0.03 0.009 0.9 0.04 0.01 0.78 2.79 0.04 0.011

ns ns ** ** ns ns ns ns ns

IL-17F ANG Eotaxin Fractalkine IFNα IP-10 ITAC MCP-1 MIG RANTES

3.45 311 0.04 1.56 0.02 71 5.12 0.49 1.94 0.07

0.01 0.001 0.04 0.001 0.001 0.11 0.89 0.01 0.000 4 0.38 48 0.02 0.48 0.01 21 1.19 0.18 0.64 0.02

FC (Ctrls vs GCD) 2 2 16 60 18 5 2 4 6

2.94 259 0.006 0.75 0.003 54.37 5.15 0.08 1.36 0.016

2.39 7556 0.12 6.08 0.08 861 4.36 0.48 1.67 0.18

0.97 2580 0.02 1.81 0.01 739 0.95 0.11 0.67 0.04

0.84 1569 0.09 2.71 0.056 38.14 3.08 0.3 0.59 0.14

*** ns ns *** * ns ns ns ns ns

Soluble cell adhesio n molecul es

ICAM-1 VCAM L-selectin P-selectin TGFBIp

6.58 0.79 6.96 0.78 2.7

2.59 0.27 1.68 0.31 0.05

0.61 0.1 7.32 0.15 2.26

21 3.53 9.03 1.04 5.7

8.72 0.97 2.81 0.25 1.8

11.07 1.65 4.28 0.49 3.98

Growth factors & others

TGFβ1 VEGF IgE

0.18 3.42 0.09

0.05 0.85 0.06

0.11 2.8 0.01

10.67 4.09 64.81

3.07 1.62 19.4

10.01 2.36 40.73

Ctrl (pg/µg) Class

Cytokin es & Chemok ines

Analytes

Mean

SEM

Median

P value

0.09 0.014 1.89 0.05 0.02 10 5.07 0.09 0.010

0.05 0.004 0.6 0.01 0.01 6 1.68 0.03 0.004

0.04 0.012 1.17 0.042 0.016 1.15 3.36 0.05 0.005

ns ns *** * ns ns ns ns ns

FC (Ctrls vs LCD) 3 1 25 25 3 19 2 5 5

-1 24 3 4 4 12 -1 1 -1 3

3.17 3857 0.1 7.21 0.09 1547 6.27 0.57 3.49 0.31

1.61 1769 0.02 3.64 0.03 1391 1.83 0.24 1.57 0.11

1.09 1950 0.093 3.27 0.059 47.4 3.99 0.32 1.11 0.18

** ns ns ** ns ns ns ns ns ns

ns ** ns ns ns

3 4 1 1 2

23 6.93 11.2 0.63 10.2

10 3.49 6.1 0.09 1.5

12.8 2.68 4.66 0.56 9.36

* ns ***

58 1 716

14 5 57

5 2 13

13.3 2.67 60.71

GCD (pg/µg)

Mean

SEM

Median

0.05 0.015 1.14 0.05 0.02 3.3 6.38 0.13 0.01

0.01 0.003 0.13 0.01 0.004 1.21 1.88 0.04 0.01

0.05 0.013 0.96 0.049 0.024 1.121 4.03 0.06 0.014

ns ns ** ** ns ns ** ns

FC (Ctrls vs MCD) 1 1 15 25 3 6 2 7 5

-1 12 2 5 4 22 1 1 2 4

2.93 3715 0.11 9.61 0.18 372 5 0.78 2.2 0.2

1.14 1226 0.02 3.84 0.06 177.7 1.05 0.23 0.84 0.05

0.88 2470 0.097 4.63 0.07 31.5 3.25 0.36 0.6 0.14

*** ns ns *** ** ns ns ns ns ns

-1 12 3 6 8 5 1 2 1 3

ns ** ns ns *

3 9 2 -1 4

23 7 16 0.75 7

6.88 3 7 0.14 3

13.3 2.65 5.65 0.64 3.35

ns * ns ns ns

3 9 2 1 3

* ns **

74 1 628

23 4.16 65

6 1.29 13

20.66 2.12 68.21

*** ns ***

123 1 723

LCD (pg/µg)

MCD (pg/µg) P value

Table describes the mean and median concentration of analytes (pg/µg) in tear fluid of control subjects (18 eyes, n=17), granular corneal dystrophy (16 eyes, n=9), lattice corneal dystrophy (9 eyes, n= 6) and macular corneal dystrophy (13 eyes, n=8). Ctrl – Controls; GCD – Granular Corneal Dystrophy; LCD – Lattice Corneal Dystrophy; MCD – Macular Corneal Dystrophy; SEM – Standard error mean; FC – Fold change; ns – not significant; *P<0.05, **P<0.01, ***P<0.001, Mann-Whitney Test (versus controls). Picogram and microgram denoted as pg and µg, respectively.

Table 6: Concentration of tear fluid soluble factors in endothelial corneal dystrophy patients Class

Cytokines & Chemokines

Soluble cell adhesion molecules

Growth factors & others

Ctrl (pg/µg)

EnCD (pg/µg)

P value

FC (Ctrls vs EnCD)

ns

-7

0.0014

**

-12

0.02

0.55

ns

7

0.003

0.0006

0.003

ns

1

0.007

0.0006

0.0002

0.0006

ns

-11

0.11

0.52

0.11

0.03

0.11

ns

-5

3.21

0.89

2.61

0.47

0.11

0.48

ns

-7

IL-13

0.02

0.01

0.005

0.014

0.0033

0.013

ns

-1

IL-17A

0.002

0.0004

0.002

0.0004

6E-05

0.0003

ns

-5

IL-17F

3.45

0.38

2.94

0.12

0.02

0.13

***

-28

ANG

311

48

259

15.6

3.62

17.14

ns

-20

Eotaxin

0.04

0.02

0.006

0.0204

0.0027

0.019

ns

-2

Fractalkine

1.56

0.48

0.75

0.51

0.03

0.49

ns

-3

IFNα

0.02

0.01

0.003

0.014

0.0037

0.01

ns

-2

IP-10

71

21

54.37

2.19

0.4

2.18

*

-32

ITAC

5.12

1.19

5.15

0.85

0.12

0.71

ns

-6

MCP-1

0.49

0.18

0.08

0.06

0.01

0.06

ns

-8

MIG

1.94

0.64

1.36

0.07

0.0035

0.07

*

-27

RANTES

0.07

0.02

0.016

0.0274

0.0047

0.0307

ns

-3

ICAM-1

6.58

2.59

0.61

2.28

0.35

2

ns

-3

VCAM

0.79

0.27

0.1

0.39

0.04

0.37

ns

-2

L-selectin

6.96

1.68

7.32

1.09

0.14

0.919

ns

-6

P-selectin

0.78

0.31

0.15

0.15

0.03

0.14

ns

-5

TGFBIp

2.7

0.05

2.26

20

8.3

17.43

***

7

TGFβ1

0.18

0.05

0.11

1.32

0.3

1.29

ns

7

VEGF

3.42

0.85

2.8

0.29

0.05

0.32

ns

-12

IgE

0.09

0.06

0.01

6.68

1.39

6.5

ns

74

Analytes IL-1α

Mean 0.03

SEM 0.01

Median 0.01

Mean 0.0048

SEM 0.0011

Median 0.005

IL-1β

0.02

0.001

0.01

0.0013

0.0003

IL-2

0.08

0.04

0.005

0.54

IL-4

0.002

0.001

0.001

IL-6

0.006

0.001

IL-8

0.54

IL12/IL23p40

Table showing the mean and median concentration of analytes (pg/µg) in tear fluid of control subjects (18 eyes, n= 17) and endothelial corneal dystrophy (6 eyes, n=3) patients. Ctrl – Controls; EnCD – Endothelial Corneal Dystrophy; SEM – Standard error mean; FC – Fold change; ns – not significant; *P<0.05, **P<0.01, ***P<0.001, MannWhitney Test (versus controls). Picogram and microgram denoted as pg and µg, respectively.

Supplementary Table 1 Corneal dystrophy Epithelial EBMD/

Reference number

Endothelial

MCD

GCD

LCD

FECD

TGFBIp

CHST6

TGFBIp

TGFBIp, GSN

Col8A2, SLC4A11, ZEB1

1, 2, 69

1, 2, 68, 70

1, 2, 68

1, 2, 68

1, 2, 71

MDFPD Implicated Gene mutations

Stromal

Table shows the genes implicated in CD and respective references. Epithelial basement membrane dystrophy – EBMD; Map-dot-fingerprint dystrophy – MDFPD, Macular corneal dystrophy – MCD, Granular corneal Dystrophy – GCD, Lattice corneal Dystrophy – LCD, Fuch’s endothelial corneal dystrophy – FECD. Transforming growth factor beta induced protein (TGFBIp) gene mutations are implicated in EBMD/ MDFPD [1, 2, 69]. Mutations in the TGFBIp and Gelsolin (GSN) gene have been associated with LCD and GCD [1, 2, 68]. Mutations in the carbohydrate sulfotransferase 6 (CHST6) gene is the underlying cause for MCD [1, 2, 68, 70]. Genes associated with FECD [1, 2, 71] are: Collagen Type VIII Alpha 2 Chain (Col8A2); sodium bicarbonate transporter-like protein 11 (SLC4A11 gene) and zinc finger E-box-binding homeo-box 1 (ZEB1).