Proteomics Differentiate Between Thyroid-Associated Orbitopathy and Dry Eye Syndrome

Immunology and Microbiology Proteomics Differentiate Between Thyroid-Associated Orbitopathy and Dry Eye Syndrome

Nina Matheis,1,2 Franz H. Grus,1,3 Matthias Breitenfeld,2 Ivo Knych,2 Sebastian Funke,1 Susanne Pitz,3 Katharina A. Ponto,3 Norbert Pfeiffer,3 and George J. Kahaly2,4

1Experimental Ophthalmology, Johannes Gutenberg University Medical Center, Mainz, Germany 2Molecular Thyroid Research Laboratory, Johannes Gutenberg University Medical Center, Mainz, Germany 3Department of Ophthalmology, Johannes Gutenberg University Medical Center, Mainz, Germany 4Department of Medicine, Johannes Gutenberg University Medical Center, Mainz, Germany

Correspondence: George J. Kahaly, PURPOSE. In patients with thyroid-associated orbitopathy (TAO), the dry eye syndrome occurs JGU Medical Center, Mainz 55101, frequently, and symptoms and signs of both disorders overlap making early and accurate Germany; differential diagnosis difficult. A differentiation via specific markers is warranted. [email protected]. METHODS. Tear fluid samples of 120 subjects with TAO, TAO þ dry eye, dry eye, and controls Submitted: February 17, 2015 were collected. The samples were measured using matrix-assisted laser desorption ionization Accepted: March 21, 2015 mass spectrometry. The identified proteins were tested with antibody microarrays. Citation: Matheis N, Grus FH, Breiten- feld M, et al. Proteomics differentiate RESULTS. Proteomics identified deregulated proteins in TAO and dry eye. Compared with dry between thyroid-associated orbitop- eye, proline-rich protein 1 (PROL1, P ¼ 0.002); uridine diphosphate (UDP)–glucose- athy and dry eye syndrome. Invest dehydrogenase (UGDH, P ¼ 0.017); calgranulin A (S10A8, P < 0.0001); transcription- Ophthalmol Vis Sci. 2015;56:2649– activator BRG1 (SMCA4, P < 0.0001); annexin A1 (P ¼ 0.007); cystatin (P ¼ 0.009); heat 2656. DOI:10.1167/iovs.15-16699 shock protein 27 (P ¼ 0.03); and galectin (P ¼ 0.04) were markedly downregulated in TAO. Compared with healthy controls, PROL1 (P < 0.05.); proline-rich protein 4 (PRP4, P < 0.05), S10A8 (P ¼ 0.004) and SMCA4 (P ¼ 0.002) were downregulated in TAO. In contrast, the proteins midasin and POTE-ankyrin–domain family-member I were upregulated in TAO versus healthy controls (P < 0.05). Protein dysregulation was associated with inflammatory response and cell death. Antibody microarray confirmed significant changes of PRP4, PROL1, and UGDH between TAO and dry eye or healthy controls (P < 0.01). The presence of these three proteins was negatively correlated with smoking (P < 0.05).

CONCLUSIONS. Proteomics of tear fluid demonstrated an upregulation of inflammatory proteins versus a downregulation of protective proteins in TAO, and a significantly different protein panel in TAO versus dry eye and/or controls. The spectrum of inflammatory and protective proteins might be a useful indicator for disease activity and ocular surface disease in patients with TAO. Keywords: proteomics, tear fluid, thyroid-associated orbitopathy, dry eye syndrome, antibody microarrays

hyroid-associated orbitopathy (TAO) is the most frequent In thyroid-associated orbitopathy, circulating pro-inflamma- T extrathyroidal manifestation in patients with autoimmune tory cytokines (e.g., TNF alpha) increase the expression of the thyroid disease.1 In patients with TAO, involvement of the Fas molecule on the surface of the lacrimal cells.13 This leads to lacrimal gland and a reduced tear production has been apoptosis of the target cells and to the release of a membrane reported.2,3 Eye lid retraction, an abnormally wide palpebral bound 264 KD protein, fodrin, which is known as the fissure, insufficient eyelid closure, proptosis, reduced blinking, autoantigen in patients with Sjögren syndrome.14 Sjögren and impaired Bell’s phenomenon are factors regarded to syndrome, frequently observed in patients with various contribute to an increased tear film evaporation.4 Also, the autoimmune diseases, encompasses symptoms and signs of composition of tear fluid is altered in TAO.5–7 Thus, patients dry eye. with TAO often exhibit symptoms and signs of the dry eye In previous studies, we have shown that protein profiles of syndrome8 and the combination of both makes an early and TAO patients differ from those in healthy controls using the accurate differential diagnosis difficult. The quality of life and surface enhanced laser desorption ionization time of flight mass visual functioning of these patients is markedly impaired.9 Dry spectrometry (SELDI-TOF MS) technology.15 These proteins eye is defined by an abnormal tear film that results in changes of were identified with the help of the matrix assisted laser the ocular surface often accompanied by ocular discomfort. Its desorption ionization time of flight (MALDI-TOF) technology.5 symptoms may include blurred vision, burning, itchiness, In the present prospective and controlled study, we aimed to redness or grittiness in the eye, and sensitivity to light.10 Dry identify specific protein patterns in the tear fluid of patients eye is a common disease and has an increased prevalence in with TAO. To gain more insight into the relation of TAO and dry people with autoimmune disease and thyroid disorders.11–12 eye on tear composition, we compared the protein expression

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2650

in the tear fluid of TAO patients with and without concomitant 2465.19, Somatostatin 28 3147.47; Bruker Daltonics). Data dry eye syndrome and in healthy controls. Thus, we analyzed processing of raw spectra, peak detection, and protein tear samples of patients with TAO, dry eye, combined TAO þ identification were performed using commercial software dry eye, and controls with the MALDI-TOF MS to detect and (Flex analysis 2.4 and BioTools 3.1; Bruker Daltonics; and identify disease specific altering proteins. To verify the MALDI MASCOT (Matrix Science, Boston, MA, USA). The MALDI results, we performed antibody microarray studies. spectra obtained were used for database searches with MASCOT using SwissProt release 11_1 (Swiss Institute of Bioinformatics, Geneva, Switzerland) database. MASCOT com- METHODS pares the peptide and lift spectra against peptide patterns in the database and searches for homologies. In this case the Subjects BioTools Software which is linked with the MASCOT-server A total of 120 subjects were included in the study. Of those, 60 was used. If the data of the spectra are matching the data in the patients had various degrees of clinical activity and severity of database, the probability is measured if this was a contingency. TAO with and without concomitant dry eye syndrome, 30 The protein queries were run under MudPIT scoring condi- patients had dry eye syndrome only, and 30 were healthy, tions with a significance threshold of P < 0.05 for protein euthyroid control persons. All patients and controls gave their identification. written informed consent. The protocol was approved by the local Ethics Committee of the state Rhineland Palatinate, Antibody Microarrays Germany, in accordance with the tenets of the Declaration of The proteins with significant associations in mass spectrome- Helsinki. At the joint thyroid eye clinic of the JGU Medical try and differing between TAO, dry eye, and controls were Center, complete endocrine and ophthalmic investigations investigated with antibody microarrays. Antibodies against the were performed prior to tear sampling. Diagnosis and detected proteins were spotted on nitrocellulose slides definition of clinical activity and severity of TAO followed the (OnCyte; Grace Bio-Labs, Bend, OR, USA) with a spotter criteria recommended by the Consensus Statement of the (Scienion AG, Berlin, Germany) with three drops per spot. The European Group on Graves’ orbitopathy.16 Further information arrays were blocked with blocking substance (Super G was obtained using a questionnaire retrieving patients’ clinical blocking buffer; Grace Bio-Labs) for 1 hour. The samples were history and previous treatment. All participants underwent the labeled with fluorescent dye cy5 at RT for 1 hour in the dark. following ophthalmic investigations: Schirmer’s test with The slides were incubated with the labeled samples for 2 hours anaesthesia (STA), measurement of tear film breakup time, at 4 C shaking. After washing with 0.5% Tween PBS, the slides staining of the conjunctiva (Oxford system), and slit lamp 8 were scanned with 10 db. We used commercial software examination of lid parallel conjunctival folds. (Imagene; BioDiscovery, Inc., Hawthorne, CA, USA) for spot analysis. Tear Fluid Collection To obtain samples of tear fluid from all 120 subjects, STA was Data Analysis performed and the Schirmer strips were stored 808C until use. 2 The Schirmer strips were eluted with 0.1% dodecyl maltoside. With the help of the P M (Proteomics Pipeline Mainz, Subsequently, the samples were precipitated with 100% ice Experimental Ophthalmology, Mainz, Germany), software intensities of identified proteins were normalized and clus- cold acetone. Per study collective, 30 samples were pooled in 17 five samples per pool resulting in six pool groups. A total of 10 tered. Corresponding cluster lists containing the normalized lg protein of each sample was pooled, allowing at least 50 lg peak intensity values and the identifications for each sample protein per pool group to be applied on a SDS Gel. were exported to a statistical analysis program (Statistica, version 6.2; StatSoft, Tulsa, OK, USA). After testing for normal distribution, we used the Kruskal-Wallis test based on MALDI Mass Spectrometry and Data Acquisition combinations of multiple peaks for analysis. This analysis The samples were separated on a 1D SDS–PAGE. Per lane and calculates the most significant proteins differing over all pool group, 50 lg(53 10 lg) were applied on the gel. An groups. Further, a post hoc test was run to perform a pairwise overnight in gel digestion and an elution of the peptides testing between the groups. For analysis of the microarray data, followed. The samples were evaporated and adjusted to pH an ANOVA and subsequently a post hoc analysis were 4 with 0.5% trifluoroacetic acid for fractionation with zip tips performed. (Zip Tip Pipette Tips; Merck Millipore, Darmstadt, Germany) according to the manufacturer’s protocol. The samples were eluted in three steps with 15%, 35%, and 50% acetonitrile RESULTS solution and then directly spotted to a steel target. The sample Demographic, Clinical, and Serological Data was cocrystallized with an energy-absorbing matrix (cinnamic acid). Data acquisition was accomplished using a MALDI-TOF/ The demographic data of all subjects included in the study are TOF mass spectrometer (Ultraflex II TOF/TOF; Bruker summarized in Table 1. Baseline serum thyroid stimulating Daltonics, Billerica, MA, USA) with a nitrogen laser. After hormone (TSH) levels were within the normal range (median acquiring the digest spectra with 100 laser shots averaged from TSH: 1.23 mU/L; range, 0.45–3.3 mU/L) in patients with dry eye five sample positions in the linear mode, peptides below m/z and in healthy controls, respectively. Table 2 also shows the dry 4000 Da with good peak intensity were selected for eye related test results in all subjects. Clinical and serological fragmentation analysis using a reflector mode. Peptide data of the 60 patients with TAO are summarized in Table 3 fragmentation was performed using collision-induced dissoci- showing no significant differences between the two groups. ation and 50 laser shots from five sample positions were summed up for each parent ion. All spectra were externally Mass Spectrometry calibrated by using the peptide calibration standard (Angio- tensin II 1047, 19 Angiotensin I 1297.49, Substance P 1348.64, A total of 69 proteins with over 400 peptides were identified Bombesin 1620.86, ACTH clip 1-17 2093.08, ACTH clip 18-39 by the Mascot method with a peptide mass tolerance of 6100

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2651

TABLE 1. Demographic Data

TAO, n ¼ 30 TAO þ Dry Eye, n ¼ 30 Dry Eye, n ¼ 30 Healthy Controls, n ¼ 30

Males, n (%) 7 (23.3) 5 (16.6) 7 (23.3) 3 (10) Females, n (%) 23 (76.6) 25 (83.3) 23 (76.6) 27 (90) Age, y (median) 45.5 (17–68) 51 (31–70) 54.5 (32–80) 47.5 (21–70) Smokers, n (%) 13 (43.3) 15 (50) 5 (16.6) 9 (30) Graves’ disease, n (%) 27 (90) 29 (96.6) Hashimoto’s thyroiditis, n (%) 3 (10) 1 (3.3)

TABLE 2. Clinical Parameters

TAO TAO þ Dry Eye Dry Eye Healthy Controls

STA, mm (normal range, ‡10) 22.5 (6.5–29.5) 6.5 (2.5–16) 6.7 (1–17.5) 16 (7.5–34.5) Tear film breakup time, s (normal range, ‡10) 12.7 (7.67–15) 5.5 (3–10.7) 5.5 (2.5–9.5) 11 (6.3–13.8) Lid parallel conjunctival folds (normal range, <2) 1.5 (0–3) 2 (0.5–4) 1.5 (0.5–3.5) 1 (0–2) Oxford system fluorescein/lissamin (normal range, <2) 1 (0–3)/1 (0–2) 2 (0–4.5)/2 (0–4) 2 (0–4)/1.5 (0–4) 0.5 (0–2)/0.5 (0–2) Related to tear ﬁlm and ocular surface abnormalities in 120 subjects with TAO without and with associated dry eye syndrome (TAO þ dry eye), patients with dry eye syndrome only, and in healthy euthyroid control subjects.

TABLE 3. Clinical and Serological Data of the 60 Patients With TAO Without and With Concomitant Dry Eye Syndrome (TAO þ dry eye)

TAO TAO þ Dry Eye P Value

TAO duration, mo (median) 12 (0–129) 28 (2–239) 0.1 Untreated TAO, n (%) 12 (40) 10 (33.3) 0.78 Glucocorticoid treatment, n (%) 16 (53.3) 17 (56.6) 1 Clinical activity score, points (median; range, 0–7) 1 (0–5) 2 (0–5) 0.36 Active TAO, n (%) 9 (30) 14 (46.7) 0.28 Inactive TAO, n (%) 21 (70) 16 (53.3) 0.28 NOSPECS-score, points (median; range, 0–18) 4 (0–9) 4 (1–7.5) 0.6 Duration of autoimmune thyroid disease, mo (median) 34.5 (2–129) 35.5 (6–239) 0.187 Euthyroid, n (%) 23 (76.7) 24 (80) 1 Hyperthyroid, n (%) 7 (23.3) 6 (20) 1 Serum TSH, mU/L (median; reference range, 0.4–4.9) 0.63 (0.01–4.9) 0.52 (0.01–4.9) 0.696

Serum FT3, pg/mL (median; reference range, 1.7–3.7) 3.1 (2.2–11.8 ) 3.0 (2.1–17.4) 0.691 Serum FT4, ng/dL (median; reference range, 0.7–1.5) 1.2 (0.8–2.0) 1.3 (0.7–2.0) 0.563 Thyroglobulin autoantibodies, IU (median; reference range, <55) 4.5 (1.0–270.8) 7.45 (1.0–1000) 0.205 Thyroid peroxidase autoantibodies, IU (median; reference range, <6) 47.0 (3–1000) 27.0 (3–1000) 0.584 TSH receptor autoantibodies, IU (median, reference range, <1.0) 3.9 (0.5–185) 6.7 (0.5–190) 0.768

TABLE 4. Deregulated Proteins and Their Regulation in the Study Groups

Post Hoc Post Hoc Post Hoc Post Hoc Post Hoc Post Hoc P Value P Value P Value P Value P Value P Value KW TAO:DE TAO:C DE:C TAO:TAO þ DE TAO þ DE:DE TAO þ DE:C P Value

ANXA1 0.006770 0.004581 0.0128 CYTN 0.008913 0.013337 0.013 HSPB1 0.032117 0.042107 0.0183 LEG3 0.039515 0.033889 0.016363 0.013987 0.001 LYSC 0.021719 0.032 MDN1 0.037940 0.037 POTEI 0.025889 0.0021 PROL1 0.002008 0.033231 0.007665 0.0082 PRP4 0.045696 0.0115 S10A8 0.000000 0.004024 0.000000 0.000000 0.0004 SMCA4 0.000000 0.002027 0.000000 0.000000 0.028626 0.0004 UGDH 0.017643 0.029198 0.005538 0.008 Study groups: TAO, TAO þ dry eye, dry eye, healthy controls. Shown are the P values obtained by the Kruskal-Wallis test over all groups and the post hoc single equation values. C, healthy controls; DE, dry eye syndrome.

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2652

FIGURE 1. Box-and-whisker plots of PROL 1. (A) Mass spectrometry data. (B) Microarray data. The x-axis represents the groups and the y-axis the measured intensity values. (C) Scatterplot of PROL 1 versus the number of pack years in patients with TAO (r ¼0.301; P ¼ 0.027).

ppm and a fragment mass tolerance 60.6 Dalton (Da). A total Using the IPA pathway software Ingenuity Systems (Inge- of 28 proteins identified in the tear fluid were significantly nuity Systems, Inc., Redwood City, CA, USA) the identified different over all four study groups (Table 4). Eighteen proteins proteins above were found to be involved in inflammatory (64%) significantly differed between TAO and dry eye, eight response, cell-to-cell signaling and interaction, cellular move- (28%) between TAO and controls and 11 (39%) between dry ment, and cell death. eye and controls. Compared with dry eye, proline-rich protein 1 (PROL1, P ¼ Antibody Microarray 0.002); uridine diphosphate (UDP)–glucose-dehydrogenase (UGDH, P ¼ 0.017); calgranulin A (S10A8, P < 0.0001); Antibody microarray confirmed significant changes of PRP4, transcription activator BRG1 (SMCA4, P < 0.0001); annexin (P PROL1, and UGDH between TAO with and without dry eye, ¼ 0.006); cystatin (P ¼ 0.008); heat shock protein 27 (P ¼ dry eye, or controls (P < 0.01). These three proteins negatively 0.032); and galectin (LEG3; P ¼ 0.039) were markedly correlated with smoking, P < 0.05 (Figs. 1–4). The higher the downregulated in TAO (Figs. 1–3). The highest downregula- number of pack years, the lower the protein intensity was. tions in TAO were noted for S10A8 (5-fold) and SMCA4 (4-fold) compared with dry eye. Also compared with controls, PROL1 was 5-fold (P < 0.05); proline-rich protein 4 (PRP4, P < 0.05) DISCUSSION 2-fold (Fig. 4); SMCA4 2-fold; and S10A8 1.8-fold downregulat- This prospective and controlled study is the first that ed in TAO. investigates the tear fluid composition of TAO patients with In contrast, the following proteins were upregulated in TAO and without concomitant dry eye syndrome in comparison to versus dry eye and/or controls. Lysozyme C was 1.9-fold dry eye only and healthy controls. Proteome analysis of tear upregulated in TAO versus TAO þ dry eye (P ¼ 0.02) and fluid can be used to identify specific proteins in patients with Midasin and POTE ankyrin domain family member I (POTEI) TAO and to differentiate between TAO and the dry eye were 1.7- and 3.9-fold upregulated in TAO versus controls (P < syndrome as well as between TAO and normal controls. 0.05), respectively. Also significantly upregulated in dry eye Protein dysregulation was associated with inflammatory versus controls (Table 4) were the proteins LEG3 (P ¼ 0.033) response, cell-to-cell signaling and interaction, cellular move- and S100A8 (P < 0.001, 2.5-fold); BRG1 (P < 0.001); and ment, and cell death. In our hands, sampling of tear fluid was HSP27 (P ¼ 0.042, 2-fold), as well as ANXA1 (P ¼ 0.004, 1.8- well-accepted by both patients and controls and proteome fold). analysis of tear samples was technically easy to perform. It thus

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2653

FIGURE 2. Box-and-whisker plots of UGDH. (A) Mass spectrometry data. (B) Antibody microarray data. The x-axis represents the groups and the y- axis the measured intensity values. (C) Scatterplot of UGDH versus the number of pack years in patients with TAO (r ¼0.287; P ¼ 0.035).

represents an ideal tool to investigate lacrimal gland involve- Further, the anti-inflammatory protein annexin A1 (ANXA1) ment in patients with TAO and dry eye. Further characteriza- was downregulated in TAO versus dry eye. Annexin A1 acts as tion of these proteins as well as their demonstration in the sera an endogenous downregulator of inflammation in cells of the of the corresponding patients may help to identify protein innate immune system as it blocks the interaction of activated regulations of the disease, especially in the various stages of neutrophils to endothelial cells. There is evidence that it also TAO. plays a role in the adaptive immune system as a tuner of T cell In this study, protective proteins (i.e., PROL1/PRP4) were receptor signaling and a subsequent differentiation of Th1/Th2 markedly downregulated in patients with TAO. Proline-rich cells over the activation of both the extracellular signal- proteins are highly expressed and secreted by lacrimal acinar regulated kinases/mitogen-activated protein kinases pathway and protein kinase B pathway via formyl peptide receptor like cells which have a large share of the cellular mass of the 23 lacrimal gland.18 Lacrimal PRPs have protective functions 1(FPRL-1). Also downregulated in TAO was UDP-a-d-glucose similar to those of salivary acidic PRPs by modulating the (UGDH) which catalyzes the reaction of UDP-a-d-glucuronic acid to UDP-a-d-glucose through a 2-fold oxidation. Uridine microflora (e.g., through agglutination and clearance of diphosphate–a-d-glucose is a precursor of glycosaminoglycans pathogens on the ocular surface).19,20 Downregulation might which are produced in activated fibroblasts and released in the be explained by the FAS ligand mediated apoptosis of lacrimal 24–26 14 florid and active phase of TAO. Hydrophilicity of these cells during the pathogenesis of TAO. This downregulation mucopolysaccharides attracts water and leads to the clinical was further verified and confirmed with the antibody micro- phenotype of proptosis. Since over 60% of the TAO patients array experiments. The results are also compliant with the included in this study were in the late and hence inactive phase previously reported downregulation of PRP4 in tears of of the disease, down-regulation of this enzyme may be 11 patients with dry eye compared to controls. In line with explained by the late stage of TAO.27 the above data was the downregulation of the ‘‘protective’’ In contrast, proteins that are involved in inflammatory cystatin in TAO. Cystatins are cysteine proteinase inhibitors processes were upregulated in TAO (e.g., the POTE isoform belonging to the cystatin superfamily.21 High concentrations of POTE-2a-actin), which is elevated in Hela cells treated with cystatins are reported in saliva and tears and have a protective proapoptotic factors (e.g., the FAS ligand, antibodies to FAS function in case of inhibition of cysteine proteases from Ligand, and TAIL). Also, overexpression of POTEI was microorganisms.22 Hence, the downregulation is a result of the demonstrated to induce apoptosis. In the present study, destruction of the lacrimal cells in the late phase of TAO. upregulation of POTEI in TAO points to an inflammatory

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2654

FIGURE 3. Box-and-whisker plots of proteins that are deregulated in TAO compared with dry eye obtained with mass spectrometry. (A) Calgranulin A. (B) Transcription activator BRG1. (C) Annexin A1. (D) Cystatin N. The x-axis represents the groups and the y-axis the measured intensity values.

process in the orbit. Furthermore, Midasin, which is required of TAO patients.32 These reduced protein concentrations in the for maturation and nuclear export of pre-60S ribosome tears of TAO patients may contribute to changes in their ocular subunits involved in the posttranslational modification of surfaces via diminished reactive oxygen species depletion and cellular proteins that regulate various cellular inflammatory adaptive immune responses. In another study,33 comparison of responses was upregulated, probably related to the increased orbital fat protein from TAO with age-matched controls cellular processes during inflammatory progression in the showed significant differences in the proteome, and upregu- orbit. Patients with TAO have an abnormally high tear film lation of specific proteins in orbital tissue from TAO was osmolarity.4 Hyperosmolarity stimulates proinflammatory cy- associated with biochemical mechanisms or capacities against tokines including interleukin 1B, tumor necrosis factor a, and endoplasmic reticulum stress, mitochondria dysfunction, and matrix metalloproteinase 9 (MMP-9) in mice.28 These cytokines cell proliferation as well as apoptosis in TAO orbital tissues. activate mitogen-activated protein kinases cascades that Also a correlation between the decreased protein concentra- stimulate further inflammatory cytokines.29 This cycle can tions of proline-rich proteins and UGDH with smoking was lead to a high amount of ocular inflammation. Evidence shown. This is in line with findings that show that smoking is a suggests that ocular inflammation mediated by T lymphocytes risk factor for developing TAO and has an impact on tear is also important in the pathogenesis of dry eye.30 Hyperos- composition.7 In our present study, a limitation lies in the small molarity may also cause pathological changes to the corneal smoker amount in the control group and a subsequent study epithelium cells, where MMP-9 can lyse substrates such as the with groups matched for smoking is foreseen in our lab. corneal epithelial basement membrane and tight junction In conclusion, proteome analysis of tear fluids demonstrat- proteins that normally have a corneal epithelial barrier ed an upregulation of inflammatory proteins and a downreg- function.31 ulation of protective proteins in TAO as well as a significantly Scarce data and only a few comparable studies are available different protein panel in TAO versus dry eye and/or controls. in the literature. One study recently showed in tear samples of Microarray investigation confirmed the proteomics results in TAO subjects an essential reduction of the protein fractions of several characterized proteins. In our lab, similar studies are inflammation-related protein immunoglobulin kappa chain C currently being performed analyzing orbital tissue of patients region (IgKC) and serum albumin, whereas a novel isoform of with various clinical stages of TAO versus orbital control tissue complement component 3 was completely absent in the tears looking for specific protein regulations of the complex

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2655

FIGURE 4. Box-and-whisker plots of PRP4. (A) Mass spectrometry data. (B) Microarray data. The x-axis represents the groups and the y-axis the measured intensity values. (C) Scatterplot of PRP4 versus the number of pack years in patients with TAO (r ¼0.31; P ¼ 0.023).

autoimmune disease. Subsequent to their identification in the 4. Gilbard JP, Farris RL. Ocular surface drying and tear film serum of patients with TAO and/or dry eye, proteomics may osmolarity in thyroid eye disease. Acta Ophthalmol (Copenh). offer a useful tool to identify specific markers of this disease. 1983;61:108–116. 5. Matheis N, Okrojek R, Grus FH, Kahaly GJ. Proteomics of tear fluid in thyroid-associated orbitopathy. Thyroid. 2012;22: Acknowledgments 1039–1045. The authors thank Tanja Diana, MSc, Molecular Thyroid Research 6. Khalil HA, De Keizer RJ, Bodelier VM, Kijlstra A. Secretory IgA Lab, JGU Medical Center, for manuscript evaluation. and lysozyme in tears of patients with Graves’ ophthalmopathy. Doc Ophthalmol. 1989;72:329–334. Disclosure: N. Matheis, None; F.H. Grus, None; M. Breitenfeld, None; I. Knych, None; S. Funke, None; S. Pitz, None; K.A. 7. Baker GR, Morton M, Rajapaska RS, et al. Altered tear composition in smokers and patients with graves ophthal- Ponto, None; N. Pfeiffer, None; G.J. Kahaly, None mopathy. Arch Ophthalmol. 2006;124:1451–1456. 8. Achtsidis V, Tentolouris N, Theodoropoulou S, et al. Dry eye in References Graves ophthalmopathy: correlation with corneal hypoesthe- sia. Eur J Ophthalmol. 2013;23:473–479. 1. Bartalena L, Fatourechi V. Extrathyroidal manifestations of 9. Ponto KA, Hommel G, Pitz S, Elflein H, Pfeiffer N, Kahaly GJ. Graves’ disease: a 2014 update. . 2014;37: J Endocrinol Invest Quality of life in a German graves orbitopathy population. Am 691–700. J Ophthalmol. 2011;152:483–490, e481. 2. Chang TC, Huang KM, Chang TJ, Lin SL. Correlation of orbital 10. Research in dry eye: report of the Research Subcommittee of computed tomography and antibodies in patients with the International Dry Eye WorkShop (2007). Ocul Surf. 2007; hyperthyroid Graves’ disease. Clin Endocrinol (Oxf). 1990; 5:179–193. 32:551–558. 11. Boehm N, Funke S, Wiegand M, Wehrwein N, Pfeiffer N, Grus 3. Eckstein AK, Finkenrath A, Heiligenhaus A, et al. Dry eye FH. Alterations in the tear proteome of dry eye patients–a syndrome in thyroid-associated ophthalmopathy: lacrimal matter of the clinical phenotype. Invest Ophthalmol Vis Sci. expression of TSH receptor suggests involvement of TSHR- 2013;54:2385–2392. specific autoantibodies. Acta Ophthalmol Scand. 2004;82: 12. Funke S, Azimi D, Wolters D, Grus FH, Pfeiffer N. Longitudinal 291–297. analysis of taurine induced effects on the tear proteome of

Downloaded from tvst.arvojournals.org on 09/30/2021 Proteomics of Tears IOVS j April 2015 j Vol. 56 j No. 4 j 2656

contact lens wearers and dry eye patients using a RP-RP- 24. Turcu AF, Kumar S, Neumann S, et al. A small molecule Capillary-HPLC-MALDI TOF/TOF MS approach. J Proteomics. antagonist inhibits thyrotropin receptor antibody-induced 2012;75:3177–3190. orbital fibroblast functions involved in the pathogenesis of 13. McArthur C, Wang Y, Veno P, Zhang J, Fiorella R. Intracellular Graves ophthalmopathy. J Clin Endocrinol Metab. 2013;98: trafficking and surface expression of SS-A (Ro), SS-B (La), 2153–2159. poly(ADP-ribose) polymerase and alpha-fodrin autoantigens 25. van Zeijl CJ, Fliers E, van Koppen CJ, et al. Thyrotropin during apoptosis in human salivary gland cells induced by receptor-stimulating Graves’ disease immunoglobulins induce tumour necrosis factor-alpha. Arch Oral Biol. 2002;47:443– hyaluronan synthesis by differentiated orbital fibroblasts from 448. patients with Graves’ ophthalmopathy not only via cyclic 14. Kahaly GJ, Bang H, Berg W, Dittmar M. Alpha-fodrin as a adenosine monophosphate signaling pathways. Thyroid. putative autoantigen in Graves’ ophthalmopathy. Clin Exp 2011;21:169–176. Immunol. 2005;140:166–172. 26. van Zeijl CJ, Fliers E, van Koppen CJ, et al. Effects of 15. Okrojek R, Grus FH, Matheis N, Kahaly GJ. Proteomics in thyrotropin and thyrotropin-receptor-stimulating Graves’ dis- autoimmune thyroid eye disease. Horm Metab Res. 2009;41: ease immunoglobulin G on cyclic adenosine monophosphate 465–470. and hyaluronan production in nondifferentiated orbital fibroblasts of Graves’ ophthalmopathy patients. Thyroid. 16. Bartalena L, Baldeschi L, Dickinson A, et al. Consensus 2010;20:535–544. statement of the European Group on Graves’ orbitopathy 27. Hansen C, Rouhi R, Forster G, Kahaly GJ. Increased sulfatation (EUGOGO) on management of GO. Eur J Endocrinol. 2008; of orbital glycosaminoglycans in Graves’ ophthalmopathy. 158:273–285. J Clin Endocrinol Metab. 1999;84:1409–1413. 17. Bohm D, Keller K, Pieter J, et al. Comparison of tear protein 28. Luo L, Li DQ, Doshi A, Farley W, Corrales RM, Pflugfelder SC. levels in breast cancer patients and healthy controls using a de Experimental dry eye stimulates production of inflammatory novo proteomic approach. Oncol Rep. 2012;28:429–438. cytokines and MMP-9 and activates MAPK signaling pathways 18. Ozyildirim AM, Wistow GJ, Gao J, et al. The lacrimal gland on the ocular surface. Invest Ophthalmol Vis Sci. 2004;45: transcriptome is an unusually rich source of rare and poorly 4293–4301. characterized gene transcripts. . Invest Ophthalmol Vis Sci 29. Luo L, Li DQ, Corrales RM, Pflugfelder SC. Hyperosmolar 2005;46:1572–1580. saline is a proinflammatory stress on the mouse ocular surface. 19. Dickinson DP, Thiesse M. A major human lacrimal gland mRNA Eye Contact Lens. 2005;31:186–193. encodes a new proline-rich protein family member. Invest 30. El Annan J, Chauhan SK, Ecoiffier T, Zhang Q, Saban DR, Dana Ophthalmol Vis Sci. 1995;36:2020–2031. R. Characterization of effector T cells in dry eye disease. Invest 20. Perumal N, Funke S, Pfeiffer N, Grus FH. Characterization of Ophthalmol Vis Sci. 2009;50:3802–3807. lacrimal proline-rich protein 4 (PRR4) in human tear 31. Gurdal C, Genc I, Sarac O, Gonul I, Takmaz T, Can I. Topical proteome. Proteomics. 2014;14:1698–1709. cyclosporine in thyroid orbitopathy-related dry eye: clinical 21. Barrett AJ, Fritz H, Grubb A, et al. Nomenclature and findings, conjunctival epithelial apoptosis, and MMP-9 expres- classification of the proteins homologous with the cysteine- sion. Curr Eye Res. 2010;35:771–777. proteinase inhibitor chicken cystatin. Biochem J. 1986;236: 32. Jiang LH, Wei RL. Analysis of Graves’ ophthalmopathy 312. patients’ tear protein spectrum. Chin Med J (Engl). 2013; 22. Magister S, Kos J. Cystatins in immune system. J Cancer. 2013; 126:4493–4498. 4:45–56. 33. Cheng KC, Huang HH, Hung CT, et al. Proteomic analysis of 23. D’Acquisto F, Merghani A, Lecona E, et al. Annexin-1 the differences in orbital protein expression in thyroid modulates T-cell activation and differentiation. Blood. 2007; orbitopathy. Graefes Arch Clin Exp Ophthalmol. 2013;251: 109:1095–1102. 2777–2787.

Downloaded from tvst.arvojournals.org on 09/30/2021