Eotaxin-1expression in tears of thyroid-associated ophthalmopathy

doi:10.21203/rs.3.rs-452257/v1

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Eotaxin-1expression in tears of thyroid-associated ophthalmopathy

https://doi.org/10.21203/rs.3.rs-452257/v1

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posted 29 Apr, 2021

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Purpose To explore changes of Eotaxin-1concentration in tears of thyroid associated

ophthalmopathy (TAO) patients

Methods This prospective observational study.16 patients with TAO (32 eyes) and 18

healthy volunteers (36 eyes) were recruited.TAO patients were divided into active TAO (16eyes) and inactive TAO derived from the active TAO patients after corticosteroid treatment(16eyes).The tear concentration of Eotaxin-1 was measured per ELISA assay. Eotaxin-1protein expression was evaluated per Western Blot .

Results The highest Eotaxin-1 concentration in tears was in inactive TAO patients, followed by patients with active TAO and the controls per ELISA. Western Blot analysis indicated that Eotaxin-1 expression in inactive TAO was also higher than in active TAO and the controls.

Conclusion The changes of Eotaxin-1 expression in tears of patients with active and inactive TAO suggested that orbital inflammation may be involved in the ocular surface damage of TAO.

Ophthalmology

Thyroid associated orbitopathy

Eotaxin-1

Ocular surface

Lacrimal gland

Tears

TAO is a inflammatory autoimmune disorder of the orbit, characterized by inflammation and proliferation of the orbital tissue caused by CD4 + T cells and orbital fibroblasts [1]. Many Patients often have symptoms of ocular surface discomfort, which are caused by the changes of tear fluid proteins, which increase the tear fluid’s osmolarity, and result in ocular surface damage༻ 2༽. Our previous study༻3༽ has been shown increased IL-7 expression in tears and Orbital tissues of inactive TAO patients, which indicates that orbital inflammation may be involved in the pathogenesis of ocular surface damage in TAO. The process of TAO is an active phase initially, followed by an inactive (fibrotic) phase༻4༽. Interventions are generally effective in active TAO patients, but are ineffective in those of inactive TAO. Therefore, the early assessment of TAO disease activity is essential for optimal outcome of medical interventions༻5༽.

Immune response plays a central role in the pathogenesis of TAO. The notion that T cell-mediated immunity contributes to TAO development has been widely accepted [6], but some studies have suggested that other potential signal molecules, such as chemokines, can be involved in the pathogenesis of TAO༻7༽. Chemokines exert their biological effects by interacting with Chemokines receptors in many homeostatic and pathological processes. Thus, an alteration of chemokine and/or chemokine receptor expression may lead to the persistence of an inflammatory reaction ༻8༽.

Eotaxin-1, encoded by the CCL11 gene, is a chemokine within the CC subfamily that is produced by a variety of cell types [9–11]. Binding of Eotaxin-1 to the chemokine receptors CCR2, CCR3, and CCR5༻12–13༽, Eotaxin-1stimulates the migration of cells༻8༽.Effects of Eotaxin-1 have been involved in variousimmune-mediated diseases༻14༽. Previous studies have shown that the level of Eotaxin-1 is increased in several chronic inflammatory diseases, including allergic rhinitis, rheumatoid arthritis,and ankylosing spondylitis༻15–17༽. Recent study reported that the reduction of the level of Eotaxin-1 significantly attenuates airway inflammation in experimental mice asthma༻18༽, which indicating the potential use of eotaxin-1 as a biomarker for the diagnosis and assessment of inflammation severity and control.

In our study, we investigated the level of Eotaxin-1 in tears of patients with active TAO, inactive TAO and the controls per ELISA. Furthermore, the expression of Eotaxin-1protein was determined per western blot analysis. This results showed that Eotaxin-1 may be related to the pathogenesis of the lacrimal gland and tear inflammation in ocular surface damage among TAO patients.

Subjects

A total of 16 patients who were diagnosed with TAO based on Bartley’s criteria［19］ ,
were enrolled in the present study. We used the Bartalena Standard classification［20］ for moderate-to-severe selection of TAO. Standard ophthalmic examinations such as visual acuity, slit-lamp examination, fundus examination, tear film break-up time, fluorescein staining and Schimer tests were performed for both TAO and control patients.

Exclusion criteria included patients suffering from ocular surface disorders, such as dry-eye syndrome, allergic ocular surface diseases or pterygium prior to development of TAO; a history of medication which may affect ocular surface parameters［21］, such as rheumatoid arthritis drugs, topical anti-glaucoma drugs; wearing of contact lenses; patients with a history of steroid treatment or radiation therapy; and patients with other diseases that are related with TAO such as diabetes. Ophthalmopathy was evaluated based on the clinical activity score (CAS) followingcomplete examination of the eyes. Patients with CAS ≥ 3/7 were defined as having active phase TAO, whereas, CAS ≤ 2/7 were defined as having inactive phase TAO［22］. Based on these criteria, 16 patients were classified as active phase TAO, while,16 patients with inactive TAO were from the same patients with active TAO after corticosteroid treatment. In addition, 18 age-matched and sex-matched healthy volunteers were enrolled as the control group. There was no signifificant difference between the ocular parameters and disease course in our experiments.

The study was approved by the Ethics Committee of Inhuman Hospital affiliated
with Shanghai Jiaotong University School of Medicine, Shanghai, China. All experiments were performed in strict accordance to the Declaration of Helsinki. Informed consent was obtained from each participating patient prior to taking part in the study.
Tear collection and protein extraction
Unstimulated tear samples were collected from 32 eyes of the 16 TAO patients and 36
eyes of 18 healthy volunteers using disposable 10μl microcapillary pipettes (Microcap 10μl; Drummond Scientific, Broomall, PA, USA) via the inferior meniscus. Tear samples were placed into 0.5 ml Eppendorf tubes and stored at -80℃ for subsequent investigation.

Eotaxin-1 concentration was measured using Human Eotaxin-1 ELISA Kit (Abcam, UK) according to the manufacturer’s instructions. The samples were divided into three groups: 16 patients with active TAO, 16 patients with inactive TAO-derived from the active TAO patients after corticosteroid treatment, and 18healthy volunteers. For the experiment, tear samples were diluted 10 times with assay diluents. A total volume of 100μl from each sample was used for the assay.

Detection of protein expression in tears via western blot assay

Western blot was performed according to standard procedures. Briefly, total protein
was quantified using BCA protein assay kit (Tiangen Biotech Co., Ltd, China) and equal amounts of protein was loaded into each well for separation via SDS-PAGE. Proteins were then transferred onto a PVDF membrane (Millipore, USA), blocked for
1 h at room temperature, then incubated with relevant antibodies at 4℃overnight. The
membranes were then incubated with the HRP-conjugated secondary antibody for 1 h
at room temperature and immunoreactive bands were reacted with ECL substrate and
visualized under chemiluminescence imaging analyzer (GE, USA). GAPDH was used
as the internal control to normalize protein loading. Eotaxin-1 primary antibodies was
purchased from Santa Cruz Biotechnology and used at 1:2000 dilution. Western blot images were analyzed using Quantity One software (Bio-Rad, USA).

Statistical analysis
Eotaxin-1concentration was expressed as mean ± standard derivation. Statistical analysis of the data was performed per multivariate ANOVA and Bonferroni’s adjusted test for multiple comparisons, automatically applied by computer program (System). The multiplication factor was calculated based on the number of comparisons applied. Statistical software (IBM SPSS Statistics 25.0; SPSS inc ChicaTAO, IL) was used for analysis, and P＜0.05 was considered as statistically significant.

Validation of protein expression
In order to verify the identified Eotaxin-1 expression, we performed western blot analysis, which indicated that the expression of Eotaxin-1 protein was significantly higher in inactive TAO and active TAO than in the control (p<0.01, respectively). Furthermore, the protein expression in inactive TAO was significantly higher than in active TAO(p<0.01).These results indicate that the occurrence of TAO was associated with dynamic changes in immune regulatory proteins (Fig. 1).

Eotaxin-1 expression in the tear sample of TAO patients
We focused on Eotaxin-1 and further analyzed its expression level in tear samples by
ELISA assay, which showed that the level of Eotaxin-1in inactive TAO and active TAO was significantly higher than in the control (p < 0.05, respectively). Furthermore,
Eotaxin-1concentration in inactive TAO was also higher than in active TAO (p <0.05). In healthy volunteers, the level of Eotaxin-1 in tears was approximately 739pg/ml. However, following the occurrence of TAO, the level of Eotaxin-1 increased rapidly, reaching an average of 1496 mg/ml in active TAO group , and as high as 1882 mg/ml in inactive TAO, indicating a 2.5-fold increase compared with the controls. These results suggested that Eotaxin may be implicated in the development and progression of the ocular surface damage of TAO (Fig. 2).

TAO has been shown to be associated with the typical signs and symptoms characteristic of dry eye. Our study found that Eotaxin-1 concentration in tears is significantly higher in inactive TAO than in active TAO and the controls, which, may
be useful as a potential biomarker for inactive TAO.

The mechanism of changes on Eotaxin-1 expression in tears of TAO was not characterized. However，accumulating data suggested that the lacrimal gland and tear fluid may be directly involved in the ocular surface damage in TAO［23］ . Danping Huang［24］ reported that the lacrimal gland is significantly enlarged in active and inactive TAO than in the control, suggesting that inflammation of the lacrimal gland might be associated with the ocular surface damage. In addition, lacrimal gland acinar cells had a common target for autoimmunity with thyroidal and orbital tissues, which thus contributed to lacrimal gland impairment and ocular surface damage［23］. lacrimal gland inflammation could increase the expressions of tear inflammatory cytokines in TAO［24］. Our previous study[3]showed that IL-7 was increased in tears of TAO patients, suggesting that orbital inflammation may be involved in the ocular surface damage of TAO.

Eotaxin-1, is a secreted protein produced by a variety of cell types, such as fibroblasts[10]. As immune modulators, chemokines exert in a wide variety of inflammatory and immune responses via the chemoattractive effects of immune cells,
which, participate in the pathogenesis or progression in various disease[25]. Our study showed that elevated eotaxin-1 concentration is significantly detected in tears of the inactive TAO compared with the controls and active TAO.The reason for increased eotaxin-1 concentration was not established. Cho [26] found higher expression of Eotaxin‐ 1 in that fibroblasts are increasingly proliferating in chronic stage, resulting in fibrosis. Furthermore, the ocular surface tissues, including lacrimal glands, cornea and conjunctiva, might be primarily responsible for the overexpression of Eotaxin‐ 1 [27, 28]. other reasons including aging,obesity,cigarette smoking can also affect Eotaxin‐ 1 concentration [29-31].

In conclusion, our result suggested that Eotaxin‐ 1 concentration in tears ofinactive TAO is significantly more higher than in inactive TAO and the control, indicating a significant role of orbital inflammation in interpreting the ocular surface damage in TAO. Eotaxin-1 may be an ideal biomarker for TAO diagnosis and prevention using a clinical diagnostic test of non-invasive patient tear samples.

Compliance with Ethical Standards

Funding: This study was funded by Medical-engineering cross fund of Shanghai Jiao Tong University (Grant No. YG2014MS72).

Conflict of Interest: HaiBo Tan declares that he has no conflict of interest. Kebo， Cai declares that he has no conflict of interest.

Ethical Approval: All procedures performed in studies involving human participants
were in accordance with the ethical standards of the Ethics Committee of Xinhua Hospital affiliated with Shanghai Jiaotong University School of Medicine, Shanghai,
China and with the 1964 Helsinki declaration and its later amendments or comparable
ethical standards.

Informed consent: Informed consent was obtained from each participating patient
prior to taking part in the study

Kishazi E, Dor M, Eperon S, Oberic A, Hamedani M, Turck N（2018）Thyroid-associated orbitopathy and tears: A proteomics study. J Proteomics 170：110-116. https:// doi: 10.1016/j.jprot.2017.09.001
Edward J Wladis, Vinay K Aakalu, Jill A Foster, Suzanne K Freitag, Rachel K Sobel, Jeremiah P Tao, Michael T Yen（2020）Intense Pulsed Light for Meibomian Gland Disease: A Report by the American Academy of Ophthalmology. Ophthalmology 127(9):1227-1233,2020. https://doi: 10.1016/j.ophtha.2020.03.009
Cai K, Wei R (2013) Interleukin-7 expression in tears and orbital tissues of patients with Graves' ophthalmopathy. Endocrine 44(1):140-144, 2013. https://doi: 10.1007/s12020-012-9840-7
TAOnnella D (2019) The Th1 chemokine MIG in Graves' ophthalmopathy.Clin Ter170: e368-e372. https:// doi: 10.7417/CT.2019.2162
Shen J, Li Z, Li W, Ge Y, Xie M, Lv M, Fan Y, Chen Z, Zhao D, Han Y(2015) Th1,Th2,and Th17 Cytokine Involvement in Thyroid Associated Ophthalmopathy. Dis Markers 2015: 609593. https:// doi: 10.1155/2015/609593
Yazhuo Huang , Sijie Fang , Dan Li , Huifang Zhou, Bin Li , Xianqun Fan (2019) The involvement of T cell pathogenesis in thyroid-associated ophthalmopathy. Eye (Lond) 33(2):176-182. https://doi: 10.1038/s41433-018-0279-9
Mishra S, Maurya VK, Kumar S, Kaur A, Saxena SK (2020) Clinical Management and Therapeutic Strategies for the Thyroid-Associated Ophthalmopathy: Current and Future Perspectives. Curr Eye Res 21:1-17. https: //doi: 10.1080/02713683.2020.1776331
Kindstedt E, Holm CK, Sulniute R, Martinez-Carrasco I, Lundmark R, Lundberg P (2017) CCL11, a novel mediator of inflammatory bone resorption.Sci Rep 7(1): 5334. https: //doi: 10.1038/s41598-017-05654-w
Garcia-Zepeda EA, Rothenberg ME, Ownbey RT, Celestin J, Leder P, Luster AD (1996) Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nature medicine 2:449–456. https: //doi: 10.1038/nm0496-449
J Bartels , C Schlüter, E Richter, N Noso, R Kulke, E Christophers, J M Schröder(1996) Human dermal fibroblasts express eotaxin: molecular cloning, mRNA expression, and identification of eotaxin sequence variants. Biochemical and biophysical research communications 225(3):1045–1051.https://doi: 10.1006/bbrc.1996.1292
Jazia Alblowi, Chen Tian, Michelle F Siqueira, Rayyan A Kayal, Erin McKenzie, Yugal Behl, Louis Gerstenfeld, Thomas A Einhorn, Dana T Graves (2013) Chemokine expression is upregulated in chondrocytes in diabetic fracture healing. Bone 53(1):294–300. https:// doi: 10.1016/j.bone.2012.12.006
M Kitaura, T Nakajima, T Imai, S Harada, C Combadiere, H L Tiffany, P M Murphy, O Yoshie (1996) Molecular cloning of human eotaxin, an eosinophil-selective CC chemokine, and identification of a specific eosinophil eotaxin receptor, CC chemokine receptor 3. The Journal of biological chemistry 271(3) :7725–7730. https:// doi: 10.1074/jbc.271.13.7725
Ye J, Kohli LL, Stone MJ (2000) Characterization of binding between the chemokine eotaxin and peptides derived from the chemokine receptor CCR3. The Journal of biological chemistry 275(35):27250–27257. https://doi: 10.1074/jbc.M003925200
Shaw OM, Nyanhanda T, McGhie TK, Harper JL, Hurst RD (2017) Blackcurrant anthocyanins modulate CCL11 secretion and suppress allergic airway inflammation. Mol Nutr Food Res 61(9): 1700324.https://doi: 10.1002/mnfr.201600868
Han MW, Kim SH, Oh I, Kim YH, Lee J (2019) Serum IL-1β can be a biomarker in children with severe persistent allergic rhinitis. Allergy Asthma Clin Immunol 15: https:// doi: 10.1186/s13223-019-0368-8
Liu X, Zhang H, Chang X, Shen J, Zheng W, Xu Y, Wang J, Gao W, He S (2017) Upregulated expression of CCR3 in rheumatoid arthritis and CCR3-dependent activation of fibroblast-like synoviocytes.Cell Biol Toxicol 33(1):15-26. https://doi: 10.1007/s10565-016-9356-7
Sohn DH, Jeong H, Roh JS, Lee HN, Kim E, Koh JH, Lee SG (2018) Serum CCL11 level is associated with radiographic spinal damage in patients with ankylosing spondylitis. Rheumatol Int 38(8):1455-1464.https://doi: 10.1007/s00296-018-4073-6
Carvalho VF, Barreto EO, Arantes ACS, Serra MF, Ferreira TPT, Jannini-Sá YAP, Hogaboam CM, Martins MA, Silva PMR (2018) Diabetes Downregulates Allergen-Induced Airway Inflammation in Mice.Mediators Inflamm 2018:6150843. https://doi: 10.1155/2018/6150843
Drui D, Du Pasquier Fediaevski L, Vignal Clermont C, Daumerie C (2018) Graves' orbitopathy: Diagnosis and treatment.Ann Endocrinol (Paris) 79(6): 656-664. https://doi: 10.1016/j.ando.2018.08.005
Bartalena, A. Pinchera, C：Marcocci. (2000) Management of Graves’ ophthalmopathy: reality and perspectives. Endocr Rev 21(2):168–199. https://doi: 10.1210/edrv.21.2.0393
Danping Huang, Nuo Xu, Yiyue Song, Peijuan Wang, Huasheng Yang (2012) Inflammatory cytokine profifiles in the tears of thyroid-associated ophthalmopathy. Graefes Arch Clin. Exp Ophthalmol 250(81):619–625. https://doi: 10.1038/s41598-018-29113-2
G. Gianoukakis, N. Khadavi, T.J. Smith (2008) Cytokines, Graves’ disease, and thyroid-associatedophthalmopathy.Thyroid18:953–958.https://doi:10.1089/thy.2007.0405
Eckstein AK, Finkenrath A, Heiligenhaus A, Renzing-Köhler K, Esser J, Krüger C Quadbeck B, Steuhl KP, Gieseler RK(2004) Dry eye syndrome in thyroid-associated ophthalmopathy: lacrimal expression of TSH receptor suggests involvement of TSHR-specific autoantibodies. Acta Ophthalmol Scand 82(3): 291–297. https:// doi: 10.1111/j.1395-3907
Danping Huang , Quan Luo, Huasheng Yang, Yuxiang Mao(2014) Changes of lacrimal gland and tear inflammatory cytokines in thyroid-associated ophthalmopathy.IOVS 55(8):4935-4943. https:// doi: 10.1167/iovs.13-13704
Nomiyama H, Osada N, Yoshie O(2010) The evolution of mammalian chemokine genes.Cytokine Growth Factor Rev 21:253-262.https://doi: 10.1016/j.cytogfr.2010.03.004
Cho, H; Lim, S.J; Won, K.Y; Bae, G.E; Kim, G.Y; Min, J.W; Noh, B.J(2016) Eosinophils in Colorectal Neoplasms Associated with Expression of CCL11 and J. Pathol. Transl. Med 50(1): 45–51. https://doi: 10.4132/jptm.2015.10.16
Elieh Ali Komi D, Rambasek T, Bielory L(2018) Clinical implications of mast cell involvement in allergic conjunctivitis. Allergy 73(3):528-539. https://doi: 10.1111/all.13334
Reksten TR, Johnsen SJ, Jonsson MV, Omdal R, Brun JG, Theander E, Eriksson P ,Wahren-Herlenius M, Jonsson R, Nordmark G(2014)Genetic associations to germinal centre formation in primary Sjogren's syndrome.Ann Rheum Dis 73(6):1253-1258. https:// doi: 10.1136/annrheumdis-2012-202500
Saul A Villeda, Jian Luo, Kira I Mosher, Bende Zou, Markus Britschgi, GreTAOr
Bieri, Trisha M Stan, Nina Fainberg, Zhaoqing Ding, Alexander Eggel (2011) The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature 477(7362): 90–94. https://doi: 10.1038/nature10357
Vasudevan AR, Wu H, Xydakis AM, Jones PH, Smith EO, Sweeney JF, Corry DB, Ballantyne CM(2006)Eotaxin and obesity. J Clin Endocrinol Metab 91(1): 256–261. https://doi: 10.1210/jc.2005-1280
LuetraTAOon T, Rutqvist LE, Tangvarasittichai O, Andersson BÅ, Löfgren S, Usuwanthim K, Lewin NL(2018) Interaction among smoking status, single nucleotide polymorphisms and markers of systemic inflammation in healthy Immunology 154(1): 98-103. https://doi: 10.1111/imm.12864

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Eotaxin-1expression in tears of thyroid-associated ophthalmopathy

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Abstract

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