Expression and significance of IL-27 and IL-35 in orbital fat in patients with severe TAO

Abstract

To detect the expression of Interleukin-27 (IL-27) and Interleukin-35 (IL-35) in orbital fat in patients with severe TAO (thyroid-related eye disease) exophthalmos, and to investigate its potential role and significance in the development of TAO. A study group of 30 patients (30 eyes) who underwent orbital decompression with severe TAO exophthalmos in the Department of Ophthalmology, Provincial Hospital affiliated to Shandong First Medical University from January 2022 to December 2023, the expression of IL-27 and IL-35 of the orbital adipose tissue was detected by western-blot, and which in 30 patients (30 eyes) underwent orbital fracture surgery and plastic repair as control group. The contents of IL-27 and IL-35 were higher in severe TAO patients than in normal controls, and the difference was significant (P < 0.01). IL-27 and IL-35 are significantly increased in the orbital fat of TAO patients with severe protrusion, suggesting that these two cytokines play key roles in the regulation of orbital fat dysplasia and the inflammatory process of TAO, and their expression has certain clinical value in the evaluation of TAO disease.

Introduction

Thyroid associated eye disease (thyroid associated ophthalmopathy, TAO) is the eye complications caused by thyroid dysfunction, is the most common clinical orbital disease, its main manifestations is eyelid swelling, eyelid withdrawal, conjunctival mixed congestion, proptosis, vision loss, strabismus, severe TAO patients will appear malignant protrusion, seriously affect the visual function [1]. The pathogenesis of TAO is complex and its clinical manifestations are diverse, and its etiology and pathogenesis are still not completely clear. Recent studies have shown that inflammatory response play a crucial role in the development of TAO.

IL-27 and IL-35, as anti-inflammatory cytokines, have received much attention in recent years [2]– [3]. IL-27 and IL-35 have the functions of inhibit the differentiation and proliferation of Th1 cells and Th17 cells. They can also inhibit the proliferation of various immune cells, including T cells, B cells and macrophages, and promote the proliferation and function of regulatory T cells (regulatory T cells, Tregs) to play immune suppression and immune regulation functions. The expression and mechanism of IL 27 and IL 35 have been studied in Respiratory, Oncology and Nephrology, but there is no report on their expression in TAO [4]. In this study, we first examined the expression of IL-27 and IL-35 in the orbital adipose tissue of TAO patients by Western blot method, explored its role in the development of TAO, and provided new ideas for the pathogenesis and course of TAO.

Materials and methods

General information

From January 2022 to December 2023, patients admitted to the Department of ophthalmology, the Provincial Hospital Affiliated to Shandong First Medical University, who diagnosed as severe exophthalmos with thyroid-related eye disease were collected. A total of 30 patients (30 eyes) were included in this study (Fig. 1-a), including 15 male 15 eyes and 15 female 15 eyes. The criteria for inclusion in the TAO case group included: complete clinical information; age between 18 and 60 years, with specific diagnostic conditions, eye processes exceeding 23 mm, clinical activity assessment (CAS) score exceeding 3 [5], and thyroid function tests within normal range before orbital decompression surgery. At the same time, 30 patients (30 eyes) were selected as the control group, who undergoing double blepharoplasty, pouch resection or orbital wall repair surgery without systemic lesions in the same period, including 15 male 15 eyes and 15 female 15 eyes. Exclusion criteria: patients with autoimmune disease or tumors; patients with other types of thyroid disease or a family history of thyroid disease; severe cardio-renal insufficiency; and patients without treatment. There were no significant differences in general data between the two groups. This study was approved by the hospital ethics committee.

Fig. 1.

Pre-and post-operative images. a Pre-decompressed surgery of the orbital wall in TAO with severe exophthalmos; b The orbital fat removed in bony decompression surgery; c The taken-out adipose tissue; d Postoperative bony orbital decompression in TAO

Study methods

Ophthalmic evaluation

Visual acuity was measured by the 1990 International Universal Vision Test (Log MAR) and accurately assessed using the YZ-S eyeball protrusion meter. Rigorous orbital imaging analysis, including CT scan and MRI technique, was performed for each participant before and after the procedure.

General body physical examination

Blood, urine routine tests, full viral infection tests, biochemical function assessment, thyroid function assessment, electrocardiogram examination, and chest X-ray to ensure that all potential factors are fully considered.

Operation method

Lateral orbital + orbital wall decompression in patients with severe TAO exophthalmos (Fig. 1-b)

All procedures were performed by the same surgeon under general anesthesia via endotracheal intubation. The lateral orbital wall was exposed, the fat of the herniated muscle space was removed carefully and taken into Liquid nitrogenbeen for the test Western-Blotting.

1.3.2 The patients in the control group underwent double eyelid plasty or pouch resection or orbital fracture repair, and the fat was removed during the operation.

The expression of IL-27 and IL-35 was confirmed by Western blotting

IL-27 and IL-35 antibody were purchased from Wuhan AIBOTEK Biotechnology Co.,Ltd. After the orbital adipose tissue was removed on the day of surgery in all patients and normal control subjects, the vascular and fiber components were removed under the microscope and stored in liquid nitrogenfor 1 h. Western blot was used to detect the expression of IL-27 and IL-35 in the orbital fat of each group, in strict accordance with the instructions of the kit. Lysates of 20 ug of each sample were electrophoresed and transferred to PVDF membranes through electroblotting. The membranes were probed with specifc primary antibodies and subsequently with secondary antibodies. Antibody binding was detected using an ECL system. The protein expression levels were quantifed using ImageJ software (version 1.6.0_24; National Institute of Health, Bethesda, MD, USA).

Statistical methods

All study data were run and processed on SPSS 20.0 software, and statistical maps were made using Graphpad Prism9. For rank data using Wilcoxon rank sum test, α = 0.05, P < 0.05 was statistically significant and P < 0.01 was very significant.

Result

All the patients in the experimental and control group completed the operation successfully, with none of them out of the group. Intraoperative fat acquisition was satisfactory and at least 2 ml of adipose tissue was obtained per patient per eye (Fig. 1-c, d).

The results of Western blot results showed that the expression of IL-27 and IL-35 in the experimental group increased significantly, and the development was clear (Fig. 2), and compared with the control group, the difference was statistically significant (P < 0.01). (Figure 3)

Fig. 2.

The expression of IL-27 in adipose tissue of normal and TAO patients was confirmed by Western Blot

Fig. 3.

The expression of IL-35 in adipose tissue of normal and TAO patients was confirmed by Western Blot

Discussion

Thyroid-associated ophthalmopathy (TAO) is the most common orbital disease in clinic. With the improvement of living standard, rhythm of life and diagnostic technique, the incidence of TAO is increasing year by year. The ocular symptoms of mild to moderate TAO are relatively mild, but severe TAO is often accompanied by severe exophthalmos, corneal ulcer, visual loss, and other malignant complications. The visual function and quality of life were affected seriously. In recent years, many researches have been devoted to the pathogenesis of thyroid-associated ophthalmopathy, but its pathogenesis is complex and its clinical manifestations are various, and it is still not completely clear [6]. Recent studies have shown that inflammatory response plays a crucial role in the development of TAO, and more than dozens of inflammatory and immune factors play a key role in the development of TAO such as serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and soluble intercellular adhesion molecule-1 (sICAM-1) et al. [7].Researches showed that about 80% of TAO patients have been found to be associated with thyroid dysfunction, lower TSH, higher FT3, FT4 in the patient indicating that the occurrence of TAO was closely related to thyroid function. TAO patients can produce a lot of Thyroid hormone, and the serum levels of IL-6, TNF-α, Sicam-1 were positively correlated with FT3 and FT4 [8]. Various inflammatory factors, orbital steatosis, proliferation, extraocular muscle thickening, resulting in orbital tissue congestion occurred malignant exophthalmos [9] Table 1.

Table 1.

Baseline information

complete clinical information
age between 18 to 60 years
with specific diagnostic conditions
eye processes exceeding 23 mm
clinical activity assessment (CAS) score exceeding 3
thyroid function tests within normal range

IL-27 and IL-35 are inflammatory factors with anti-inflammatory and immunosuppressive effects, which have received increasing attention in recent years [2]– [3]. IL-27 is a cytokine composed of IL-12 subunit p28 and IL-27 βchain. It can inhibit the differentiation and proliferation and reduce the production of IFN-γ, thus inhibiting the inflammatory response. It can also inhibit the differentiation of Th17 cells, thus reducing the production of inflammatory cytokines; Meanwhile, some studies have shown that IL-27 exerts its anti-inflammatory effects by inhibiting the production of inflammatory mediators and regulating the proliferation and differentiation of immune cells [10]. IL-35 is composed of IL-12 subunits p35 and EBI 3, which can inhibit the differentiation of initial CD4 + T cells into Th17 cells, promote the proliferation and function of regulatory T cell Tregs, and exerts immunosuppression and immune regulation functions by coordinating the ratio of Th17 to Treg cells [11]. Some studies showed that IL-27 + CD4 T cells exert a regulatory function, inhibiting the IL-2 production and proliferation of other T cells during malaria infection. These regulatory T cells are Foxp3 − CD11a + CD49d + antigen-specific cells and are distinct from Foxp3 + regulatory or IL-10-producing Tr1 T cells [12]. A recent study supported that IL-35 suppressed antigen-induced production of IL-5 and IL-13 by CD4 + CD25-effector T cells from patients with grass pollen AR. IL-35 also inhibited IL-5 and IL-13 production by ILC2 in the presence of IL-25 or IL-33, and CD40 ligand-, IL-4-and IL-21-mediated IgE production by B cells [13]. In recent years, there have been some studies on IL-27 and IL-35 in the department of respiratory, oncology, nephrology, rheumatism and immunology, but in ophthalmology has not been reported. In this experiment, we detected the expression of IL-27 and IL-35 in the orbital fat of TAO patients with malignant exophthalmos for the first time. The results showed that the two inflammatory factors were significantly higher (P < 0.01) Compared with the control group, suggesting that these two factors also participate in the occurrence and development of thyroid-related eye diseases, and played a role in the degeneration and proliferation of the condition and prognosis of TAO patients, and provided a new idea for the study of the pathogenesis and clinical treatment of TAO.

There were some limitations in our study. Due to the complexity and variability of TAO, more samples may provide more accurate results. Furthermore, the difference between the two groups should be verified from different levels, such as qPCR and immunohistochemical staining in the follow-up studies.

Authors’ contributions

All authors contributed to the study conception and design. Data collection were performed by BW, and analysis were performed by BW, ZX-X and XF-Z. The first draft of the manuscript was written by BW, LJ-Z and HZ made substantial contributions to the conception or design of the work. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.