Expressions of Type I–III Interferons in Sjögren’s Syndrome and Non-Sjögren’s Dry Eye

Dry eye disease is a condition of the ocular surface characterized by dryness of the eyes due to inadequate tear production or excessive tear evaporation and contribute to a host of symptoms ranging from mild to severe itching, burning, irritation, eye fatigue, and ocular inflammation that may lead to night blindness, conjunctival and corneal dryness, corneal ulcers, and even vision loss irreversible blindness if left untreated [1, 2]. Dry eye disease, at a minimum, causes discomfort, disabling pain, and vision disturbance, significantly limiting vision-related activities for extended periods as driving and reading, as well as recreation [3, 4]. Dry eye disease affects one in 11 people on a global scale [5]. More than 16 million individuals have diagnosed with dry eye disease in the USA, and the incidence is higher among women than men among those aged 18–34 years [6]. In Asians, the prevalence of dry eye disease is also increased in females compared with males, and its severity tends to increase with advancing age [7].

Dry eye disease is broadly classified as aqueous-deficient dry eye (ADDE) occurring due to reduced tear production and evaporative dry eye as a result of meibomian gland dysfunction [8]. One chronic inflammatory autoimmune disease causing significant ADDE is Sjögren syndrome (SS) that is characterized by immune-mediated damage to the salivary and lacrimal glands [9]. The occurrence of ADDE remains an integral part of the diagnostic criteria for SS [10]. However, the diagnosis and management of dry eye patients with underlying SS is delayed and unsatisfactory, mainly due to the diversity of patient symptoms and signs, as well as poor recognition of dry eye subtypes [11]. Significant differences with regard to cytokine levels and correlation patterns between dry eye patients with underlying SS and those without reveal the involvement of different inflammatory processes as causes of dry eye subtypes [12, 13].

Interferons (IFNs) represent a group of multifaceted cytokines that elicit significant immunomodulatory, antimicrobial, and antitumorigenic properties, which include three major subsets designated as type I IFN (IFN-α and IFN-β), type II IFN (IFN-γ), and type III IFN (IFN-λ1/IL-29, IFN-λ2/IL-28, and IFN-λ3/IL-28B) depending on their receptor usage and structural features [14‒16]. Over the last decade, robust evidence has demonstrated the essential roles of IFNs in the pathogenesis of various autoimmune diseases including primary SS [17]. Accordingly, expression patterns of IFN signatures, such as type I–II IFNs, may differ between SS-related dry eye and other dry eye subtypes [18, 19]. Although the immunoregulatory role of type III IFN has been studied in SS, limited evidence has pointed to SS-related dry eye disease.

To enhance understanding of the IFN system in dry eye disease caused by SS and provide potential diagnostic targets for underlying SS among patients with significant dry eye, the study determined and compared the serum levels of IFN-α, IFN-β, IFN-γ, IFN-λ1/IL-29, IFN-λ2/IL-28, and IFN-λ3/IL-28B in dry eye patients with or without SS.

In this prospective, observational, case-control study, ADDE patients with definitively diagnosed SS (SS group) or without underlying SS (non-SS group) were consecutively recruited from April 2021 to April 2023. The diagnosis of SS was made based on the 2012 American College of Rheumatology criteria [20] that requires positive serology (Sjögren-specific antibody A, Sjögren-specific antibody B, or a titer of rheumatoid factor vs. antinuclear antibody ≥1:320), positive labial salivary gland biopsy (focal lymphocytic sialadenitis with a focus score ≥1 per 4 mm²), and keratoconjunctivitis sicca with ocular staining score ≥3. The inclusion criteria for dry eye in both groups were reduced tear production (Schirmer’s test ≤5 mm at 5 min) [21]. Participants in the non-SS ADDE group must have no previous history of SS and negative results for serum Sjögren-specific antibody A, Sjögren-specific antibody B, showing no symptoms and positive findings in tear function tests. The exclusion criteria were a history of ocular surgery including refractive or cataract surgery, a recent ocular infection, wearing contact lenses, lid abnormalities, use of sex-steroid medicine within the previous 24 months, use of any eye drops within the previous 7 days before the examination, diagnosis of other diseases potentially inducing dry eye, such as vitamin A deficiency, Steven-Johnson syndrome, Wegener’s granulomatosis, and malignancies, infection by hepatitis B, hepatitis C or human immunodeficiency, uncontrolled hypertension, diabetes mellitus.

Eye discomfort in included patients was evaluated by using two previously validated questionnaires, the Standard Patient Evaluation of Eye Dryness (SPEED) and the Ocular Surface Disease Index (OSDI) [22]. The SPEED questionnaire serves as a frequency and severity-based questionnaire that can identify a patient’s dry eye symptoms over a period of 3 months, which inquires symptoms including dryness, grittiness or scratchiness, soreness or irritation, burning or watering, and eye fatigue, with a total score ranging from 0 to 28. The OSDI questionnaire assesses the severity of dry eye symptoms, covering 3 subscores, ocular discomfort (items 1–5), vision-related function (items 6–9), and environmental triggers (items 10–12). The total score ranges from 0 to 100, with higher scores indicating more severe dry eye symptoms. Scores of 0–12, 13–22, and 23–32 are classified as mild, moderate, and severe dry eye, respectively.

Tear stability was evaluated by noninvasive tear breakup time (NITBUT) that was recorded as the time between the last blink and the first sign of distortion of the ring pattern and measured using the Keratograph 5M (OCULUS, Wetzlar, Germany). Included patients were instructed to blink three times, successively, then, to hold their eyes open for as long as possible.

Tear secretion test was performed by using the Schirmer I test. The standard Schirmer test strips are placed at the junction of the inner and outer thirds of the lower conjunctival sac. Included patients are instructed to gently close their eyes, and after 5 min, the length of the strip moistened by tears was read.

A strip (Jingming, Tianjin, China) impregnated with fluorescent dye was wet with a drop of normal saline without preservative and then placed in the lower conjunctiva sac. The dye was observed with blue light illumination from a slit-lamp. According to the Sjögren International Collaborative Clinical Alliance scoring system [23], the corneal fluorescein staining was graded as 0 for no staining, 1 for less than 5 dots; 2 for 1–3, and 3 for bulk or strip staining for each quadrant of corneas (superior temporal, inferior temporal, superior nasal, and inferior nasal), with a total score ranging from 0 to 12.

Each participant was required to provide venous blood (5 mL) after overnight fasting. The sera was obtained after centrifugation (2,000 g for 10 min) of venous blood and stored until measurement with commercially available ELISA kits specific for IFN-α (BMS216, Affymetrix eBioscience), IFN-β (CSB-E09889h; Cusabi, Wuhan, China), IFN-γ (ab174443; Abcam, Cambridge, UK), IFN-λ1/IL-29 (DY7246; R&D Systems, Minneapolis, MN, USA), IFN-λ2/IL-28A (DY1587; R&D Systems), and IFN-λ3/IL-28B (D28B00; R&D Systems) according to the manufacturer’s instructions.

The reporter cell assay was performed to determine serum type I IFN activity. The reporter cells WISH (#CCL-25; ATCC, Rockville, MD, USA) were exposed to the obtained sera of each patient for 6 h and then lysed. After extracting total RNA from sera-exposed WISH cells by using the Trizol reagents (Invitrogen, Carlsbad, CA, USA) to synthesize cDNA by using the PrimeScript RT Reagent kit (Takara, Dalian, China), expressions of canonical type I IFN-induced genes (PKR, MX1, and IFIT1) were quantified by the qRT-PCR (Table 1 lists the primer sequences) using the SYBR Master Mixture (Takara, Tokyo, Japan) and the LightCycler 480 II System (Roche Diagnostics, Indianapolis, IN, USA). GADPH was used for loading control. The relative expressions of MX1, PKR, and IFIT1 in the sera of dry eye patients with SS were normalized to those without SS and then summed to a score reflecting serum type I IFN activity.

The distribution of continuous data was determined by the Shapiro-Wilk normality test. Statistical differences regarding continuous data between the SS and non-SS ADDE groups were examined by the Student’s t test (for normally distributed data; mean ± standard deviation) and the Mann-Whitney non-parametric test (for nonnormally distributed data; median [quantile 1, quantile 3]). Area under the curve (AUC) using receiver operating characteristic method with cut-off points identified for the highest sensitivity and specificity as assessed by Youden’s index were used to estimate the diagnostic values of type I–III IFNs for underlying SS from dry eye patients. All statistical analyses and figure visualization were performed with the aid of SPSS Statistics version 23 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA) for Windows.

Among 144 dry eye patients, those with definitively diagnosed SS were regarded as the SS group (78 women; mean age: 48.74 ± 15.04 years) and those without underlying SS as the non-SS ADDE group (65 women and 1 man; mean age: 50.12 ± 15.12 years). Two groups were gender- and age-matched. The dry eye measures of patients in these two groups are summarized in Table 2. Two groups showed only a significant difference in the value of NIBUT (p = 0.006).

The mean values of IFN-α and IFN-β levels were 83.13 pg/mL and 52.38 pg/mL in the sera of dry eye patients with SS, respectively. The mean values of IFN-α and IFN-β levels were 60.52 pg/mL and 42.19 pg/mL in the sera of dry eye patients without underlying SS, respectively. Data obtained from ELISA detection suggested that the serum levels of IFN-α and IFN-β were notably higher in dry eye patients with SS than those without underlying SS (p < 0.0001, Fig. 1a). The functional assay for serum type I IFN activity showed the mean summed scores in dry eye patients with SS were remarkably increased compared to those without underlying SS (p < 0.0001, Fig. 1b). After Pearson correlation analysis, it was found that the serum type I IFN activity shared positive correlations with the serum levels of IFN-α (r = 0.735, p < 0.0001) and IFN-β (r = 0.650, p < 0.0001) of dry eye patients with SS (Fig. 1c).

The mean value of IFN-γ level was 5.16 pg/mL in the sera of dry eye patients with SS and the mean value of IFN-γ level was 3.49 in the sera of dry eye patients without underlying SS. Data obtained from ELISA detection suggested that the serum level of IFN-γ was notably higher in dry eye patients with SS than those without underlying SS (p < 0.0001, Fig. 2a).

We were unable to detect the levels of IFN-λ2/IL-28A and IFN-λ3/IL-28B in the sera of dry eye patients either with or without SS. The mean value of IFN-λ1/IL-29 level was in the sera of dry eye patients with SS and the mean value of IFN-λ1/IL-29 level was in the sera of dry eye patients without underlying SS. The IFN-λ1/IL-29 level seemed higher in dry eye patients with SS than those without underlying SS (p < 0.0001, Fig. 2b).

We employed the receiver operating characteristic method to rank the expression levels of type I–III IFNs in their ability to differentiate underlying SS from dry eye patients. The serum level of type I IFN (IFN-α level combined with IFN-β level) used as a test to predict underlying SS among dry eye patients produced an AUC of 0.86 (95% CI: 0.80–0.92) (sensitivity: 72%; specificity: 88%) (Fig. 3a). The serum level of type II IFN (IFN-γ level) applied as a test to predict underlying SS among dry eye patients yielded an AUC of 0.73 (95% CI: 0.65–0.81) (sensitivity: 62%; specificity: 81%) (Fig. 3b). The serum level of type III IFN (IFN-λ1/IL-29 level) utilized as a test to predict underlying SS among dry eye patients generated an AUC of 0.94 (95% CI: 0.90–0.98) (sensitivity: 85%; specificity: 95%) (Fig. 3c).

This is the first demonstration of the expressions of type I–III IFNs in serum samples of dry eye patients with or without SS as well as their ability to differentiate underlying SS from dry eye patients. As anticipated, the serum levels of type I IFN (IFN-α combined with IFN-β), type II IFN (IFN-γ level), and type III IFN (IFN-λ1/IL-29) was found to be higher in SS dry eye than non-SS dry eye patients. This pattern could be supported by the AUCs they yielded when used as a test to predict underlying SS among dry eye patients.

Although five classes of type I IFN, IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω, have been identified, IFN-α and IFN-β are the most extensively studied type I IFN subtypes that appears to induce a more systemic effect against viruses and other intracellular pathogens [24]. Thus, most of the biological mechanisms ascribed to type I IFN system in rheumatic diseases including SS have been limited to IFN-α and IFN-β [25]. In our study, the serum levels of IFN-α and IFN-β were notably higher in dry eye patients with SS than those without underlying SS. IFN-α and IFN-β interact with that IFN-α/β receptor (IFNAR) that has two subunits, IFNAR1 and IFNAR2. This binding leads to auto-phosphorylation of Janus protein kinases 1 (JAK1) and tyrosin kinase 2 (Tyk2) followed by activation of STAT1 and 2 as well as the formation of a STAT2/STAT1 heterodimer [26]. The subsequent binding to IFN-regulatory factors (IRFs), namely IRF3 and IRF7, induces the formation of IFN-stimulated gene factor 3 (ISG3) [27]. Then, ISG3 translocation into the nucleus promotes ISG activation and transcription, which was evident in the context of SS [28]. An upregulation of ISGs was noted in minor salivary gland tissues and ocular epithelial cells of SS patients compared to healthy controls [29]. In addition to minor salivary gland tissues, enhanced expressions of type I IFN inducible genes or proteins were demonstrated in peripheral blood mononuclear cells [30], plasmacytoid dendritic cells [31], and B cells [32]. In isolated CD14 monocytes, type I IFN signature was related to more pronounced production of autoantibodies and enhanced B-cell-activating factor gene expression in SS patients [33]. Our results revealed an enhanced ability of SS dry eye patient sera to induce IFN-induced gene expression, namely an elevated serum type I IFN activity, compared to non-SS dry eye patient sera, which, in part, were in line with another study in which a greater type I IFN activity noted in the peripheral blood of SS patients than healthy individuals [34]. Type I IFN activation in neutrophils in the context of SS contributed to mitochondrial damage as well as the production of reactive oxygen species, thus leading to the generation of neutrophil extracellular traps [35].

Apart from type I IFN, recent data pointed toward a prevailing role of type II IFN-IFN-γ in SS [36]. IFN-γ is predominantly generated in T lymphocytes and natural killer cells, to a lesser extent by B cells, dendritic cells, and macrophages. Similarly binding with IFNGR1 and IFNGR2, IFN-γ induces the activation of the JAK/STAT signaling pathway. The key proteins in this pathway, JAK1 and JAK2, interact with IFNGR1 and IFNGR2 in turn, leading to STAT1 phosphorylation followed by the activation of the promoter region of IFN-γ-induced genes [37]. In a previous pilot study, an increased level of IFN-γ was found in 82% of SS patients and associated with disease activity [38]. An increased level of IFN-γ in SS could induce the death of salivary gland epithelial cells [39]. As shown in our study, the serum level of IFN-γ was notably higher in dry eye patients with SS than those without underlying SS.

Type I and type III IFNs share many properties, including induction by viral infection, activation of shared signaling pathways, and transcriptional programs. Apart from anti-viral and antitumor function, type III IFNs activate diverse types of cells, such as plasmacytoid dendritic cells, macrophages and natural killer cells, and inhibit the T helper type 1 (Th1) or Th17 responses or neutrophils recruitment [40]. The present study obtained the finding that IFN-λ1/IL-29, the major effector of type III IFN, were elevated in dry eye patients with SS compared to those without. Concurring with this pattern, in the study performed by Apostolou et al. [41] only IFN-λ1/IL-29 was detected in the sera and was elevated in SS patients.

Several limitations of the current study should be noted when the data are interpreted: (i) more large-scale prospective studies including male patients are required to explore distinct differences among dry eye groups; (ii) tear measurements of type I–III IFN are necessary to validate serum measurements; (iii) the lack of well-understood detection limits has hindered broader applications of type I–III IFN measurements; and (iv) further validations by using digital PCR, droplet digital PCR, and next-generation sequencing to obtain greater accuracy of such analyses are required.

The study demonstrates elevated serum levels of type I IFN (IFN-α and IFN-β), type II IFN (IFN-γ), and type III IFN (IFN-λ1/IL-29) may be associated with the presence of underlying SS among dry eye patients, which aids in early identification of dry eye pathogenesis to achieve definitive diagnostic tests for underlying SS. We believe that the expression pattern of IFN signature deserves further investigations in other sites, such as minor salivary gland tissues and plasmacytoid dendritic cells and their clinical correlations.

This study protocol was reviewed and approved by the Ethics Committee of the First People’s Hospital of Lanzhou City, Approval No. 2024A-21. A written informed consent was obtained from participants. The data used for the study are available in the present study.

None of the authors have a conflict of interest to disclose.

The study was supported by the Lanzhou Municipal Guiding Project of the Lanzhou Science and Technology Bureau (No. 2022-5-123).

N.S. and L.J. conceived the study and wrote the first draft of the manuscript. Y.W. contributed to data collection. Y.D. and Y.Y. were responsible for data analysis and visualization, K.Y. and S.G. completed manuscript revisions. All authors reviewed the manuscript.

Nan Su and Lei Jiang contributed equally to this work.

All data generated or analyzed during this study are included in this published article. Further inquiries can be directed to the corresponding author.