Background
Sjögren’s Syndrome (SS) is an autoimmune condition characterized by epithelial inflammation of the salivary and lacrimal glands, causing dysfunction of these exocrine glands [
1]. The spectrum of symptoms seen in SS ranges from dry eyes and mouth to diverse, systemic extraglandular manifestations. Glandular inflammatory infiltrates, comprised of B-, T-, natural killer and other immune cells, along with cytokines, are key drivers of autoimmunity. Importantly, an activated type I interferon signature characterized by the up-regulation of many interferon inducible genes has been identified in SS patients through gene expression profiling of the salivary gland [
2,
3], peripheral blood monocytes [
4,
5], and plasmacytoid dendritic cells [
5]. While low levels of interferon-α are found in the serum of SS patients, interferon-α-producing cells are enriched in the salivary gland, consistent with an increased local cytokine production [
6]. In addition, other autoimmune conditions, including systemic lupus erythematosus (SLE), dermatomyositis, and psoriasis, also show an interferon signature [
7‐
10]. In SLE patients, elevated interferon-α levels in blood correlate with disease flares [
11‐
15].
Naturally occurring anticytokine autoantibodies are increasingly being linked to autoimmune-mediated immunodeficiency and other conditions [
16‐
18]. In patients with pulmonary alveolar proteinosis (PAP), high levels of anti-GMCSF autoantibodies neutralize the activity of this cytokine and cause macrophage and neutrophil dysfunction leading to pulmonary pathology [
19]. Similarly, patients with neutralizing anti-interferon-γ autoantibodies block signaling of this cytokine and are often associated with disseminated non-tuberculous mycobacterial (DNTM) infections [
20‐
23]. Thymoma patients with elevated levels of autoantibodies against multiple cytokines, including interferon-α, anti-interferon-ω, and interleukin-12, have immune deficiency-like infections such as chronic mucocutaneous candidiasis, disseminated varicella zoster virus infection, and/or other opportunistic infections [
24,
25]. Increasing evidence suggests that neutralizing anticytokine autoantibodies, in some cases, may also alter autoimmune disease activity. Notably, SLE patients harboring high levels of anti-interferon-α autoantibodies demonstrate less severe disease activity [
26‐
29]. Furthermore, a subset of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients exhibiting anticytokine autoantibodies did not develop type I diabetes [
30]. Despite these findings, a mechanistic understanding of how anticytokine autoantibodies are generated in autoimmune diseases and their association with clinical symptoms remains poorly understood.
Luciferase immunoprecipitations systems (LIPS) utilizes light-emitting recombinant antigens to detect autoantibodies with high sensitivity and specificity in different autoimmune conditions [
31] including SLE [
28] and SS [
32‐
36]. This is because LIPS is a fluid-phase immunoassay that presents autoantigens such as cytokines in solution, allowing them to adopt native structures. Numerous studies have shown LIPS to generate highly informative anticytokine autoantibody profiles in multiple human disorders [
20,
25,
28,
30,
37‐
40]. In one study, LIPS was shown to strongly track the anticytokine autoantibodies observed by ELISA and protein array, but LIPS demonstrated autoantibody levels spanning a larger dynamic range of detection [
30]. Based on the interferon-α gene expression signature found in SS, patients with this autoimmune condition were examined for anticytokine autoantibodies to determine if they correlated with clinical symptoms.
Methods
Healthy volunteer and SS subjects
All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Sera from fifty-seven well-characterized patients diagnosed with primary SS and twenty-five healthy volunteers (HV) were evaluated as part of a natural history study under Institutional Review Board-approved protocol (IRB-D-0172) at the SS clinic of the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD. The diagnosis of SS was established using the 2002 American-European consensus criteria [
41]. These same serum samples were also used in a previous study for LIPS profiling of autoantibodies to Ro52, Ro60, and other autoantigens [
32]. Most SS cases were from the time of diagnosis in the SS clinic.
In addition to the SS cases and healthy controls, selected positive control sera containing high levels of known anticytokine autoantibodies were collected under Institutional Review Board-approved protocols (NCT02190266) at the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. These clinical samples included two PAP patients with high titer GM-CSF autoantibodies [
42], two DNTM patients with high levels of interferon-γ autoantibodies [
20], and two thymoma patients with levels of autoantibodies against interferon-α, interferon-λ, and interferon-ω [
25]. Serum samples from these subjects were tested in parallel by LIPS as positive controls and in some cases for the study of serum anticytokine neutralizing activity.
Besides autoantibody data, other clinical information for the SS patients and volunteers included standardized tests for salivary flow rate, lacrimal gland function (Schirmer’s tests), and histopathological focus scores from minor salivary gland biopsy. The values for the focus score, a marker of clustered lymphocytic infiltrate in the salivary biopsy range from 0 (no infiltrate) to 12 (confluent). Rheumatoid factor (RF), extractable nuclear antigen (ENA), and antinuclear antibodies (ANA) were determined by ELISA in the Laboratory of Clinical Medicine, Clinical Center, NIH.
Luciferase immunoprecipitations systems (LIPS) assays
Based on a previous study of anticytokine autoantibodies in SS [
29], a select LIPS panel of five autoantigen targets was employed. The five cytokine targets included GM-CSF, IFN-γ, IFN- λ1, IFN-ω and IFN-α1 and have been previously described [
20,
25,
28]. For LIPS autoantibody testing, serum samples were diluted 1:10 in assay buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM MgCl
2, 1% Triton X-100), arrayed in 96 deep well microtiter plates, and tested as described [
43]. Buffer blanks were used to monitor the performance and background binding activity of the LIPS assays. LUs were measured using a Berthold luminometer and all LU data were obtained from the average of at least two separate experiments.
Analysis of anticytokine autoantibody neutralization activity
Using an established in vitro assay [
20,
25,
29,
37,
44,
45], the neutralizing capacity of specific anti-interferon autoantibodies, were evaluated using control peripheral blood mononuclear cells (PBMC). In these experiments, PBMC were incubated in the presence of 10% healthy volunteer or patient sera and left unstimulated or stimulated with the cytokine recognized by the specific anticytokine autoantibody found in the serum samples. The PBMC were then fixed and permeabilized. To detect intracellular phosphorylation of the specific downstream Signal Transducer and Activator of Transcription-1 (STAT-1) the PBMC were immunostained using a monoclonal antibody to phospho-STAT-1 (BD Biosciences) and analyzed by flow cytometry. In the case of patients harboring interferon-α autoantibodies, cells were stimulated with interferon-α (1000 U/ml) and assessed for the corresponding interferon-α-induced pSTAT-1 in CD14+ monocytes. Data were collected using FACSCanto (BD Biosciences) and analyzed using FlowJo Version 9.1 software (TreeStar). Using this method, the amount of pSTAT production due to cytokine stimulation was extremely reproducible and sensitive to varying amounts of the cytokine added. Serum samples sera from the different subjects were then classified as non-neutralizing, partially neutralizing and neutralizing.
Statistical and data analysis
GraphPad Prism software (San Diego, CA) was used for analysis of LIPS autoantibody data and for plotting values. For each test, LU were determined from the average of at least two separate measurements. Cut-off values were determined from the mean plus five standard deviations (SD) of the healthy volunteers and are indicated for each autoantibody test in the figures.
Discussion
While high levels of anticytokine autoantibodies are often identified in the setting of opportunistic infection [
16,
17], here we report that 16% (9/57) of our SS cohort demonstrated statistically significant levels of autoantibodies against one or more cytokines, including GM-CSF and interferon-α, -γ, and -ω. Despite employing a different immunoassay for cytokine autoantibody detection, the prevalence of anticytokine autoantibodies is in general agreement with a recent larger study examining several different autoimmune diseases, including SS [
29]. The finding that one SS patient showed autoantibodies against GM-CSF and two others harbored autoantibodies against interferon-γ are consistent with the activation of these pathways in SS [
4]. Particularly intriguing was the high prevalence of autoantibodies to type I interferons. Similar to our previous findings in SLE [
28], interferon-ω autoantibody seropositivity in the SS cohort showed the highest prevalence (11%; 6/57), suggesting this cytokine may play an important role in driving immune dysfunction in both autoimmune diseases. It is important to point out that interferon-ω is historically listed as separate interferon subtype, but structurally belongs it to subfamily of interferon-α [
46]. Consistent with this observation, two of the interferon-ω subjects were seropositive for interferon-α autoantibodies. While interferon-α1 is known to be overexpressed within the salivary gland of SS patients [
6], the exact tissue source of the multiple different cytokines, including interferon-ω, and interferon-α, driving autoantibody production in SS is not known. The finding that all nine SS patients with seropositive anti-cytokine autoantibodies also harbored additional autoantibodies against multiple other autoantigens including ANA, RF, Ro52, Ro60, and La, highlights the heightened B cell activation seen in the individuals. Since not all SS patients seropositive for these SS-related autoantibodies were seropositive for anticytokine autoantibodies, additional factors such as the timing/stage of SS, HLA differences, or concurrent infection by certain pathogens, are likely to play a role.
A study of rheumatoid arthritis, SS, and SLE patients found that SLE patients had the broadest and highest range of serum anticytokine autoantibodies [
29]. In SLE, interferon-γ autoantibodies were found to correlate with the most severe disease activity. In our SS cohort, one of two anti-interferon-γ autoantibody seropositive cases had peripheral neuropathy. In addition, several of the anti-interferon-ω seropositive cases had Hashimoto’s thyroiditis and lymphoma, pulmonary hypertension, and anti-centromere autoantibodies. Due to the anticytokine autoantibody heterogeneity and the relatively small size of our cohort, a larger cohort of samples is needed to determine whether these anticytokine autoantibodies are associated with any specific sets of clinical findings.
SLE is another autoimmune disease characterized by activation of the type I interferon system [
47]. Multiple studies have demonstrated that SLE subjects harboring high levels of anti-interferon-α autoantibodies, but not anti-interferon-ω autoantibodies, were associated with more clinically quiescent disease activity [
26‐
29]. In our cohort, two SS cases exhibited autoantibodies against interferon-α, yet only one patient had relatively high levels of anti-interferon-α autoantibodies. Consistent with this observation, in vitro testing revealed that this SS subject (#56) had partially neutralizing serum autoantibodies to interferon-α. Although only serum was tested for anticytokine neutralizing activity, it is possible that high levels of interferon-α autoantibodies are present in the salivary gland. This subject fulfilling the diagnostic criteria of SS with SSA/SSB seropositivity and a low but positive focus score (i.e. 2 of 12), the subject displayed a normal unstimulated salivary flow rate and was negative for ocular problems as determined by the Schirmer’s test. Although two different serum samples taken 1 year apart from this SS subject were similarly positive for interferon-α autoantibodies, the exact onset of the anti-cytokine autoantibodies in this subject and other cases is not known. Potentially confounding this analysis is the recognition that SSA autoantibodies and other autoantibodies are often present over 18 years before clinical diagnosis of SS [
48], and it is unclear when the anticytokine autoantibodies might arise. Nevertheless, it is tempting to speculate that after the initial onset of SS in this patient that induction of anti-interferon-α autoantibodies may have later dampened interferon signaling resulting in improved clinical features. As previously proposed, the production of certain cytokine autoantibodies may be caused by by-stander autoimmunization and/or be part of a natural feedback loop to decrease cytokine signaling in chronic inflammation [
49,
50]. Recently, anti-interferon-α therapy has shown promising clinical results by reducing SLE disease activity [
51,
52]. By extension, our unique findings that the one SS patient harboring naturally, partially neutralizing anti-interferon-α activity exhibited milder sicca symptoms is potentially consistent with the idea that blocking the interferon-α pathway might show efficacy for the treatment of SS. Future studies exploring whether anticytokine autoantibodies exist in saliva derived from the salivary gland and the cell/tissue origin of the interferons involved in autoimmunization may provide additional insights into the functional significance of these autoantibodies in SS.
Authors’ contributions
PDB conceived of the study, performed LIPS assays, analyzed the data and wrote the paper. SB performed in vitro neutralization experiments. SMH provided serum samples for anticytokine positive controls. MJI contributed to study design and editing of paper. IA was director of SS clinic whose Sjögren’s syndrome and healthy volunteers were studied. All authors read and approved final manuscript.