Background
Factors involved in the development of various autoimmune diseases including type I diabetes (T1D), rheumatoid arthritis, myositis, and systemic lupus erythematosus (SLE) remain poorly defined. Studies of monozygotic twins with autoimmune conditions including multiple sclerosis, T1D, rheumatoid arthritis and SLE show a concordance rate of 20-70%, suggesting that the genetic makeup alone cannot completely explain the pathogenesis of autoimmunity[
1]. Consistent with these findings, genetic linkage studies have identified a limited number of susceptibility genes responsible for different autoimmune conditions, many of which play important roles in immune function[
2]. Increasing evidence suggests that environmental factors, including epigenetic DNA methylation, chemical exposures, and host-pathogen interactions, may trigger autoimmune conditions in genetically susceptible individuals[
3‐
5]. Specific environmental exposures, including pathogens, as well as with the timing of exposure or infection, are of particular interest because they may profoundly impact immune function and may promote autoimmunity[
6].
Systemic autoimmune diseases including systemic sclerosis, rheumatoid arthritis, SLE and idiopathic inflammatory myopathies (IIM) or myositis, are a group of immune disorders characterized by immune activation, autoantibody production and tissue destruction involving multiple organs. Although the target tissues and symptoms are markedly different among these diseases, a common molecular feature is the increased expression of interferon-regulated genes[
7]. For example, gene expression studies have shown that SLE, Sjögren’s syndrome and dermatomyositis (DM, a subtype of IIM with characteristic cutaneous findings) share up-regulated expression of type 1 interferon response genes[
8‐
11]. In some cases, increased interferon-α has been reported in patients with systemic autoimmune disease, whereby this cytokine likely drives inflammation in target tissues[
12‐
14].
Autoantibodies are another common feature of these diseases, which have important clinical and prognostic utility[
15]. A number of autoantibodies such as anti-DNA, anti-Smith (Sm) and antinuclear antibodies (ANA) have been selected as classification criteria for SLE[
16]. Some autoantibodies are myositis-specific and include those reactive against aminoacyl-tRNA synthetases, signal recognition particle, Ku, Mi-2, p155/140, and PM/Scl[
17]. However, autoantibodies against other targets, such as SSA and SSB, are not disease-specific and can be detected in patients with most systemic autoimmune conditions. Besides assisting with clinical diagnosis, the levels of autoantibodies can correlate with specific symptoms and disease severity[
15]. In some cases, autoantibodies have also been detected before clinical symptoms, thereby providing insight into the temporal onset of autoimmune disease[
15]. In one seminal study in SLE, autoantibodies against several targets, including DNA, SSA, SSB and Sm antigens, were detected before the clinical onset of SLE[
18].
However, despite the widespread applications of autoantibody testing, only a limited number of studies have utilized autoantibody profiling to study cohorts of twins to dissect genetic and environmental factors that may contribute to systemic autoimmune disorders. In one small study of seven twin pairs discordant for SLE, 50% of the affected twins were found to have anti-DNA antibodies[
19]. Other studies have used first degree relatives of SLE patients instead of twins and have found higher ANA autoantibody levels in those unaffected, related subjects than those seen in unrelated, healthy controls[
20,
21].
Recently, we have employed a liquid phase immunoassay, luciferase immunoprecipitation systems (LIPS), which utilizes light-emitting recombinant antigens to efficiently detect antibodies against linear and conformational epitopes associated with a variety of human autoantigens and infectious agents[
22]. Due to the wide dynamic range of detection and low backgrounds, LIPS has been highly informative in characterizing autoantibody levels in multiple autoimmune conditions, including patients with opportunistic infections[
23], Sjögren’s syndrome[
24], T1D[
25], Stiffman syndrome[
26], myasthenia gravis[
27] and SLE[
28]. In T1D, Sjögren’s syndrome and SLE, LIPS identified unique patient autoantibody profiles that potentially associated with disease subsets[
24,
25,
28]. Here we describe our findings profiling antibodies against a panel of autoantigens in a cohort of 31 twin pairs discordant for myositis or SLE along with control subjects.
Discussion
While autoimmune diseases have high morbidity and mortality, little is known about the cause of most autoimmune disorders[
39]. Here autoantibody profiles were used to study a cross-sectional cohort of mono-and dizygotic twins discordant for myositis and SLE. The overall data provides compelling evidence that autoantibodies selectively segregated in the twins with autoimmune disease and were not prevalent in the corresponding unaffected twin or in matched controls. While the presence of autoantibodies may indicate sustained disease activity in the affected twins, the reduced seropositivity in the unaffected twins may indicate a lack of detectable subclinical disease. Additionally, there were no significant differences in autoantibody levels between the unaffected twins and healthy controls. These findings suggest that there are no intermediate autoantibody levels in the unaffected twins between the affected twins and healthy controls.
Although three of the unaffected twins had autoantibody responses, the autoantibody levels were low and directed against three autoantigens, TPO, TGM2 and IFN-γ, which are not typically associated with systemic autoimmunity. Autoantibodies against TPO and TGM2 are also common in the general population, while the low levels of autoantibodies against IFN-γ in one unaffected twin was unlikely to have neutralizing cytokine activity. Moreover, the corresponding affected twins in each case were not found to harbor these three autoantibodies. The presence of autoantibodies in three unaffected twins may reflect normal, acute inflammatory conditions, such as those seen in response to infection, or possibly other immune-mediated abnormalities observed among relatives of patients with autoimmune conditions. A key finding in our study was that 31.8% of the monozygotic affected twins were autoantibody seropositive vs. only 4.5% of the unaffected, monozygotic twins (
P = 0.046) suggesting that the production of autoantibodies likely involves more than genetic risk factors requiring additional epigenetic or environmental factors for inducing disease[
4,
5].
Based on our previous study, approximately 90% of SLE patients demonstrated autoantibodies against five of the candidate autoantigens[
28]. Surprisingly, we detected only 44% seropositivity for the SLE affected twins (4/9) and 41% seropositivity for the myositis affected twins (9/22) against this larger autoantigen panel. The lower number of seropositive affected twins may have been due to two features of the cohort. First, the subjects used in the present study were younger (i.e. mean age of 14.2 years) compared to adults that were used in the previous study. Children with these autoimmune manifestations may represent different subsets of patients and would have had the disease for a relatively shorter period of time. Secondly, approximately 90% of the affected twins were receiving treatment, which may have attenuated autoantibody responses. Another potential explanation for the observed relative lack of autoantibodies is that our autoantigen panel did not include either anti-DNA or anti-phospholipids antigens for SLE[
40] or other DM-specific autoantigens[
41]. A more extended autoantigen panel including several of these additional autoantigens may provide further insights into the nature of the disease-discordant twin pair cohort. Our current findings that affected twins with high autoantibody levels had increased disease activity illustrate the possible clinical usefulness of the LIPS assay approach in serial analyses.
One novel finding of our study was the detection of autoantibodies directed against the gastric ATPase in several myositis patients that were also positive for other autoantigens. Previous studies have identified autoantibodies against the gastric ATPase in other autoimmune conditions including autoimmune gastritis[
42], Sjögren’s Syndrome[
24], and T1D[
25,
43]. The finding of gastric autoantibodies in some myositis patients is perhaps consistent with the gastrointestinal symptoms experienced in some patients. A variety of organ-specific autoantibodies have recently been described in JDM patients from Brazil including those against TPO and T1D autoantigens[
44]. The autoantibodies observed in our cohort against the gastric ATPase may reflect the B-cell immune dysfunction or epitope spreading in more severe cases of myositis. It would be of interest to further examine longitudinal samples from gastric ATPase seropositive myositis patients to determine the temporal relationship between seroconversion and myositis disease onset. Based on recent findings concerning the role of the intestinal microbiome in autoimmune disease[
45], further studies are needed to explore the possibility that the gastrointestinal infections might trigger these autoantibodies.
In contrast to the study by Reichlin et al.[
19], we found little evidence for the presence of autoantibodies in otherwise healthy unaffected SLE twins. Another study of ANA autoantibodies in SLE and first-degree relatives (FDR) suggested measurable ANA titers in family members[
20]. In a related SLE study, only a small percentage of unaffected family members had autoantibodies against SSA (49.57% with SLE vs. 4.9% FDR) and DNA (79.1% with SLE vs. 4.58% FDR)[
21]. Studies with adult discordant twins (i.e. average age > 40 years) for rheumatoid arthritis[
46] and Hashimoto’s thyroiditis[
47] have also found autoantibodies in the unaffected twins. There are some important differences in these published studies compared to our study. First, our cohort consisted of primarily myositis patients and a smaller number of SLE patients. Most importantly, the age of the subjects from our cross-sectional study was considerably younger (i.e. mean age of 14.2 years) than other published studies. If the genetic background is necessary but not sufficient for autoantibody production, the younger twins examined in our study may have had insufficient time to develop higher titer autoantibodies. Moreover, our study used a highly quantitative assay that employed a panel of autoantigens that focused on specific protein targets. In this study, we did not examine ANA or anti-DNA autoantibodies, which have been characterized more extensively in other studies. It is possible that more subtle clinical findings like ANA or altered proteomic and gene expression profiles are present in genetically-related individuals with and without autoimmune disease[
29,
30]. For example, intermediate gene expression profiles have been observed in unaffected twins compared with affected twins and healthy controls[
30]. Lastly, it is important to point out that our study has several limitations, including the small sample size and combination of adults and children, resulting from the challenges of identifying and recruiting qualified twins discordant for IIM and SLE. Additional studies in larger cohorts will be needed to more completely assess our findings.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Contribution: LG contributed to study design, sample preparation, data analyses, and manuscript preparation. TPO’H contributed to study design, sample preparation, and manuscript editing. AG contributed to LIPS test, data analysis and manuscript editing. LGR contributed to patient recruitment, clinical assessments, and manuscript editing. FWM contributed to study design, patient recruitment, clinical assessments, and manuscript editing. P.D.B contributed to study design, LIPS test, data analyses, and manuscript drafting and editing. All authors read and approved the final manuscript.