The Roles of Anti-RNP Antibodies in Primary Sjögren’s Syndrome Pathogenesis and Severity May Be Underestimated

Background Associations between anti-ribonucleoprotein (RNP) antibodies status and distinct clinical primary Sjögren’s syndrome (pSS) subtypes have not yet been rmly established. The aim of our study is to determine whether associations exist between RNP antibody status and the clinical manifestations, laboratory features, or disease activity in pSS. Methods A retrospective cohort of 39 anti-RNP antibody-positive and 294 anti-RNP antibody-negative pSS patients was assembled. Data regarding demographic information, glandular and extraglandular manifestations, laboratory ndings, and disease activity (scored according to the European League Against Rheumatism SS disease activity index (ESSDAI)) were extracted from patient records. Univariate methods followed by multivariable logistic regression analysis were used to evaluate potential associations between anti-RNP antibody status and clinicopathologic features. Results Patients with anti-RNP antibody-positive pSS had a higher prevalence of Raynaud’s phenomenon (RP) and hematological, pulmonary, lymphatic system, and mucocutaneous involvement; higher erythrocyte sedimentation rates and serum IgG levels; lower lymphocytes counts; and signicantly higher ESSDAI scores (median (interquartile range): 13 (7–18) versus 7 (3–12), p < 0.001). No signicant differences were observed between groups for C-reactive protein levels and rheumatoid factor or anti-Ro/SSA or -La/SSB antibody positivity. Multivariate analysis identied RP, interstitial lung disease (ILD), and lymphatic system involvement as independent predictors of anti-RNP antibody positivity in pSS patients. Conclusions In this cohort, anti-RNP antibodies were associated with several clinicopathologic features of severe pSS, such as RP and hematologic, lymphatic, and pulmonary disorders. Therefore, anti-RNP antibodies may play an important role in the pathogenesis and severity of pSS.


Introduction
Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic in ltration and dysfunction of lacrimal and salivary glands, resulting in xerophthalmia and xerostomia.
However, the clinical features of pSS are heterogeneous and vary from localized sicca symptoms to systemic involvement. Extra-glandular manifestations occur in approximately one third of patients [1,2].
In addition, the variety of autoantibody pro les observed in pSS patients is associated with differential clinical manifestations [1,3]. Anti-Ro/SSA and -La/SSB antibodies are diagnostic hallmarks of pSS [4], but patients may also exhibit a number of other autoantibody types including rheumatoid factor (RF), anti-centromere antibodies (ACA), anti-Ro52 antibodies, and anti-ribonucleoprotein (RNP) antibodies. However, the existence of associations between different autoantibodies and particular clinical features is controversial [5]. Nevertheless/For example, ACA antibodies are associated with a higher mean age at disease onset, and increased frequencies of Raynaud's phenomenon (RP), sclerodactyly, peripheral neuropathy, and concomitant autoimmune disorders (e.g., autoimmune thyroiditis and primary biliary cirrhosis) [6][7][8][9]. Similarly, RF is associated with more severe ocular symptoms; increased frequencies of articular manifestations, cutaneous vasculitis, salivary gland enlargement, cytopenias, RP, renal involvement, and central nervous system involvement; and higher European League Against Rheumatism SS disease activity index (ESSDAI) scores [10][11][12].
High titers of anti-RNP antibodies are characteristically associated with mixed connective tissue disease (MCTD), originally described in 1972 [13,14]. However, anti-RNP antibodies are also detectable across a broad spectrum of autoimmune diseases, including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), myositis, rheumatoid arthritis (RA), and pSS [15][16][17]. Clinical signi cance of anti-RNP antibodies is a matter of debate. In the context of SLE, anti-RNP antibodies occur in 20-33 % of patients and are associated with speci c clinical manifestations (e.g., scleroderma-like features) [18][19][20]. However, the utility of anti-RNP antibodies in classifying clinical pSS subgroups remains unknown. In the context of pSS, anti-RNP antibodies occur in 1.5-13.8 % of patients, depending on study population demographic characteristics [21,22]. Despite this relatively high prevalence, anti-RNP antibody-positive pSS patients are poorly described, although anti-RNP antibodies may be associated with more active systemic disease, more frequent muscular and pulmonary involvement, and increased gammaglobulin levels [15].
Interpreting the clinical signi cance of anti-RNP antibody positivity in pSS patients is challenging, given the uncertain role of these autoantibodies in disease pathogenesis and their potential association with distinct clinicopathologic features. Moreover, these autoantibodies may have prognostic value in pSS patients. Finally, no study has yet examined anti-RNP antibody positivity in pSS patients of Asian descent. Because studies evaluating the potential association between anti-RNP antibody presence and clinical manifestations (including severity, organ involvement, and disease activity) are urgently required, the present study retrospectively assessed a large pSS patient cohort to determine whether associations exist between anti-RNP antibody status and clinicopathologic manifestations, including disease activity.

Statistical analysis
All statistical analyses were performed using SPSS version 25.0 (SPSS Inc., IL, USA). Data were expressed as median (interquartile range) for continuous variables and number (%) for categorical variables. Anti-RNP antibody-positive and -negative patient groups were compared using the Mann-Whitney U test for continuous variables and the χ 2 test or Fisher's exact test for categorical variables.
Differences were considered statistically signi cant at p < 0.05 (two-tailed). Variables identi ed as differing signi cantly between groups by univariate analysis were considered for multivariate logistic regression analysis. Odds ratios (ORs) and 95 % con dence intervals (CIs) were calculated.

Discussion
Findings of the present study suggest that anti-RNP antibody-positive pSS -despite sharing many common (especially pathological) features with anti-RNP antibody-negative pSS -constitutes a clinical subgroup distinct from other well-known pSS subgroups.Speci cally, anti-RNP antibody-positive patients demonstrated signi cantly higher frequencies of extra-glandular manifestations more commonly associated with MCTD and SLE, including RP, and pulmonary, lymphatic system, mucocutaneous, and hematological involvement. Additionally, anti-RNP antibody-positive patients exhibited signi cantly higher ESSDAI scores.
In the present cohort, anti-RNP antibody positivity prevalence was approximately 12 %, which is consistent with that reported in a previous study. In an Israeli pSS cohort (N = 82), 13.41 % of patients exhibited anti-RNP antibody positivity [24]. However, in a pSS cohort of Hispanic descent (N = 402), anti-RNP antibody positivity prevalence was only 2.49 % [25], and a French pSS cohort (N = 467) exhibited 4.50 % anti-RNP antibody positivity prevalence [15]. This relatively broad range of prevalence is likely attributable to ethnicity-related genetic factors.
Although the presence of anti-RNP antibodies is not included in American-European Consensus Group 2002 classi cation criteria for pSS [4], they do seem to be associated with a speci c subset of system involvements and disease activity. Notably, the present study demonstrated that such patients exhibited a higher frequency of RP, and that RP was independently predictive for anti-RNP antibody positivity. This is consistent with anti-RNP antibody positivity association with RP across a broad spectrum of connective tissue disorders (e.g., MCTD, SSc, SLE, and RA) [26,27]. This is interesting considering that ANA, ACA, anti-RNP antibody, anti-Sm antibody, and anti-Scl-70 antibody status is apparently not of utility in elucidating the pathology underlying RP [28]. However, our previous research also suggested that pSS patients with RP represent a distinct subset characterized by more frequent presence of ACA or anti-RNP antibodies, and more frequent pulmonary involvement. Furthermore, it has been hypothesized that RP may be associated with antibodies targeting oxidized U1-70kDa (a common target of anti-RNP antibodypositive autoimmune sera), which may be generated due to the formation of reactive oxygen species during ischemia-reperfusion [29,30].
Regarding pulmonary manifestations of pSS, anti-RNP antibody positivity is known to be associated with pulmonary hypertension and ILD. Pulmonary brosis is more prevalent in anti-RNP antibody-positive than in anti-RNP antibody-negative pSS (51.28 % versus 15.65%). Moreover, we previously demonstrated that pSS patients with ILD exhibited a signi cantly higher frequency of anti-RNP antibody positivity than those without ILD (i.e., that the presence of anti-RNP antibodies was signi cantly associated with the risk of ILD). This is consistent with other studies reporting a higher incidence of ILD in the anti-RNP antibodypositive pSS patient cluster. Within such a patient cohort (N = 188), 26 % who underwent a computed tomography (CT) scan of the chest had ILD [31]. Interestingly, ILD (one of the most common extramuscular features of myositis) occurs in 45 % of anti-U1-RNP antibody-positive myositis patients, but in signi cantly lower proportions of DM patients (13 %) and IMNM patients (6 %) (all: p < 0.01) [32]. It was hypothesized that anti-RNP antibodies may contribute to connective tissue disorder-associated ILD by recognizing pulmonary endothelial antigens and stimulating in ammatory cytokine production [33][34][35][36]. In contrast, a meta-analysis revisiting antisynthetase syndrome patients demonstrated a lower frequency of ILD in patients with anti-U1-RNP autoantibodies [37]. It is possible that anti-U1-RNP antibodies contribute to disease pathogenesis via distinct mechanisms in different types of connective tissue disorder [34,38]. Considering the seriousness of pulmonary brosis (which is generally asymptomatic during its early stages), screening by means of pulmonary CT may be prudent in anti-RNP antibody-positive pSS. In contrast, the present study found no signi cant difference in pulmonary hypertension frequency (12.82 % versus 5.78 %, p = 0.16) on the basis of anti-RNP antibody status.
Anti-RNP antibody positivity also correlated with hematological involvement in the present study: the proportion of patients exhibiting leukocytopenia, lymphopenia, and thrombocytopenia was signi cantly higher in the anti-RNP antibody-positive group. It has previously been suggested that anti-Sm/RNP antibodies (but not anti-Sm antibodies) are strong correlates of hematological disorders in SLE patients of European-American and African-American (but not Hispanic) descent. Relatedly, autoantibodies targeting RNP (but not Sm) were independently enriched in SLE patients of Hispanic descent exhibiting lymphopenia [39]. There may be an inter-individual difference in mechanisms by which anti-RNP antibodies contribute to pSS pathology. However, mechanisms underlying the above associations require further elucidation. Regarding muscular involvement, anti-RNP antibody positivity is associated with more frequent muscular involvement in pSS patients [15]. However, the present study did not con rm the association between anti-RNP antibody positivity and myositis.
Several potential limitations of the present study are acknowledged. First, the retrospective nature of the study precluded inclusion of comprehensive patient clinical data. For example, electromyography and muscle biopsy ndings were not available for all patients, thereby preventing conclusions regarding the true prevalence of myositis in this population. A longitudinal prospective study would address such shortcomings. Second, the proportion of anti-RNP antibody-positive pSS patients was small relative to that of anti-RNP antibody-negative pSS patients. However, it was large enough to facilitate conclusions regarding statistical signi cance of between-group differences. Third, an insu cient number of anti-RNP antibody-positive patients had long-term follow-up data available. Finally, we did not evaluate associations between the presence of anti-RNP antibodies and other autoantibodies commonly found in pSS patients. Larger, multicenter, prospective studies (including systematic evaluation of autoantibody pro les) are recommended to elucidate the mechanistic and clinical signi cance of autoantibodies in pSS.

Conclusions
Taking together, our ndings suggest that anti-RNP antibodies, long underestimated, may play key roles in the pathogenesis of pSS, resulting in distinct clinical and immunological characteristics of the anti-RNP antibody-positive patient group. In particular, RP, thrombocytopenia, and lymphatic system involvement are independently associated with anti-RNP antibody positivity. These observations highlight the potential mechanistic and clinical signi cance of anti-RNP antibody positivity, and suggest that knowledge of anti-RNP antibody status may have utility in risk strati cation, diagnosis, and prognostication (e.g., enhancing early diagnosis of severe organ dysfunction, such as pulmonary brosis). To the best of our knowledge, this is the rst study exploring the association between anti-RNP antibody status and clinicopathologic features in pSS patients of Asian descent, and the rst study to demonstrate that RP, thrombocytopenia, and lymphatic system involvement are independently associated with anti-RNP antibody positivity.

Consent for publication
Not applicable Availability of data and materials All data generated or analysed during this study are included in this published article and its supplementary information les. The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Lin Wei, Xin Zhifei, and Ning Xiaoran were involved in drafting the manuscript. All authors participated in the study conception and design, participated in the acquisition of data, and analysis and interpretation of data. All authors were involved in revising it critically for important intellectual content, and revising the nal version to be submitted for publication.

Figure 2
Multivariate Analysis of features predicting anti-RNP positive in pSS patients.