Rethinking Sjögren Beyond Inflammation: Considering the Role of Nerves in Driving Disease Manifestations.

ABSTRACT: Sjögren syndrome (SS) is a chronic inflammatory autoimmune disease characterized by destruction of mucosal glands resulting in dry eye and dry mouth. Ocular presentations can be heterogenous in SS with corneal nerves abnormalities that are structural, functional, or both. Some individuals present with corneal hyposensitivity, with a phenotype of decreased tear production and epithelial disruption. Others present with corneal hypersensitivity, with a phenotype of neuropathic pain including light sensitivity and pain out of proportion to signs of tear dysfunction. A similar correlate can be found outside the eye, with dry mouth predominating in some individuals while pain conditions predominate in others. Understanding how nerve status affects SS phenotype is an important first step to improving disease management by targeting nerve abnormalities, as well as inflammation.

Association of Combined Anti-Ro52/TRIM21 and Anti-Ro60/SSA Antibodies With Increased Sjögren Disease Severity Through Interferon Pathway Activation.

OBJECTIVE: The biologic diagnosis of primary Sjögren disease (SjD) mainly relies on anti-Ro60/SSA antibodies, whereas the significance of anti-Ro52/TRIM21 antibodies currently remains unclear. The aim of this study was to characterize the clinical, serological, biologic, transcriptomic, and interferon profiles of patients with SjD according to their anti-Ro52/TRIM21 antibody status. METHODS: Patients with SjD from the European PRECISESADS (n = 376) and the Brittany Diagnostic Suspicion of primitive Sjögren's Syndrome (DIApSS); (n = 146) cohorts were divided into four groups: double negative (Ro52-/Ro60-), isolated anti-Ro52/TRIM21 positive (Ro52+), isolated anti-Ro60/SSA positive (Ro60+), and double-positive (Ro52+/Ro60+) patients. Clinical information; EULAR Sjögren Syndrome Disease Activity Index, a score representing systemic activity; and biologic markers associated with disease severity were evaluated. Transcriptome data obtained from whole blood by RNA sequencing and type I and II interferon signatures were analyzed for PRECISESADS patients. RESULTS: In the DIApSS cohort, Ro52+/Ro60+ patients showed significantly more parotidomegaly (33.3% vs 0%-11%) along with higher ß2-microglobulin (P = 0.0002), total immunoglobulin (P < 0.0001), and erythrocyte sedimentation rate levels (P = 0.002) as well as rheumatoid factor (RF) positivity (66.2% vs 20.8%-25%) compared to other groups. The PRECISESADS cohort corroborated these observations, with increased arthritis (P = 0.046), inflammation (P = 0.005), hypergammaglobulinemia (P < 0.0001), positive RF (P < 0.0001), leukopenia (P = 0.004), and lymphopenia (P = 0.009) in Ro52+/Ro60+ patients. Cumulative EULAR Sjögren Syndrome Disease Activity Index results further confirmed these disparities (P = 0.002). Transcriptome analysis linked anti-Ro52/TRIM21 antibody positivity to interferon pathway activation as an underlying cause for these clinical correlations. CONCLUSION: These results suggest that the combination of anti-Ro52/TRIM21 and anti-Ro60/SSA antibodies is associated with a clinical, biologic, and transcriptional profile linked to greater disease severity in SjD through the potentiation of the interferon pathway activation by anti-Ro52/TRIM21 antibodies.

EZH2 Promotes T Follicular Helper Cell Differentiation Through Enhancing STAT3 Phosphorylation in Patients With Primary Sjögren's Syndrome.

Objectives: Enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator that plays an essential role in immune system development and autoimmune diseases. This study aimed to characterize the role of EZH2 in the pathogenesis of primary Sjögren's syndrome (pSS). Methods: We analyzed EZH2 expression in two transcriptomic datasets of peripheral blood mononuclear cells (PBMCs) from pSS patients and healthy controls. We measured EZH2 expression in CD4+ T cells, CD8+ T cells, and CD19+ B cells from pSS patients and healthy controls and correlated EZH2 expression with clinical parameters. We also examined the activation, proliferation, and T-cell differentiation of CD4+ T cells using the EZH2 inhibitor GSK126, EZH2 siRNA, and EZH2-expressing vector. We further examined the STAT3 signaling pathway after EZH2 inhibition and detected Tfh differentiation in EZH2-overexpressed CD4+ T cells with STAT3 knocked down. Results: EZH2 was upregulated in GSE164885 and GSE48378. EZH2 expression was higher in pSS CD4+ and CD8+ T cells, and EZH2 expression in circulating pSS CD4+ T cells was positively correlated with IgG, IgA, ESR, RF, and the circulating Tfh population. EZH2 inhibition and silencing EZH2 suppressed activation, proliferation, and Tfh differentiation. Furthermore, overexpressing EZH2 promoted activation, proliferation, and Tfh differentiation in CD4+ T cells. EZH2 inhibition attenuated STAT3 phosphorylation in CD4+ T cells. STAT3 knockdown abrogated EZH2-promoted Tfh differentiation. Conclusions: EZH2 expression was abnormally elevated in pSS CD4+ T cells, which facilitated Tfh differentiation of CD4+ T cells by enhancing STAT3 phosphorylation. EZH2 promotes Tfh differentiation and might be implicated in pSS pathogenesis.

CCL5 Release by CCR9+ CD8 T Cells: A Potential Contributor to Immunopathology of Primary Sjögren's Syndrome.

Introduction: Increased CCL5 expression and CD8 T cells have been shown to be pivotal regulators of immunopathology in primary Sjögren's syndrome (pSS) and pSS-like disease. Increased CCL5 expression by CCR9+ CD4 T cells has previously been implicated as a contributor to immunopathology in pSS. The role of CD8 T cells and in particular CCR9+ CD8 T cells and their potential to secrete CCL5 has not previously been studied in pSS. In this study we investigated both CCR9 and CCL5 expression by CD8 T cells in pSS patients compared to healthy controls (HC). Methods: CCR9 expression on CD8 T cells from peripheral blood was compared between patients with pSS and HC by flow cytometry. Intracellular CCL5 expression by naive, memory and effector CCR9- and CCR9+ CD8 T cells was assessed. In addition, the capacity and pace of CCL5 release upon T cell activation was determined for all subsets and compared with CD4 T cells. Results: The frequency of circulating CCR9+ CD8 T cells in pSS patients is increased compared to HC. Antigen-experienced CD8 T cells, especially CCR9+ effector CD8 T cells, express the highest CCL5 levels, and release the highest levels of CCL5 upon activation. Memory and effector CD8 T cells of pSS patients express significantly less CCL5 and subsequently release less CCL5 upon stimulation compared to HC. CCR9+ CD8 T cells rapidly release CCL5 and significantly more than CCR9+ CD4 T cells. Conclusion: CCR9+ CD8 T cells express more CCL5 than CCR9- CD8 T cells. CCL5 is rapidly released upon activation, resulting in reduced intracellular expression. Reduced CCL5 expression by an elevated number of antigen-experienced CCR9-expressing CD8 T cells in pSS patients points towards increased release in vivo. This suggests that CCL5 release by CCR9+ CD8 T cells contributes to immunopathology in pSS.

Proteogenomic analysis of the autoreactive B cell repertoire in blood and tissues of patients with Sjögren's syndrome.

OBJECTIVE: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow. METHODS: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS. RESULTS: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification. CONCLUSION: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab.

Pharmacological Inhibition of Glutaminase 1 Normalized the Metabolic State and CD4+ T Cell Response in Sjogren's Syndrome.

Previous studies have shown that abnormal metabolic reprogramming in CD4+ T cells could explain the occurrence of several autoimmune disorders, including Sjogren's syndrome (SS). However, therapeutic targets of the abnormal metabolism of CD4+ T cells remain to be explored. Here, we report that glutaminase 1 (Gls1), a pivotal factor in glutaminolysis, might be involved in the pathogenesis of SS. The expression of Gls1 was upregulated in infiltrated labial CD4+ T cells and circulating CD4+ T cells of SS patients. Inhibiting Gls1 with BPTES significantly abolished the proliferation rate, as indicated by EdU, CFSE, and Western blot analyses. Additionally, BPTES downregulated the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) values of activated CD4+ T cells from SS mice. In vivo, we injected different doses of BPTES into SS-like NOD/Ltj mice and found that 10 mg/kg BPTES significantly restored the salivary flow rate. Histological and qRT-PCR analyses showed that this concentration of BPTES attenuated lymphocytic infiltration and the numbers of PCNA-positive cells and CD4+ T cells. The proportions of IFNγ-producing cells and IL-17A-producing cells and the expression of several proinflammatory cytokines, including IFNγ and IL-17A, were also affected in the salivary glands of SS-like mice. Cytokine production in circulating serum was analyzed and showed that BPTES downregulated the effector functions of Th17 cells and Th1 cells. Collectively, these results indicate a positive relationship between Gls1 and SS development. Pharmacological inhibition of Gls1 with BPTES could normalize the effector functions of CD4+ T cells and effectively attenuate the symptoms of SS.

Immunoregulatory effects of dental mesenchymal stem cells on T and B lymphocyte responses in primary Sjögren's syndrome.

Background: In this article, the authors investigate the modulatory effects of dental mesenchymal stem cells (MSCs) on lymphocyte responses in primary Sjögren's syndrome (pSS), which is an autoimmune disease resulting from keratoconjunctivitis sicca and xerostomia. Methods: Mononuclear cells isolated from pSS patients cultured with or without dental MSCs and analyzed for lymphocyte responses via flow cytometry. Results: Dental-follicle (DF)- and dental-pulp (DP)-MSCs downregulated CD4+ T lymphocyte proliferation by increasing Fas-ligand expression on T lymphocytes and FoxP3 expressing Tregs, and decreasing intracellular IFN-γ and IL-17 secretion in pSS patients. DF-MSCs decreased the plasma B cell ratio in the favor of naive B cell population in pSS patients' mononuclear cells. Conclusion: DF- and DP-MSCs can be the new cellular therapeutic candidates for the regulation of immune responses in pSS.

Plain language summary In this article, the authors investigate the modulatory effects of dental mesenchymal stem cells (dental MSCs) on lymphocyte responses in primary Sjögren's syndrome (pSS), which is characterized by the infiltration of lymphocytes in exocrine glands. Lymphocyte proliferation, apoptosis, Tregs and total Bregs, intracellular cytokine secretion, total memory, plasma and naive B cell subsets were analyzed in pSS patients and compared them with healthy individuals. Dental follicle- and dental pulp-MSCs modulated CD4⁺ T lymphocyte responses in pSS patients' mononuclear cells by increasing Fas-ligand expression, enhancing FoxP3-expressing Tregs, and decreasing pro-inflammatory cytokine secretion. Our findings provide evidence for the potential role of dental-MSCs as a cellular therapy option in the treatment of pSS.

Disease mechanisms in Sjögren's syndrome: What do we know?

Why should we explore and study disease mechanisms? This is particularly important when we are dealing with complex pathogenesis without a direct causal agent, for example, syndromes with multiple organ involvements. Sjögren's syndrome is definitely such an entity. Also, there are a number of reasons for such studies such as disclosing the aetiology, to identify biomarkers for diagnosis and assessment of the disease process and monitor response to treatment, to determine targets for treatment, to define critical items in classification criteria, amongst others. Samples available for the study of disease mechanisms in Sjögren's syndrome have included serum (autoantibodies, cytokines), DNA (gene profiling, GWAS), cells (phenotypes/flow cytometry, proportion of cells/CyTOF), tissue (focal inflammation, germinal centres, mass cytometry), and saliva (proteomics, biochemistry, mucosal immunity). An original explanatory concept for the pathogenesis of Sjögren's syndrome proposed a specific and self-perpetuating immune-mediated loss of exocrine tissue as the principal cause of glandular hypofunction. This hypothesis however falls short of accommodating several Sjögren's syndrome-related phenomena and experimental findings. Today, the emergence of advanced bio-analytical platforms has further enabled the identification of central pathogenic processes and potential biomarkers. The purpose of this minor review is to highlight a selection of previous but also recent and novel aspects on the disease mechanisms in Sjögren's syndrome.

Does HTLV-1 Infection Show Phenotypes Found in Sjögren's Syndrome?

Viruses are a possible cause for Sjögren's syndrome (SS) as an environmental factor related to SS onset, which exhibits exocrine gland dysfunction and the emergence of autoantibodies. Although retroviruses may exhibit lymphocytic infiltration into exocrine glands, human T-cell leukemia virus type 1 (HTLV-1) has been postulated to be a causative agent for SS. Transgenic mice with HTLV-1 genes showed sialadenitis resembling SS, but their phenotypic symptoms differed based on the adopted region of HTLV-1 genes. The dominance of tax gene differed in labial salivary glands (LSGs) of SS patients with HTLV 1-associated myelopathy (HAM) and adult T-cell leukemia. Although HTLV-1 was transmitted to salivary gland epithelial cells (SGECs) by a biofilm-like structure, no viral synapse formation was observed. After infection to SGECs derived from SS patients, adhesion molecules and migration factors were time-dependently released from infected SGECs. The frequency of the appearance of autoantibodies including anti-Ro/SS-A, La/SS-B antibodies in SS patients complicated with HAM is unknown; the observation of less frequent ectopic germinal center formation in HTLV-1-seropositive SS patients was a breakthrough. In addition, HTLV-1 infected cells inhibited B-lymphocyte activating factor or C-X-C motif chemokine 13 through direct contact with established follicular dendritic cell-like cells. These findings show that HTLV-1 is directly involved in the pathogenesis of SS.

TLR7 agonism accelerates disease in a mouse model of primary Sjögren's syndrome and drives expansion of T-bet+ B cells.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by chronic inflammation of exocrine tissue, resulting in loss of tears and saliva. Patients also experience many extra-glandular disease manifestations. Treatment for pSS is palliative, and there are currently no treatments available that target disease etiology. Previous studies in our lab demonstrated that MyD88 is crucial for pSS pathogenesis in the NOD.B10Sn-H2b (NOD.B10) pSS mouse model, although the way in which MyD88-dependent pathways become activated in disease remains unknown. Based on its importance in other autoimmune diseases, we hypothesized that TLR7 activation accelerates pSS pathogenesis. We administered the TLR7 agonist Imiquimod (Imq) or sham treatment to pre-disease NOD.B10 females for 6 weeks. Parallel experiments were performed in age and sex-matched C57BL/10 controls. Imq-treated pSS animals exhibited cervical lymphadenopathy, splenomegaly, and expansion of TLR7-expressing B cells. Robust lymphocytic infiltration of exocrine tissues, kidney and lung was observed in pSS mice following treatment with Imq. TLR7 agonism also induced salivary hypofunction in pSS mice, which is a hallmark of disease. Anti-nuclear autoantibodies, including Ro (SSA) and La (SSB) were increased in pSS mice following Imq administration. Cervical lymph nodes from Imq-treated NOD.B10 animals demonstrated an increase in the percentage of activated/memory CD4+ T cells. Finally, T-bet+ B cells were expanded in the spleens of Imq-treated pSS mice. Thus, activation of TLR7 accelerates local and systemic disease and promotes expansion of T-bet-expressing B cells in pSS.

Blocking IAg7 class II major histocompatibility complex by drug-like small molecules alleviated Sjögren's syndrome in NOD mice.

BACKGROUND: Sjögren's syndrome (SjS) is an autoimmune disease with a strong genetic association. To date, no vaccine or therapeutic agent exists to cure SjS, and patients must rely on lifelong therapies to treat symptoms. Human leukocyte antigens (HLA) are primary susceptibility loci that form the genetic basis for many autoimmune diseases, including SjS. In this study, we sought to determine whether blocking MHC class II IAg7 antigen presentation in the NOD mouse would alleviate SjS by preventing the recognition of autoantigens by pathogenic T cells. METHODS: Mapping of the antigenic epitopes of Ro60 autoantigen to IAg7 of the NOD mice was performed using structural modeling and in-vitro stimulation. Tetraazatricyclo-dodecane (TATD) and 8-Azaguanine (8-Aza) were previously identified as potential binders to IAg7 of the NOD mice using in silico drug screening. Mice were treated with 20mgs/kg via IP every day five days/week for 23 weeks. Disease profiling was conducted. FINDINGS: Specific peptides of Ro60 autoantigen were identified to bind to IAg7 and stimulated splenocytes of the NOD mice. Treating NOD mice with TATD or 8-Azaguanine alleviated SjS symptoms by improving salivary and lacrimal gland secretory function, decreasing the levels of autoantibodies, and reducing the severity of lymphocytic infiltration in the salivary and lacrimal glands. INTERPRETATION: This study presents a novel therapeutic approach for SjS by identifying small molecules capable of inhibiting T cell response via antigen-specific presentation. FUNDING: CQN is supported financially in part by PHS grants AI130561, DE026450, and DE028544 from the National Institutes of Health.

Salivary free light chains and salivary immunoglobulins as potential non-invasive biomarkers in primary Sjögren's syndrome.

BACKGROUND: B cells contribute significantly to the pathogenesis of primary Sjögren's syndrome (pSS). Free light chains (FLCs) are generated during the production of immunoglobulins (Igs) and are surrogates of B cell activity. We hypothesized that salivary FLCs and salivary Igs could represent salivary gland inflammation and therefore, serve as biomarkers in pSS. METHODS: Patients >18 years old fulfilling the American College of Rheumatology / European League Against Rheumatism (EULAR) 2016 criteria for pSS and age-matched healthy and disease controls (sicca non-pSS, rheumatoid arthritis, systemic lupus erythematosus) were recruited for this cross-sectional study. FLCs in saliva and serum were measured by immunoturbidimetry. Serum and salivary Igs were measured by nephelometry and enzyme-linked immunosorbent assay, respectively. Area under the receiver operator characteristic curve was determined. The factors influencing the serum and salivary FLCs in pSS were determined using backward linear regression. RESULTS: A total of 78 patients with pSS, 76 healthy controls and 62 disease controls were recruited. Median EULAR SS disease activity index (interquartile range) was 1 (3.75). Serum FLCκ and FLCλ, salivary FLCλ, serum and salivary IgG, salivary IgM was significantly higher in the pSS group compared to the controls. Areas under the curve for salivary FLCλ, serum FLCκ, serum and salivary IgG were 0.75, 0.72, 0.78 and 0.77, respectively. Regression analysis showed that salivary FLCκ, salivary FLCλ and salivary IgG were associated with positive salivary gland histopathology. Use of immunosuppressants and glucocorticoids was associated with lower values of salivary parameters. CONCLUSION: Salivary FLCλ and salivary IgG were significantly different between pSS and control groups and could be potential non-invasive biomarkers in pSS. These findings should be confirmed in larger longitudinal studies.

In vitro effects of curcumin on proinflammatory cytokines and expression of their genes in minor salivary gland tissue of patients with Sjogren's syndrome.

Curcumin reduces disease severity and ameliorates lupus-like/Sjögren's Syndrome-like disease in mice model. The immunological basis of these effects is largely unknown. This study examined the effects of curcumin on pro-inflammatory cytokines secreted by minor salivary glands in patients with primary Sjögren's syndrome (pSS). Minor salivary gland (MSG) tissue samples were collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30 µM) and cultured in RPMI 1640 medium for 48 h at 37 °C in CO2 incubator. After the incubation period, culture supernatant and tissues were stored in the freezer (-80 °C). IL-6 levels were measured in supernatant by ELISA kit. Gene expressions of pro-inflammatory cytokines, namely IL-6, IL-8, TNF-α, IL-1ß, IL-4, IL-10, IL-17, IL-21, and IFN-γ, were measured by qPCR. IL-6 secretion levels and gene expressions were compared statistically between groups by Student's t test. Forty-seven patients were screened. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serology constituted sicca controls. These 15 subjects were included in final analysis. In pSS group, but not in controls, median IL-6 levels in supernatant were less in curcumin-treated as compared to PHA-alone subset [5.5 (0.7-13.34) vs 18.3 (12-32) ng/ml; p = 0.0156]. mRNA expression levels of IL-6 were also lower in curcumin-treated samples as compared to PHA alone, when cases and controls were analyzed together as well as in cases alone (p = 0.0009 and p = 0.0078, respectively); however, mRNA expression of IL-1ß was lower in curcumin-treated samples as compared to PHA alone, only when cases and controls were analyzed together (p = 0.0215). There was no difference in other cytokine gene expression levels between the subsets under the in-vitro experimental conditions. In conclusion, curcumin reduced mRNA expression as well as secretion of IL-6 levels by salivary gland tissue of patients with pSS. Curcumin also suppressed PHA-induced mRNA expression levels of IL-6 and IL-1ß in MSG tissue of patients with pSS and controls when analyzed together as a combined group.

Patients with early-onset primary Sjögren's syndrome have distinctive clinical manifestations and circulating lymphocyte profiles.

OBJECTIVES: To further investigate the clinical characteristics and circulating lymphocyte profiles of patients with early-onset primary Sjögren's syndrome (pSS). METHOD: Data of 333 patients with pSS were analysed retrospectively. Early onset was defined as a pSS diagnosis at an age of 35 years or younger. The clinical, laboratory and immunophenotypic profiles of peripheral blood lymphocyte subsets were compared between early- and later-onset pSS. RESULTS: Thirty-six (10.81%) patients matched the definition of early-onset pSS, with age at disease onset being 28.97 (5.53) years. Elevated serum IgG level (77.14% vs 31.16%, P <0.001), low C3 (41.67% vs 20.20%, P =0.004) and C4 levels (27.78% vs 6.40%, P <0.001), anti-SSA positivity (91.67% vs 51.85%, P <0.001) and anti-SSB positivity (50% vs 20.54%, P <0.001) were more frequent in early-onset patients. The frequencies of hematological (80.56% vs 52.53%, P =0.001), renal (19.44% vs 5.05%, P =0.005) and mucocutaneous involvement (50% vs 22.56%, P <0.001) were significantly higher in the early-onset pSS group, which showed a higher 2010 EULAR SS Disease Activity Index (ESSDAI) [11(6.25-17) vs 7(3-12); P =0.003], compared with the later-onset group. In addition, profound CD4+ T-cell lymphopenia was found in patients with early-onset. CONCLUSIONS: Patients with early-onset pSS have distinctive clinical manifestations and greater activation of the cellular immune system, present with more severe clinical symptoms and immunological features, have increased activation of circulating T cells and have an unfavourable prognosis. Thus, they require more positive treatment with glucocorticoids and/or immunosuppressants and merit closer follow-up and regular monitoring.

Significance of Sjögren's syndrome and anti-cN1A antibody in myositis patients.

OBJECTIVE: We recently recorded a high prevalence of inclusion body myositis (IBM) in patients with Sjögren's syndrome (SS). Whether myositis patients with SS differ from myositis patients without SS in terms of the characteristics of the myositis is currently unknown. Anti-cytosolic 5'-nucleotidase 1 A (cN1A) has recently been proposed as a biomarker for IBM but is also frequent in SS. Whether anti-cN1A is independently associated with IBM is still an open question. We aimed to assess the significance of SS and anti-cN1A in myositis patients. METHODS: Cumulative data on all myositis patients (EULAR/ACR 2017 criteria) screened for SS (ACR/EULAR 2016 criteria) in a single centre were analysed. Ninety-nine patients were included, covering the whole spectrum of EULAR/ACR 2017 myositis subgroups and with a median follow-up of 6 years (range 1.0-37.5). The 34 myositis patients with SS (myositis/SS+) were compared with the 65 myositis patients without SS (myositis/SS-). RESULTS: . IBM was present in 24% of the myositis/SS+ patients vs 6% of the myositis/SS- group (P = 0.020). None of the IBM patients responded to treatment, whether they had SS or not. Anti-cN1A was more frequent in myositis/SS+ patients (38% vs 6%, P = 0.0005), independently of the higher prevalence of IBM in this group (multivariate P value: 0.02). Anti-cN1A antibody specificity for IBM was 0.96 (95% CI: 0.87, 0.99) in the myositis/SS- group but dropped to 0.70 (95% CI: 0.48, 0.85) in the myositis/SS+ group. INTERPRETATION: In myositis patients, SS is associated with IBM and with anti-cN1A antibodies, independently of the IBM diagnosis. As a consequence, anti-cN1A has limited specificity for IBM in myositis patients with SS.

Rheumatoid arthritis, systemic lupus erythematosus and primary Sjögren's syndrome shared megakaryocyte expansion in peripheral blood.

OBJECTIVES: Rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (pSS) share many clinical manifestations and serological features. The aim of this study was to identify the common transcriptional profiling and composition of immune cells in peripheral blood in these autoimmune diseases (ADs). METHODS: We analysed bulk RNA-seq data for enrichment of biological processes, transcription factors (TFs) and deconvolution-based immune cell types from peripheral blood mononuclear cells (PBMCs) in 119 treatment-naive patients (41 RA, 38 pSS, 28 SLE and 12 polyautoimmunity) and 20 healthy controls. The single-cell RNA-seq (scRNA-seq) and flow cytometry had been performed to further define the immune cell subsets on PBMCs. RESULTS: Similar transcriptional profiles and common gene expression signatures associated with nucleosome assembly and haemostasis were identified across RA, SLE, pSS and polyautoimmunity. Distinct TF ensembles and gene regulatory network were mainly enriched in haematopoiesis. The upregulated cell-lineage-specific TFs PBX1, GATA1, TAL1 and GFI1B demonstrated a strong gene expression signature of megakaryocyte (MK) expansion. Gene expression-based cell type enrichment revealed elevated MK composition, specifically, CD41b+CD42b+ and CD41b+CD61+ MKs were expanded, further confirmed by flow cytometry in these ADs. In scRNA-seq data, MKs were defined by TFs PBX1/GATA1/TAL1 and pre-T-cell antigen receptor gene, PTCRA. Cellular heterogeneity and a distinct immune subpopulation with functional enrichment of antigen presentation were observed in MKs. CONCLUSIONS: The identification of MK expansion provided new insights into the peripheral immune cell atlas across RA, SLE, pSS and polyautoimmunity. Aberrant regulation of the MK expansion might contribute to the pathogenesis of these ADs.

Antineutrophil Cytoplasmic Antibody-Associated Vasculitis Accompanied by IgG4-Tubulointerstitial Nephritis with Underlying Sjögren Syndrome: A Case Report and Review of Literature.

OBJECTIVE: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune multisystemic diseases characterized by necrotizing inflammation of small vessels and the presence of circulating ANCA. The prevalence of overlap AAV with other autoimmune diseases was low. CASE REPORT: We report a case of a 54-year-old woman who presented with a 20-year-history of sicca symptoms, the presence of anti-Ro/SS-A, anti-La/SS-B antibodies, myeloperoxidase -ANCA (MPO-ANCA), significant increase of serum IgG4 level, microscopic hematuria, non-nephrotic proteinuria, and progressive renal dysfunction. A renal biopsy showed pauci-immune necrotizing glomerulonephritis with crescents with severe tubulointerstitial nephritis (TIN) which shows extensive infiltration of IgG4-positive plasma cells. Considering these findings and the clinical course, the disease was considered more likely to be MPO-ANCA-associated vasculitis accompanied by IgG4-TIN with underlying primary Sjögren syndrome (pSS). CONCLUSION: This report shows a possible unusual disease overlap of MPO-ANCA-associated vasculitis and IgG4-TIN with underlying pSS.

Transcriptomic and Single-Cell Analysis Reveals Regulatory Networks and Cellular Heterogeneity in Mouse Primary Sjögren's Syndrome Salivary Glands.

Sjögren's Syndrome (SS) is a chronic autoimmune disease of unknown etiology which primarily affects the salivary and lacrimal glands resulting in the loss of secretory function. Treatment options for SS have been hampered due to the lack of a better understanding of the underlying gene regulatory circuitry and the interplay between the myriad pathological cellular states that contribute to salivary gland dysfunction. To better elucidate the molecular nature of SS, we have performed RNA-sequencing analysis of the submandibular glands (SMG) of a well-established primary Sjögren's Syndrome (pSS) mouse model. Our comprehensive examination of global gene expression and comparative analyses with additional SS mouse models and human datasets, have identified a number of important pathways and regulatory networks that are relevant in SS pathobiology. To complement these studies, we have performed single-cell RNA sequencing to examine and identify the molecular and cellular heterogeneity of the diseased cell populations of the mouse SMG. Interrogation of the single-cell transcriptomes has shed light on the diversity of immune cells that are dysregulated in SS and importantly, revealed an activated state of the salivary gland epithelial cells that contribute to the global immune mediated responses. Overall, our broad studies have not only revealed key pathways, mediators and new biomarkers, but have also uncovered the complex nature of the cellular populations in the SMG that are likely to drive the progression of SS. These newly discovered insights into the underlying molecular mechanisms and cellular states of SS will better inform targeted therapeutic discoveries.

Therapeutic Potential of Anti-Interferon α Vaccination on SjS-Related Features in the MRL/lpr Autoimmune Mouse Model.

Sjögren's syndrome (SjS) is a frequent systemic autoimmune disease responsible for a major decrease in patients' quality of life, potentially leading to life-threatening conditions while facing an unmet therapeutic need. Hence, we assessed the immunogenicity, efficacy, and tolerance of IFN-Kinoid (IFN-K), an anti-IFNα vaccination strategy, in a well-known mouse model of systemic autoimmunity with SjS-like features: MRL/MpJ-Faslpr/lpr (MRL/lpr) mice. Two cohorts (with ISA51 or SWE01 as adjuvants) of 26 female MRL/lpr were divided in parallel groups, "controls" (not treated, PBS and Keyhole Limpet Hemocyanin [KLH] groups) or "IFN-K" and followed up for 122 days. Eight-week-old mice received intra-muscular injections (days 0, 7, 28, 56 and 84) of PBS, KLH or IFN-K, emulsified in the appropriate adjuvant, and blood samples were serially collected. At sacrifice, surviving mice were euthanized and their organs were harvested for histopathological analysis (focus score in salivary/lacrimal glands) and IFN signature evaluation. SjS-like features were monitored. IFN-K induced a disease-modifying polyclonal anti-IFNα antibody response in all treated mice with high IFNα neutralization capacities, type 1 IFN signature's reduction and disease features' (ocular and oral sicca syndrome, neuropathy, focus score, glandular production of BAFF) improvement, as reflected by the decrease in Murine Sjögren's Syndrome Disease Activity Index (MuSSDAI) modelled on EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). No adverse effects were observed. We herein report on the strong efficacy of an innovative anti-IFNα vaccination strategy in a mouse model of SjS, paving the way for further clinical development (a phase IIb trial has just been completed in systemic lupus erythematosus with promising results).