B cell-activating factor (BAFF) has a key role in promoting B-lymphocyte activation and survival in primary Sjögren's syndrome (pSS). The cellular origin of BAFF overexpression in salivary glands of patients with pSS is not fully known. We investigated whether salivary gland epithelial cells (SGECs), the main targets of autoimmunity in pSS, could produce and express BAFF. We used quantitative RT-PCR, ELISA and immunocytochemistry in cultured SGECs from eight patients with pSS and eight controls on treatment with IL-10, tumor necrosis factor α (TNF-α), IFN-α and IFN-γ. At baseline, BAFF expression in SGECs was low in pSS patients and in controls. Treatment with IFN-α, IFN-γ and TNF-α + IFN-γ increased the level of BAFF mRNA in pSS patients (the mean increases were 27-fold, 25-fold and 62-fold, respectively) and in controls (mean increases 19.1-fold, 26.7-fold and 17.7-fold, respectively), with no significant difference between patients and controls. However, in comparison with that at baseline, stimulation with IFN-α significantly increased the level of BAFF mRNA in SGECs of pSS patients (p = 0.03) but not in controls (p = 0.2), which suggests that SGECs of patients with pSS are particularly susceptible to expressing BAFF under IFN-α stimulation. Secretion of BAFF protein, undetectable at baseline, was significantly increased after IFN-α and IFN-γ stimulation both in pSS patients (40.8 ± 12.5 (± SEM) and 47.4 ± 18.7 pg/ml, respectively) and controls (24.9 ± 8.0 and 9.0 ± 3.9 pg/ml, respectively), with no significant difference between pSS and controls. Immunocytochemistry confirmed the induction of cytoplasmic BAFF expression after stimulation with IFN-α and IFN-γ. This study confirms the importance of resident cells of target organs in inducing or perpetuating autoimmunity. Demonstrating the capacity of SGECs to express and secrete BAFF after IFN stimulation adds further information to the pivotal role of these epithelial cells in the pathogenesis of pSS, possibly after stimulation by innate immunity. Our results suggest that an anti-BAFF therapeutic approach could be particularly interesting in pSS.
Noninvasive objective tests are needed to diagnose primary Sjogren’s syndrome (pSS) and to evaluate treatment responses. Ultrasound imaging of the salivary glands is rapid and noninvasive. Recent open-label studies suggested that anti-CD20 (rituximab) may be effective in pSS. The purpose of this study was to look for ultrasound evidence of the effects of rituximab in pSS.
We compared 16 patients fulfilling the new American-European consensus group criteria for pSS to 9 controls, using B-mode ultrasound features (parenchymal homogeneity and gland size) and Doppler waveform analysis of the transverse facial artery of parotid glands. We compared the same parameters in the patients before and after 12 weeks of intravenous rituximab therapy.
Compared to controls, untreated patients had significant abnormalities in salivary gland structure (p < 0.0001) and parotid size (2.05 ± 0.33 cm versus 1.70 ± 0.28 cm; p = 0.001). Doppler waveform analysis showed significant differences before, but not after, lemon stimulation between untreated patients and controls. After rituximab treatment, significant size reductions were noted in the parotids (2.05 ± 0.3 cm at baseline and 1.86 ± 0.27 cm at week 12; p = 0.002) and submandibular glands (2.02 ± 0.54 cm at baseline and 1.66 ± 0.34 cm at week 12; p = 0.001). Doppler resistive indices after lemon stimulation were significantly increased after rituximab treatment.
Salivary gland measurements and blood inflow responses to salivary stimulation as assessed by ultrasound hold promise as objective noninvasive tools for evaluating rituximab effects in patients with pSS.
ultrasonography; primary Sjögren’s syndrome; rituximab
The main purpose of this study was to determine the expression of interleukins-17/-23 (ILs-17/-23) and receptors of interleukins-17/-23 (IL-17R, IL-23R) in minor salivary glands (MSGs) of patients with primary Sjögren's syndrome (pSS). Expression of IL-17, IL-23 and receptors of IL-17/-23 was analyzed in MSGs from 25 patients with pSS, 25 patients with probable preclinical pSS, and 25 patients with nonautoimmune sicca syndrome by immunohistochemistry. Comparison of the expression of IL-17, IL-23 and receptors of IL-17, IL-23 in MSG of patients with pSS with probable preclinical pSS, and with nonautoimmune sicca syndrome showed significant differences between three groups. However, the expression of IL-17, IL-23 and receptors of IL-17/-23 in MSG was comparable in pSS and probable preclinical pSS patients. We did not find correlation between the expression of IL-17 and IL-23 and of IL-17R and IL-23R in patients with pSS. These results demonstrate an involvement of IL-17/-23 system in the early pSS pathogenesis.
Id3−/− mice represent a model for T cell mediated primary Sjogren's syndrome (PSS). An intriguing feature of this disease model is the early appearance of impaired salivary function or exocrinopathy prior to lymphocytic infiltration of the salivary glands. This phenomenon prompted us to examine the role of cytokines produced by T cells in the systemic regulation of gland function. A comprehensive examination of serum cytokine profiles revealed elevated levels of IL-13 in Id3−/− mice. We found that the increase in serum IL-13 levels in Id3−/− mice was largely dependent on αβ T cells. Removal of αβ T cells in Id3−/− mice also eliminates disease symptoms, including lymphocytic infiltration in the gland tissues, and impaired saliva production. We further show that the number of mast cells in the salivary glands of Id3−/− mice is significantly increased, in a trend inversely related to the saliva production. This increase in the number of mast cells is also dependent on the presence of αβ T cells. Treatment of young Id3−/− mice with anti-IL-13 antibodies over a two-month period resulted in a reduction of both serum IL-13 levels and the number of mast cells in the salivary gland tissues, as well as correspondingly improved saliva production. These findings indicate a potentially important role for IL-13 in gland regulation and disease pathology.
Id3 deficient mice; IL-13; Exocrinopathy; Mast cells
The purpose of our study was to understand if Toll-like receptor 9 (TLR9) activation could contribute to the control of inflammation in Sjogren's syndrome. To this end, we manipulated TLR9 signaling in non-obese diabetic (NOD) and TLR9−/− mice using agonistic CpG oligonucleotide aptamers, TLR9 inhibitors, and the in-house oligonucleotide BL-7040. We then measured salivation, inflammatory response markers, and expression of proteins downstream to NF-κB activation pathways. Finally, we labeled proteins of interest in salivary gland biopsies from Sjogren's syndrome patients, compared to Sicca syndrome controls. We show that in NOD mice BL-7040 activates TLR9 to induce an alternative NF-κB activation mode resulting in increased salivation, elevated anti-inflammatory response in salivary glands, and reduced peripheral AChE activity. These effects were more prominent and also suppressible by TLR9 inhibitors in NOD mice, but TLR9−/− mice were resistant to the salivation-promoting effects of CpG oligonucleotides and BL-7040. Last, salivary glands from Sjogren's disease patients showed increased inflammatory and decreased anti-inflammatory biomarkers, in addition to decreased levels of alternative NF-κB pathway proteins. In summary, we have demonstrated that activation of TLR9 by BL-7040 leads to non-canonical activation of NF-κB, promoting salivary functioning and down-regulating inflammation. We propose that BL-7040 could be beneficial in treating Sjogren's syndrome and may be applicable to additional autoimmune syndromes.
OBJECTIVE—To localise and characterise follicular dendritic cells (FDC) present in autoimmune lesions of primary Sjogren syndrome.
METHODS—Cryostat sections of labial salivary glands from 15 patients with primary Sjogren syndrome were examined by an indirect immunoperoxidase technique and monoclonal antibodies to a panel of dendritic cell markers. Tonsils from two controls were also examined for the same markers.
RESULTS—FDC were localised in the centre of 75% of lymphoid focal structures in labial salivary gland biopsies. FDC in labial salivary glands of patients with primary Sjogren syndrome expressed CD35, CD11c, and CD106 (VCAM-1) in a pattern similar to FDC in tonsils, but they did not express either CD14 or CD11b. This indicates that they may not be of myeloid origin, while FDC in tonsillar lymphoid follicles strongly expressed both CD14 and CD11b. FDC in labial salivary glands of patients also lacked VLA-2α and VLA-3α, which were expressed by FDC in tonsils.
CONCLUSIONS—The characteristic phenotype and origin of these cells may be of importance in the immune responses involved in Sjogren syndrome and the retention of infiltrating lymphocytes in the glands.
Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a type I transmembrane protein belonging to the TNFR superfamily. After activated by its ligand GITRL, GITR could influence the activity of effector and regulatory T cells, participating in the development of several autoimmune and inflammatory diseases included rheumatoid arthritis and autoimmune thyroid disease. We previously reported that serum GITRL levels are increased in systemic lupus erythematosus (SLE) patients compared with healthy controls (HC). Here, we tested serum soluble GITR (sGITR) and GITRL levels in 41 primary Sjögren's syndrome (pSS) patients and 29 HC by ELISA and correlated sGITR and GITRL levels with clinical and laboratory variables. GITR and GITRL expression in labial salivary glands was detected by immunohistochemistry. pSS patients had significantly increased serum levels of sGITR and GITRL compared with controls (GITR: 5.66 ± 3.56 ng/mL versus 0.50 ± 0.31 ng/mL; P < 0.0001; GITRL: 6.17 ± 7.10 ng/mL versus 0.36 ± 0.28 ng/mL; P < 0.0001). Serum sGITR and GITRL levels were positively correlated with IgG (GITRL: r = 0.6084, P < 0.0001; sGITR: r = 0.6820, P < 0.0001) and ESR (GITRL: r = 0.8315, P < 0.0001; sGITR: r = 0.7448, P < 0.0001). Moreover, GITR and GITRL are readily detected in the lymphocytic foci and periductal areas of the LSGs. In contrast, the LSGs of HC subjects did not express GITR or GITRL. Our findings indicate the possible involvement of GITR-GITRL pathway in the pathogenesis of pSS. Further studies may facilitate the development of targeting this molecule pathway for the treatment of pSS.
The presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in the classification criteria for primary Sjögren's syndrome (pSS). Plasma cells producing these autoantibodies are mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma cells are the long-lived plasma cell subset.
Single, double and triple immunohistochemistry as well as immunofluorescence staining was performed on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects.
We detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6 survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells. Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that the environment of salivary glands with high FS provide factors vital for plasma cell survival.
Plasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment provided niches rich in factors vital for plasma cell survival.
The objective of this study was to analyse levels of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with primary Sjögren's syndrome (pSS) and to examine associations of MIF with clinical, serological and immunological variables. MIF was determined by ELISA in the sera of 76 patients with pSS. Further relevant cytokines (IL-1, IL-6, IL-10, IFN-γ and TNF-α) secreted by peripheral blood mononuclear cells (PBMC) were determined by ELISPOT assay. Lymphocytes and monocytes were examined flow-cytometrically for the expression of activation markers. Results were correlated with clinical and laboratory findings as well as with the HLA-DR genotype. Healthy age- and sex-matched volunteers served as controls. We found that MIF was increased in patients with pSS compared with healthy controls (p < 0.01). In particular, increased levels of MIF were associated with hypergammaglobulinemia. Further, we found a negative correlation of MIF levels with the number of IL-10-secreting PBMC in pSS patients (r = -0.389, p < 0.01). Our data indicate that MIF might participate in the pathogenesis of primary Sjögren's syndrome. MIF may contribute to B-cell hyperactivity indicated by hypergammaglobulinemia. The inverse relationship of IL-10 and MIF suggests that IL-10 works as an antagonist of MIF in pSS.
Immunohistological studies on salivary and lacrimal glands have yielded conflicting results on the Th1/Th2 balance in primary Sjögren's syndrome (pSS).
To establish whether pSS is a Th1 or Th2 directed autoimmune disease by analysing the polymorphism of the genes encoding for cytokines involved in the regulation of Th1/Th2 differentiation.
The polymorphisms of the genes encoding for interleukin 4 (IL4) −590 C/T, interleukin 13 (IL13) +2044 G/A, and interferon γ (IFNG) +874 T/A were analysed in 63 white Finnish patients with pSS (61 female, two male) and in 63 healthy controls. The clinical and immunological data on the pSS patients were analysed in relation to these cytokine gene polymorphisms.
There were no significant differences in the genotype or allele frequencies of IL4 −590, IL13 +2044, or IFNG +874 between pSS patients and controls. The erythrocyte sedimentation rate and concentrations of serum IgA and serum β2 microglobulin were lower in pSS patients carrying the IL4 −590 T allele or the IL13 +2044 A allele than in those not carrying the respective alleles. The IL4 −509 T allele and IL13 +2044 A allele carriers less often had purpura than the corresponding non‐carriers.
The frequencies of the cytokine genotypes regulating Th1/Th2 differentiation did not differ between pSS patients and controls. However, the presence of cytokine genotypes with increased susceptibility to atopic and other Th2 diseases was associated with signs of a milder form of pSS. This finding would favour a hypothesis envisaging pSS as primarily a Th1 mediated autoimmune disease.
gene polymorphism; interleukin 4; interleukin 13; primary Sjögren's syndrome; Th2 genotypes
Recent studies have demonstrated the expression of Toll-like receptor 3 (TLR3) in salivary glands and epithelial cell lines derived from Sjögren’s syndrome (SS) patients. Since viral infections are considered to be a trigger for SS, in this study we investigated whether in vivo engagement of TLR3 affects salivary gland function.
Female NZB/WF1 mice were repeatedly injected with polyinosinic:polycytidylic acid (poly(I:C)). TLR3 expression within submandibular glands was studied by immunohistochemistry. RNA levels of inflammatory cytokines in the submandibular glands were determined by real time PCR. Pilocarpine induced saliva volume was used as an index of glandular function.
Immunohistochemical analysis of submandibular glands showed TLR3 expression in epithelium of serous and mucous acini, granular convoluted tubules and ducts. Poly(I:C) treatment rapidly upregulated the mRNA levels of type I IFN and inflammatory cytokines in the submandibular glands. By one week after treatment, the saliva volumes in poly(I:C) treated mice were significantly reduced in comparison with the PBS treated mice. Hematoxylin and eosin staining showed that salivary gland histology was normal and lymphocytic foci were not detected. Glandular function recovered after poly(I:C) treatment was stopped.
Our results demonstrate that engagement of TLR3 within the salivary glands results in a rapid loss of glandular function. This phenomenon is associated with the production of type I IFN and inflammatory cytokines in the salivary glands. Restoration of glandular function suggests that for viral etiology of SS, a chronic infection of salivary glands might be necessary.
Inflammatory Cytokines; Poly(I:C); Salivary Gland; Sjögren’s Syndrome TLR3
Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathological features. A hallmark of pSS is B-cell hyperactivity as manifested by the production of autoantibodies, hypergammaglobulinemia, formation of ectopic lymphoid structures within the inflamed tissues, and enhanced risk of B-cell lymphoma. Changes in the distribution of peripheral B-cell subsets and differences in post-recombination processes of immunoglobulin variable region (IgV) gene usage are also characteristic features of pSS. Comparison of B cells from the peripheral blood and salivary glands of patients with pSS with regard to their expression of the chemokine receptors CXCR4 and CXCR5, and their migratory capacity towards the corresponding ligands, CXCL12 and CXCL13, provide a mechanism for the prominent accumulation of CXCR4+CXCR5+ memory B cells in the inflamed glands. Glandular B cells expressing distinct features of IgV light and heavy chain rearrangements, (re)circulating B cells with increased mutations of cμ transcripts in both CD27- and CD27+ memory B-cell subsets, and enhanced frequencies of individual peripheral B cells containing IgV heavy chain transcripts of multiple isotypes indicate disordered selection and incomplete differentiation processes of B cells in the inflamed tissues in pSS. This may possibly be related to a lack of appropriate censoring mechanisms or different B-cell activation pathways within the ectopic lymphoid structures of the inflamed tissues. These findings add to our understanding of the pathogenesis of this autoimmune inflammatory disorder and may result in new therapeutic approaches.
Altered Toll-like receptor (TLR) signaling has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The present study was undertaken to characterize responses of B cells from SLE patients to TLR7 and TLR9 stimulation and to explore the potential role of single immunoglobulin interleukin-1 receptor related molecule (SIGIRR) in the regulation of TLR-mediated responses of SLE B cells.
Peripheral blood mononuclear cells (PBMC) were isolated from 64 patients with SLE and 37 healthy donors. CD19+ B cells purified using microbeads were cultured with TLR7 or TLR9 agonists. Cell proliferation was measured by thymine incorporation and the frequency of antibody-secreting cells was determined by ELISPOT assay. SIGIRR expression in PBMCs and B cells was analyzed using flow cytometry analysis. In contrast to the enhanced proliferation following B cell receptor (BCR) engagement, B cells from SLE patients exhibited a virtually normal proliferative response to TLR7 or TLR9 stimulation. Moreover, B cells from SLE patients and healthy donors were almost equally competent to differentiate into antibody-secreting cells upon TLR engagement except for a reduction in the generation of IgG-secreting cells by TLR9-stimulated lupus B cells. In line with these somehow unexpected observations, SLE B cells were found to express a significantly higher level of SIGIRR than normal B cells.
Despite the reported upregulation of TLR7 and TLR9 expression in B cell from SLE patients, their responses to TLR stimulation were largely normal. The increased expression of the negative regulator SIGIRR may be partly responsible for the “balance of terror”.
In healing wounds, some monocytes enter the wound and differentiate into fibroblast-like cells called fibrocytes. Since Toll-like receptors (TLRs) are present on monocytes, and pathogens that can infect a wound have and/or release TLR agonists, we examined whether TLR agonists affect fibrocyte differentiation.
When human peripheral blood mononuclear cells (PBMCs) were cultured with TLR3, TLR4, TLR5, TLR7, TLR8 or TLR9 agonists, there was no significant effect on fibrocyte differentiation, even though enhanced extracellular tumor necrosis factor (TNF)-α accumulation and/or increased cell surface CD86 or major histocompatibility complex (MHC) class II levels were observed. However, all TLR2 agonists tested inhibited fibrocyte differentiation without any significant effect on cell survival. Adding TLR2 agonists to purified monocytes had no effect on fibrocyte differentiation. However, some TLR2 agonists caused PBMCs to secrete a factor that inhibits the differentiation of purified monocytes into fibrocytes. This factor is not interferon (IFN)-α, IFN-γ, interleukin (IL)-12, aggregated immunoglobulin G (IgG) or serum amyloid P (SAP), factors known to inhibit fibrocyte differentiation. TLR2 agonist-treated PBMCs secrete low levels of IL-6, TNF-α, IFN-γ, granulocyte colony-stimulating factor and tumor growth factor β1, but combinations of these factors had no effect on fibrocyte differentiation from purified monocytes.
Our results indicate that TLR2 agonists indirectly inhibit fibrocyte differentiation and that, for some TLR2 agonists, this inhibition involves other cell types in the PBMC population secreting an unknown factor that inhibits fibrocyte differentiation. Together, these data suggest that the presence of some bacterial signals can inhibit fibrocyte differentiation and may thus slow wound closure.
Several studies showed signs of autonomic dysfunction in patients with primary Sjögren's syndrome (pSS). Adrenomedullary function might be of importance for pSS pathogenesis by affecting salivary gland functions and modulating immune responses. The aim of the study was to evaluate the adrenomedullary hormonal system in patients with pSS.
The glucagon test (1 mg i.v.) was performed in 18 pSS patients and 13 control subjects. During the testing each patient had electrocardiographic and impedance cardiographic monitoring. Plasma epinephrine and norepinephrine were assayed by liquid chromatography with electrochemical detection after batch alumina extraction.
Baseline concentrations of epinephrine and norepinephrine were comparable between pSS and controls. Glucagon administration induced a significant increase in systolic blood pressure, diastolic blood pressure, heart rate, cardiac output (p < 0.01), stroke volume; however the changes were comparable between pSS and controls. Epinephrine levels increased (p < 0.01) in response to glucagon administration while norepinephrine concentration did not change. There was no significant difference in neurochemical responses to glucagon between pSS and controls. In conclusion, the present results suggest normal adrenomedullary function in pSS.
Primary Sjögren’s syndrome; epinephrine; adrenal medulla; norepinephrine
the classification criteria for primary Sjögren's syndrome (pSS) include a number of oral components. In this study we evaluated if salivary flow and composition as well as dental caries are oral markers of disease severity in pSS.
in 20 patients fulfilling the American-European Consensus criteria for pSS and 20 age-matched healthy controls whole and parotid saliva flow rates and composition, measures of oral dryness, scores of decayed, missing and filled tooth surfaces (DMFS), periodontal indices, oral hygiene, and dietary habits were examined.
in pSS, salivary flow rates, pH, and buffer capacities were lower, and DMFS, salivary sodium and chloride concentrations higher than in the healthy controls. DMFS also correlated inversely to salivary flow rates and positively to oral dryness. Apart from slightly increased gingival index, and more frequent dental visits in pSS, the periodontal condition, oral hygiene or sugar intake did not differ between these two groups. In pSS, findings were correlated to labial salivary gland focus score (FS) and presence of serum-autoantibodies to SSA/SSB (AB). The patients having both presence of AB and the highest FS (>2) also had the highest salivary sodium and chloride concentrations, the lowest salivary phosphate concentrations, lowest salivary flow rates, and highest DMFS compared to those with normal salivary concentrations of sodium and chloride at a given flow rate.
the salivary changes observed in some pSS patients reflect impaired ductal salt reabsorption, but unaffected acinar transport mechanisms, despite low salivary secretion. Our results suggest that changes in salivary flow and composition as well as dental caries may serve as potential markers of the extent of autoimmune-mediated salivary gland dysfunction in pSS. The study also indicates that the ductal epithelium is functionally affected in some pSS patients, which calls for future pathophysiological studies on the mechanisms underlying this impaired salt reabsorption.
To analyse B cell activating factor (BAFF) receptor (BAFF‐R) expression on peripheral lymphocytes from patients with primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE).
Patients and methods
Peripheral blood mononuclear cells from 20 patients with pSS, 19 patients with SLE and 15 controls were examined by flow cytometry to investigate BAFF‐R mean fluorescence intensity (MFI) on lymphocytes. BAFF‐R mRNA level from isolated blood B cells of nine patients with pSS and eight controls was assessed by real‐time quantitative reverse transcription‐PCR. BAFF serum level was determined by ELISA.
In all subjects, BAFF‐R was expressed on all naïve CD27− and memory CD27+ B‐cells and was present on <0.5% of T cells. The expression of BAFF‐R on B cells was significantly decreased in patients with pSS as compared with controls (MFI = 7.8 vs 10.6, p = 0.001), and was intermediate in patients with SLE (MFI = 9.5). Serum BAFF level was inversely correlated with BAFF‐R MFI (p = 0.007), but not because of competition between endogenous BAFF (at observed concentrations in patients) and the monoclonal antibody (11C1) detecting BAFF‐R. BAFF‐R mRNA levels did not differ between patients with pSS and controls (p = 0.48). BAFF‐R MFI decreased after overnight culture with recombinant human BAFF (from 32.5 to 25.4, p = 0.03). Contrary to the serum BAFF level, BAFF‐R expression was correlated with extraglandular involvement in pSS and SLE Disease Activity Index.
BAFF‐R expression is reduced on peripheral B cells of patients with pSS and SLE. This down‐regulation occurs through a post‐transcriptional mechanism and could be the consequence of chronic increase in BAFF. BAFF‐R levels on B cells could be a novel activity biomarker in autoimmune diseases.
Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease with complex etiopathogenesis. Despite extensive studies to understand the disease process utilizing human and mouse models, the intersection between these species remains elusive. To address this gap, we utilized a novel systems biology approach to identify disease-related gene modules and signaling pathways that overlap between humans and mice.
Parotid gland tissues were harvested from 24 pSS and 16 non-pSS sicca patients and 25 controls. For mouse studies, salivary glands were harvested from C57BL/6.NOD-Aec1Aec2 mice at various times during development of pSS-like disease. RNA was analyzed with Affymetrix HG U133+2.0 arrays for human samples and with MOE430+2.0 arrays for mouse samples. The images were processed with Affymetrix software. Weighted-gene co-expression network analysis was used to identify disease-related and functional pathways.
Nineteen co-expression modules were identified in human parotid tissue, of which four were significantly upregulated and three were downregulated in pSS patients compared with non-pSS sicca patients and controls. Notably, one of the human disease-related modules was highly preserved in the mouse model, and was enriched with genes involved in immune and inflammatory responses. Further comparison between these two species led to the identification of genes associated with leukocyte recruitment and germinal center formation.
Our systems biology analysis of genome-wide expression data from salivary gland tissue of pSS patients and from a pSS mouse model identified common dysregulated biological pathways and molecular targets underlying critical molecular alterations in pSS pathogenesis.
Sjogren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into lacrimal and salivary glands leading to symptomatic dry eyes and mouth. Immunohistological studies have clarified that the majority of infiltrating lymphocytes around the lacrimal glands and labial salivary glands are CD4 positive alphabeta T cells. To analyze the pathogenesis of T cells infiltrating into lacrimal and labial salivary glands, we examined T cell clonotype of these cells in both glands from four SS patients using PCR-single-strand conformation polymorphism (SSCP) and a sequencing method. SSCP analysis showed that some infiltrating T cells in both glands expand clonally, suggesting that the cells proliferate by antigen-driven stimulation. Intriguingly, six to sixteen identical T cell receptor (TCR) Vbeta genes were commonly found in lacrimal glands and labial salivary glands from individual patients. This indicates that some T cells infiltrating into both glands recognize the shared epitopes on autoantigens. Moreover, highly conserved amino acid sequence motifs were found in the TCR CDR3 region bearing the same TCR Vbeta family gene from four SS patients, supporting the notion that the shared epitopes on antigens are limited. In conclusion, these findings suggest that some autoreactive T cells infiltrating into the lips and eyes recognized restricted epitopes of a common autoantigen in patients with SS.
Previously, a dominant role of the adaptive immune system in the pathogenesis of Sjögren's syndrome was suspected. Recent advances, however, have revealed a major role of the type I IFN pathway, documented by an increased circulating type I IFN activity and an IFN 'signature' in peripheral blood mononuclear cells and minor salivary gland biopsies from the patients. Polymorphisms in the genes IRF5 and STAT4 leading to increased IFN activation are associated with disease susceptibility. In the pathogenesis of Sjögren's syndrome, the activation of salivary gland epithelial cells appears to be the initial event. Once intrinsically activated, they express costimulatory and Toll-like receptors (TLRs) and MHC class I and II molecules, can present autoantigens and produce proinflammatory cytokines. The subsequent activation of plasmacytoid dendritic cells induces the production of high levels of proinflammatory cytokines in individuals with the risk alleles of the susceptibility genes IRF5 and STAT4. Under the influence of the high IFN concentration in the glands and through TLR ligation, B-cell activating factor is produced by epithelial cells and, together with autoantigen presentation on salivary gland epithelial cells, stimulates the adaptive immune system. In view of the central role of IFNalpha in at least the initiation of the pathogenesis of Sjögren's syndrome, blockade of this cytokine may be a rational therapeutic approach.
To clarify the clinicopathological characteristics of primary Sjögren's syndrome (pSS) with anti-centromere antibody (ACA).
Characteristics of 14 patients of pSS with ACA were evaluated. All patients were anti-SS-A/Ro and SS-B/La antibodies negative (ACA+ group) without sclerodactyly. The prevalence of Raynaud's phenomenon (RP), titer of IgG and focus score (FS) in the minor salivary glands (MSGs) were determined. Quantification analysis of Azan Mallory staining was performed to detect collagenous fiber. Forty eight patients in whom ACA was absent were chosen as the conventional (ACA-) pSS group.
Prevalence of ACA+ SS patients was 14 out of 129 (10.85%) pSS patients. RP was observed in 61.5% of the patients with ACA. The level of IgG in the ACA+ group was significantly lower than that of the ACA- group (p = 0.018). Statistical difference was also found in the FS of MSGs from the ACA+ group (1.4 ± 1.0) as compared with the ACA- group (2.3 ± 1.6) (p = 0.035). In contrast, the amount of fibrous tissue was much higher in the ACA+ group (65052.2 ± 14520.6 μm2 versus 26251.3 ± 14249.8 μm2 ) (p = 1.3 × 10-12).
Low cellular infiltration but with an increase in fibrous tissues may explain the clinical feature of a high prevalence of RP and normal IgG concentration in ACA+ pSS.
To identify key target genes and activated signal pathways associated with the disease pathogenesis by conducting a systems analysis of parotid gland manifesting primary Sjögren’s syndrome (pSS) and pSS/mucosa-associated lymphoid tissue (pSS/MALT) lymphoma phenotypes.
A systems biologic approach was used to analyze parotid gland tissues obtained from non-pSS, pSS and pSS/MALT lymphoma patients. Concurrent expression microarray profiling and proteomic analysis were performed followed by weighted gene co-expression network analysis (WGCNA).
Gene co-expression modules related to pSS and pSS/MALT lymphoma are significantly enriched with genes known to be involved in immune/defense response, apoptosis, cell signaling, gene regulation, and oxidative stress. A detailed functional pathway analysis indicates that the pSS-associated modules are enriched with genes involved in proteasome degradation, apoptosis, signal peptides (MHC) class I, complement activation, cell growth and death, and integrin-mediated cell adhesion. The pSS/MALT-associated modules are enriched with genes involved in translation, ribosome, protease degradation, signal peptides (MHC) class I, G13 signaling pathway, complement activation, and Integrin-mediated cell adhesion. The combined analysis of gene expression and proteomics data implicates six highly connected hub genes for distinguishing pSS from non-pSS controls, and eight hub genes for distinguishing pSS/MALT lymphoma from pSS.
Systems biologic analysis of pSS and pSS/MALT parotid glands reveals pathways and molecular targets associated with the disease pathogenesis. The identified gene modules/pathways provide further insights into the molecular mechanisms of pSS and pSS/MALT lymphoma. The identified disease hub genes represent promising targets for therapeutic intervention, diagnosis, and prognosis.
The development of non-Hodgkin's lymphoma (NHL) confers a high risk of mortality in primary Sjögren's syndrome (pSS) patients, but the sensitivity and specificity of proposed lymphoma predictors are insufficient for practical use. The performance of lymphoid organisation in the form of germinal centre (GC)-like lesions was evaluated in labial salivary gland biopsies taken at pSS diagnosis as a potential lymphoma-predicting biomarker.
Labial salivary gland tissue biopsies available from two Swedish pSS research cohorts (n=175) were re-evaluated by light microscopy in a blind study in order to identify GC-like structures as a sign of ectopic lymphoid tissue formation and organisation. A linkage study was performed with the Swedish Cancer Registry for lymphoma identification. The risk of developing NHL in GC-positive patients in comparison with GC-negative patients was evaluated using Kaplan–Meier statistics and log-rank test. Associations between GC-like structures and clinical and/or laboratory disease markers were also determined using χ2 or Fisher's exact tests.
At diagnosis, 25% of pSS patients had GC-like structures in their salivary glands. Seven of the 175 patients studied (14% GC+ and 0.8% GC−) developed NHL during 1855 patient-years at risk, with a median onset of 7 years following the initial diagnostic salivary gland biopsy. Six of the seven patients had GC-like structures at diagnosis; the remaining patient was GC negative at the time of diagnosis (p=0.001).
The detection of GC-like structures by light microscopy in pSS diagnostic salivary biopsies is proposed as a highly predictive and easy-to-obtain marker for NHL development. This allows for risk stratification of patients and the possibility to initiate preventive B-cell-directed therapy.
Expression profile of the toll like receptors (TLRs) on PBMCs is central to the regulation of proinflammatory markers. An imbalance in the TLRs expression may lead to several types of inflammatory disorders. Furthermore, the dynamic regulation of inflammatory activity and associated impaired production of cytokines by peripheral blood mononuclear cells (PBMCs) in obese individulas remain poorly understood. Therefore, we determined the perturbation in TLRs (TLR2 and TLR4), their adaptor proteins (MyD88, IRAK1 and TRAF6) expression in PBMCs/subcutaneous adipose tissue (AT) as well as inflammatory cytokines changes in obese individuals.
mRNA expression levels of TLR2, TLR4, IL-6, TNF-α and adaptor proteins were determined by RT-PCR. TLR2, TLR4 and adaptor proteins expression in AT was determined by immunohistochemistry.
Obese and overweight individuals showed significantly increased expression of TLR2, TLR4 and MyD88 in both PBMCs and AT as compared with lean individuals (P < 0.05). Interestingly, we found a remarkably higher expression of TLRs in obese and overweight individuals with type 2 diabetes (P < 0.05). Increased expression of TLR2, TLR4, MyD88 and IRAK1 correlated with body mass index (BMI) (TLR2: r = 0.91; TLR4: r = 0.88, P <0.0001; MyD88: r = 0.95, P < 0.0001; IRAK1 r = 0.78, P < 0.002). TLRs’ expression was also correlated with fasting blood glucose (FBG) (TLR2: r = 0.61, P < 0.002; TLR4: r = 0.52, P < 0.01) and glycated haemoglobin (HbA1c) ( TLR2: r = 0.44, P <0.03; TLR4: r = 0.48, P < 0.03). Transcript levels of IL-6 and TNF-α were highly elevated in obese subjects compared to lean subjects. There was a strong association of TLRs’ expression in PBMCs with TNF-α (TLR2: r = 0.92; TLR4: r = 0.92; P < 0.0001) and IL-6 (TLR2: r = 0.91, P < 0.0001; TLR4: r = 0.81; P < 0.001). Similarly adaptor proteins were significantly correlated with TNF-α (MyD88: r = 0.9, P < 0.0001; IRAK1: r = 0.86; P < 0.0002) and IL-6 (MyD88: r = 0.91, P < 0.0001; IRAK1: 0.77; P < 0.002).
TLRs and adapter proteins were overexpressed in PBMCs from obese subjects, which correlated with increased expression of TNF-α and IL-6. This association may explain a potential pathophysiological link between obesity and inflammation leading to insulin resistance.
Sjögren's syndrome (SS) is a chronic, progressive autoimmune disease primarily affecting women. Diagnosis of SS requires an invasive salivary gland tissue biopsy and a long delay from the start of the symptoms to final diagnosis has been frequently observed. In this study, we aim to identify salivary autoantibody biomarkers for primary SS (pSS) using a protein microarray approach. Immune-response protoarrays were used to profile saliva autoantibodies from patients with pSS (n=14), patients with systemic lupus erythematosus (SLE, n=13), and healthy control subjects (n=13). We identified 24 potential autoantibody biomarkers that can discriminate patients with pSS from both patients with SLE and healthy individuals. Four saliva autoantibody biomarkers, anti-transglutaminase, anti-histone, anti-SSA, and anti-SSB, were further tested in independent pSS (n=34), SLE (n=34), and healthy control (n=34) subjects and all were successfully validated with ELISA. This study has demonstrated the potential of a high-throughput protein microarray approach for the discovery of autoantibody biomarkers. The identified saliva autoantibody biomarkers may lead to a clinical tool for simple, noninvasive detection of pSS at low cost.
Autoantibody biomarker; Protein arrays; Protein microarray; Sjögren's syndrome