Downstream activation of NF‐κB in the EDA‐A1/EDAR signalling in Sjögren's syndrome and its regulation by the ubiquitin‐editing enzyme A20

Sjögren's syndrome (SS) is an autoimmune disease and the second most common chronic systemic rheumatic disorder. Prevalence of primary SS in the general population has been estimated to be approximately 1–3%, whereas secondary SS has been observed in 10–20% of patients with rheumatoid arthritis, systemic lupus erythematosus (SLE) and scleroderma. Despite this, its exact aetiology and pathogenesis are largely unexplored. Nuclear factor‐kappa B (NF‐κB) signalling mechanisms provide central controls in SS, but how these pathways intersect the pathological features of this disease is unclear. The ubiquitin‐editing enzyme A20 (tumour necrosis factor‐α‐induced protein 3, TNFAIP3) serves as a critical inhibitor on NF‐κB signalling. In humans, polymorphisms in the A20 gene or a deregulated expression of A20 are often associated with several inflammatory disorders, including SS. Because A20 controls the ectodysplasin‐A1 (EDA‐A1)/ectodysplasin receptor (EDAR) signalling negatively, and the deletion of A20 results in excessive EDA1‐induced NF‐κB signalling, this work investigates the expression levels of EDA‐A1 and EDAR in SS human salivary glands epithelial cells (SGEC) and evaluates the hypothesis that SS SGEC‐specific deregulation of A20 results in excessive EDA1‐induced NF‐κB signalling in SS. Our approach, which combines the use of siRNA‐mediated gene silencing and quantitative pathway analysis, was used to elucidate the role of the A20 target gene in intracellular EDA‐A1/EDAR/NF‐κB pathway in SS SGEC, holding significant promise for compound selection in drug discovery.     

Viruses induce high expression of BAFF by salivary gland epithelial cells through TLR- and type-I IFN-dependent and -independent pathways

B cell activating factor (BAFF) plays a key role in promoting B lymphocyte activation. We investigated whether danger signals induce BAFF secretion by cultured salivary gland epithelial cells (SGEC), which are the target of primary Sjögren's syndrome, a prototypic systemic autoimmune disease. SGEC cultures were established from minor salivary glands obtained from ten patients with pSS or sicca symptoms. BAFF mRNA and protein were measured after stimulation of the different Toll‐like receptors (TLR) by agonists or viruses. The expression of TLR2, –3, and –7 was detected in SGEC. Poly (I:C) (a synthetic TLR3 agonist) and reovirus‐1 (a dsRNA virus) induced high expression of BAFF mRNA (multiplied by a factor of 246 ± 39 (SEM) and 347 ± 66, respectively) and of BAFF protein secretion (58.49 ± 4.34 pg/mL and 69.73 ± 5.67). Inhibition of both the endosomal (by chloroquine) and IFN (by anti‐IFNAR antibody) pathways partly inhibited BAFF expression. Treatment with both dsRNA virus and poly (I:C) induced high levels of BAFF mRNA and protein expression by SGEC, through pathways dependent on and independent of TLR and dependent on and independent of IFN. BAFF induction by target organs of autoimmune diseases after viral infection may be a link between innate immunity and autoimmunity.     

T follicular‐like helper cells in the peripheral blood of patients with primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease characterized by exocrine gland dysfunction, mainly causing sicca symptoms. B cells have a prominent role in SS, and the T follicular helper (T_FH) cells provide B cells with survival and specialization signals in germinal centres. Here, we investigate peripheral T_FH cells in pSS. Sixteen pSS patients and healthy controls were enrolled in the study, with 13 women and 3 men in each group. Whole blood was collected and separated into PBMC and plasma, followed by cryopreservation. Plasma samples were analysed for Ro52, Ro60 and La48 autoantibodies by indirect ELISA. For flow cytometric analysis, we defined 4 subsets of TFH‐like cells within the CD3⁺CD4⁺CXCR5⁺ population, namely the ICOS⁻PD‐1⁻, ICOS⁻PD‐1⁺, ICOS⁺PD‐1⁻ and ICOS⁺PD‐1⁺ (“TFH”) cells. We also investigated 4 CD19⁺ B cell subsets, the CD20⁺CD27⁺CD38⁻ memory B cells, CD20⁺CD27⁺CD38⁺ memory B cells, CD20⁻CD27⁺CD38⁺⁺CD138⁻ plasmablasts and CD20⁻CD27⁺CD38⁺⁺CD138⁺ plasma cells. We observed higher fractions of ICOS⁺PD‐1⁻ cells, ICOS⁺PD‐1⁺ (“T_FH”) cells and plasmablasts in pSS patients compared to controls, and lower frequencies of both types of memory B cells. The number of T_FH cells correlated positively with the levels of plasmablasts and plasma cells in the pSS patients, but not in the controls. The pSS patients were stratified according to Ro52/Ro60/La48 serology, and a positive association was found between autoantibody levels and increased level of T_FH cells, plasmablasts and plasma cells and lowered levels of memory B cells. We observed a higher response to Ro/La stimulation in pSS patients compared to controls of the memory B cells, although only significantly for the CD38⁻ memory B cells. Overall, a pathological relation between the ICOS⁺ T follicular‐like helper cells and B cells in pSS was observed, but further work should be conducted to explore their overall impact upon disease progression.     

Toll‐like receptor 3 stimulation promotes Ro52/TRIM21 synthesis and nuclear redistribution in salivary gland epithelial cells,partially via type I interferon pathway

Up‐regulated expression of Ro52/tripartite motif‐containing protein 21 (TRIM21), Ro60/TROVE domain family, member 2 (TROVE2) and lupus LA protein/Sjögren's syndrome antigen B (La/SSB) autoantigens has been described in the salivary gland epithelial cells (SGEC) of patients with Sjögren's syndrome (SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll‐like receptor (TLR)‐3, whereas Ro52/TRIM21 negatively regulates TLR‐3‐mediated inflammation. Herein, we investigated the effect of TLR‐3‐signalling on the expression of Ro52/TRIM21, as well as Ro60/TROVE2 and La/SSB autoantigens, by SGECs. The effect of TLR‐3 or TLR‐4 stimulation on autoantigen expression was evaluated by polyI:C or lipopolysaccharide (LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non‐SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. PolyI:C, but not LPS, resulted in a two‐step induction of Ro52/TRIM21 mRNA expression by SGECs, a 12‐fold increment at 6 h followed by a 2·5‐fold increment at 24–48 h, whereas it induced a late two‐fold up‐regulation of Ro60/TROVE2 and La/SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up‐regulation of Ro52/TRIM21 mRNA was accompanied by protein redistribution, from nucleolar‐like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon (IFN)‐β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor (IRF)3 degradation. TLR‐3‐signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these autoantigens in HeLa cells. TLR‐3 signalling regulates the expression of autoantigens by SGECs, implicating innate immunity pathways in their over‐expression in inflamed tissues and possibly in their exposure to the immune system.     

Rituximab‐mediated Raf kinase inhibitor protein induction modulates NF‐κB in Sjögren syndrome

Primary Sjögren syndrome (pSS) is an autoimmune disorder characterized by an epithelial injury surrounded by dense lymphocytic infiltrates. The conditions for the long‐term maintenance of human salivary gland epithelial cells from pSS patients and a co‐culture system with pSS lymphocytes were used to assess the effect of Rituximab (RTX) on the inflammatory condition and progression in pSS. Quantitative real‐time PCR, genes and protein array analysis, Western blot, flow cytometry, small interfering RNA transfection and nuclear factor‐κB (NF‐κB) DNA binding assays were used as methods. Supporting the benefits of RTX, this study demonstrates that RTX decreases NF‐κB activity and interrupts the NF‐κB signalling pathway through the up‐regulation of the Raf‐1 kinase inhibitor protein (RKIP). Over‐expression of RKIP down‐regulates interleukins, their receptors and the expression of genes encodes proteins that attracted lymphocytes. Silencing of the RKIP gene leads to significantly increased expression and release of pro‐inflammatory mediators supporting that RKIP expression could be involved in the suppression of NF‐κB activation in pSS salivary gland epithelial cells.     

Involvement of REG Iα protein in the regeneration of ductal epithelial cells in the minor salivary glands of patients with Sjögren's syndrome

The regenerating gene (Reg) was originally isolated from regenerating rat pancreatic islets and revealed recently to constitute a multi‐gene family in humans. REG Iα protein is known to be overexpressed not only in various human inflammatory diseases but also in various experimental models of inflammation in animal tissues. However, its involvement in pathophysiology of the minor salivary gland (MSG) is not clear. We investigated REG Iα expression in the MSG of patients with primary Sjögren's syndrome (SS) and assessed its role in ductal epithelial cell proliferation in such tissues. Lip biopsy specimens were obtained from 40 patients with primary SS and examined using immunohistochemistry for REG Iα protein, Ki67 and single‐strand DNA (ssDNA). The relationships among clinicopathological factors and expression of REG Iα protein, Ki67 and ssDNA in the MSG were then analysed. REG Iα protein was expressed rarely in ductal epithelial cells of the normal MSG but was apparently overexpressed in those of patients with SS. The labelling indices for both Ki67 and ssDNA in the ductal cells of the MSGs were significantly higher in SS patients than in controls. Moreover, these labelling indices were significantly higher in REG Iα‐positive than in negative SS patients. REG Iα protein may play a role in the regeneration of ductal epithelial cells in the MSGs of patients with SS.     

The rheumatic disease‐associated FAM167A‐BLK locus encodes DIORA‐1, a novel disordered protein expressed highly in bronchial epithelium and alveolar macrophages

Triggering of autoimmunity that leads to rheumatic disease has been suggested to depend upon gene–environment interactions occurring in epithelial barriers and associated immune cells. Genetic studies have identified associations of the FAM167A‐BLK locus with rheumatoid arthritis, systemic lupus erythematosus (SLE) and Sjögren's syndrome. While BLK (B lymphocyte kinase) has a well‐established role in B cells, family with sequence similarity to 167 member A (FAM167A) and its gene family remain uncharacterized. To begin to understand the role of FAM167A in rheumatic disease pathogenesis, we explored this gene family and cloned and investigated the gene products. Expression of quantitative trait locus analysis was performed in immune cells. FAM167A and FAM167B were cloned from human peripheral blood mononuclear cells (PBMC). Gene conservation and protein properties were analysed by online tools, mRNA expression measured in mouse organs by quantitative polymerase chain reaction (qPCR) and protein expression investigated in human tissues by immunohistochemistry. We found that autoimmune risk genotypes within the FAM167A‐BLK locus lead to increased expression of FAM167A. The FAM167 gene family includes two members, FAM167A and FAM167B, which are not homologous to any other annotated gene but are evolutionarily conserved. The encoded proteins, which we denote ‘disordered autoimmunity’ (DIORA)‐1 and DIORA‐2, respectively, are characterized by a high content of intrinsic disorder. Notably, DIORA‐1 has its highest expression in the lung, detectable in both bronchial epithelium and alveolar macrophages with an endosomal localization pattern. In summary, the FAM167A gene is associated with several rheumatic diseases and encodes a novel disordered protein, DIORA‐1, which is expressed highly in the lung, consistent with a potential role in disease pathogenesis.     

The development of autoimmune features in aging mice is closely associated with alterations of the peripheral CD4+ T‐cell compartment

Some signs of potential autoimmunity, such as the appearance of antinuclear antibodies (ANAs) become prevalent with age. In most cases, elderly people with ANAs remain healthy. Here, we investigated whether the same holds true for inbred strains of mice. Indeed, we show that most mice of the C57BL/6 (B6) strain spontaneously produced IgG ANA at 8–12 months of age, showed IgM deposition in kidneys and lymphocyte infiltrates in submandibular salivary glands. Despite all of this, the mice remained healthy. ANA production is likely CD4⁺ T‐cell dependent, since old (40–50 weeks of age) B6 mice deficient for MHC class II do not produce IgG ANAs. BM chimeras showed that ANA production was not determined by age‐related changes in radiosensitive, hematopoietic progenitor cells, and that the CD4⁺ T cells that promote ANA production were radioresistant. Thymectomy of B6 mice at 5 weeks of age led to premature alterations in T‐cell homeostasis and ANA production, by 15 weeks of age, similar to that in old mice. Our findings suggest that a disturbed T‐cell homeostasis may drive the onset of some autoimmune features.     

A comprehensive investigation on the distribution of circulating follicular T helper cells and B cell subsets in primary Sjögren's syndrome and systemic lupus erythematosus

Follicular T helper (Tfh) cells have a crucial role in regulating immune responses within secondary lymphoid follicles by directing B cell differentiation towards memory B cells and plasma cells. Because abnormal humoral responses are key features in both primary Sjögren's syndrome (pSS) and systemic lupus erythematosus (SLE), the aim of this study was to profile the pathological connection between peripheral Tfh cells and B cells in the two diseases. Twenty‐five pSS patients, 25 SLE patients and 21 healthy controls were enrolled into the study. We determined the ratio of circulating Tfh‐like cells, their interleukin (IL)‐21 production and different B cell subsets by flow cytometry. We observed higher percentages of naive B cells in both diseases, while non‐switched and switched memory B cells showed decreased frequencies. The proportions of double‐negative B cells and plasmablasts were elevated in SLE and decreased in pSS. The percentages of transitional B cells and mature‐naive B cells were higher in SLE. Patients with more severe disease course had an elevated ratio of T_FH‐like cells and increased IL‐21 production. Moreover, expansion of Tfh‐like cells correlated positively with parameters related to antibody secretion, including serum immunoglobulin (Ig)G, immune complexes (ICs) and autoantibodies. Correlation analysis between Tfh‐like cells and certain B cell subsets revealed possible defects during B cell selection. In conclusion, our observations on the profound expansion of circulating Tfh‐like cells and their IL‐21 production, along with the characteristic aberrant peripheral B cell distribution in both pSS and SLE, indicate the prominent role of Tfh cell in the regulation of B cell selection.     

Significant involvement of nuclear factor‐κB‐inducing kinase in proper differentiation of αβ and γδ T cells

Nuclear factor‐κB‐inducing kinase (NIK) is known to play a critical role in maintaining proper immune function. This is exemplified in the spontaneous mutant mouse lacking functional NIK, alymphoplasia (aly), which is simultaneously immune‐compromised and autoimmune‐prone. To investigate the role of NIK in αβ T‐cell repertoire formation, we analysed T‐cell development in aly/aly mice bearing a transgenic T‐cell receptor (TCR). Although there were no apparent abnormalities in the mature αβ T cells of non‐transgenic aly/aly mice, the maturation efficiency of idiotype^high+ T cells in the TCR‐transgenic mice was lower in aly/aly mice compared with those found in aly/+ mice, suggesting that the mature αβ T‐cell repertoire could be altered by the absence of functional NIK. In one strain of TCR‐transgenic aly/aly mice with a negatively selecting H‐2 background, the proportion of CD8^low+ idiotype^high+ cells, which are thought to potentially represent the γδ lineage of T cells, was markedly decreased. When the γδ T cells in non‐transgenic aly/aly mice were investigated, the proportion of γδ T cells in the peripheral organs of aly/aly mice was found to be one‐half to one‐fifth of those in aly/+ mice. Analyses of bone marrow chimera mice indicated that NIK in host cells, rather than in donor cells was important for generating a normal number of peripheral γδ T cells. Collectively, these results suggest that NIK could be involved in thymic positive selection of some αβ T cells and that NIK in non‐haematopoietic cells is important for the optimal development and/or maintenance of γδ T cells.     

Salt‐inducible kinase 3 deficiency exacerbates lipopolysaccharide‐induced endotoxin shock accompanied by increased levels of pro‐inflammatory molecules in mice

Macrophages play important roles in the innate immune system during infection and systemic inflammation. When bacterial lipopolysaccharide (LPS) binds to Toll‐like receptor 4 on macrophages, several signalling cascades co‐operatively up‐regulate gene expression of inflammatory molecules. The present study aimed to examine whether salt‐inducible kinase [SIK, a member of the AMP‐activated protein kinase (AMPK) family] could contribute to the regulation of immune signal not only in cultured macrophages, but also in vivo. LPS up‐regulated SIK3 expression in murine RAW264.7 macrophages and exogenously over‐expressed SIK3 negatively regulated the expression of inflammatory molecules [interleukin‐6 (IL‐6), nitric oxide (NO) and IL‐12p40] in RAW264.7 macrophages. Conversely, these inflammatory molecule levels were up‐regulated in SIK3‐deficient thioglycollate‐elicited peritoneal macrophages (TEPM), despite no impairment of the classical signalling cascades. Forced expression of SIK3 in SIK3‐deficient TEPM suppressed the levels of the above‐mentioned inflammatory molecules. LPS injection (10 mg/kg) led to the death of all SIK3‐knockout (KO) mice within 48 hr after treatment, whereas only one mouse died in the SIK1‐KO (n = 8), SIK2‐KO (n = 9) and wild‐type (n = 8 or 9) groups. In addition, SIK3‐KO bone marrow transplantation increased LPS sensitivity of the recipient wild‐type mice, which was accompanied by an increased level of circulating IL‐6. These results suggest that SIK3 is a unique negative regulator that suppresses inflammatory molecule gene expression in LPS‐stimulated macrophages.     

Assessment of intracellular cytokines and regulatory cells in patients with autoimmune diseases and primary immunodeficiencies — Novel tool for diagnostics and patient follow-up

Serum and intracytoplasmic cytokines are mandatory in host defense against microbes, but also play a pivotal role in the pathogenesis of autoimmune diseases by initiating and perpetuating various cellular and humoral autoimmune processes.     

Optimizing conventional DMARD therapy for Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is an auto immune disorder characterized by exocrine dysfunction as a result of chronic inflammation of the glands. Part of the patients also develops inflammation in other organs. In a complex interplay of different cell types such as T-cells, B-cells, dendritic cells, monocytes/macrophages and NK cells and their effector molecules, all contribute to one of the ultimate hallmarks of pSS: B-cell hyperactivity, subsequent autoantibody production and eventually formation of germinal center-like structures in the salivary gland. Effective treatment options for this disease are currently lacking.Biological DMARDs (bDMARDs) including those targeting B-cells or B-cell activation (directly or indirectly) have been studied, so far with limited efficacy. Besides that, their high costs provide a major drawback for implementation. Relatively inexpensive conventional DMARDs (cDMARDs) with well-known safety profiles have been shown efficacious in numerous clinical studies in multiple (rheumatic) diseases. cDMARDs target several pathways that are crucial in pSS immunopathology and some have proven to effectively inhibit B-cell hyperactivity and immune activation when given to patients. However, strong conclusions about potential efficacy are hampered by lack of standardization of inclusion criteria and outcome measures, dosing and validated biomarkers for patient selection. Proper implementation of these could help to optimize the use of cDMARDs in pSS treatment. In analogy with effective treatment strategies in for example rheumatoid arthritis, combination of two cDMARDs targeting different dysregulated pathways might result in additive or synergistic inhibition of immune activation. In view of this and the unique and potent mechanisms of action to target immunopathology in pSS, optimizing cDMARDs for treatment of pSS is worthwhile.     

Eating ourselves to death (and despair): The contribution of adiposity and inflammation to depression

Obesity and related metabolic conditions are of epidemic proportions in most of the world, affecting both adults and children. The accumulation of lipids in the body in the form of white adipose tissue in the abdomen is now known to activate innate immune mechanisms. Lipid accumulation causes adipocytes to directly secrete the cytokines interleukin (IL) 6 and tumor necrosis factor α (TNFα), but also monocyte chemoattractant protein 1 (MCP-1), which results in the accumulation of leukocytes in fat tissue. This sets up a chronic inflammatory state which is known to mediate the association between obesity and conditions such as cardiovascular disease, type 2 diabetes, and cancer. There is also a substantial literature linking inflammation with risk for depression. This includes the observations that: (1) people with inflammatory diseases such as multiple sclerosis, cardiovascular disease, and psoriasis have elevated rates of depression; (2) many people administered inflammatory cytokines such as interferon α develop depression that is indistinguishable from depression in non-medically ill populations; (3) a significant proportion of depressed persons show upregulation of inflammatory factors such as IL-6, C-reactive protein, and TNFα; (4) inflammatory cytokines can interact with virtually every pathophysiologic domain relevant to depression, including neurotransmitter metabolism, neuroendocrine function, and synaptic plasticity. While many factors may contribute to the association between inflammatory mediators and depression, we hypothesize that increased adiposity may be one causal pathway. Mediational analysis suggests a bi-directional association between adiposity and depression, with inflammation possibly playing an intermediary role.     

Insight into the role of TSLP in inflammatory bowel diseases

Proinflammatory cytokines are thought to modulate pathogeneses of various inflammatory bowel diseases (IBDs). Thymic stromal lymphopoietin (TSLP), which has been studied in various allergic diseases such as asthma, atopic dermatitis (AD) and eosinophilic esophagitis (EoE), has been less considered to be involved in IBDs. However, mucosal dendritic cells (DCs) induced by various cytokines including TSLP were reported to cause polarization of T cell toward Th2 response, the differentiation of regulatory T-cell (Treg), and secretion of IgA by B cells. In this review, we discuss the concept that decreased TSLP has the potential to accelerate the development of Th1 response dominant diseases such as the Crohn's disease (CD) while increased TSLP has the potential to lead to a development of Th2 cell dominant diseases such the ulcerative colitis (UC). To examine TSLP's role as a potential determining factor for differentiating UC and CD, we analyzed the effects of other genes regulated by TSLP in regards to the UC and CD pathogeneses using data from online open access resources such as NetPath, GeneMania, and the String database. Our findings indicate that TSLP is a key mediator in the pathogenesis of IBDs and that further studies are needed to evaluate its role.