Experimental Sjögren's Syndrome Research Articles

Iguratimod suppresses plasma cell differentiation and ameliorates experimental Sjögren's syndrome in mice by promoting TEC kinase degradation.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease with an unclear pathogenesis, and there is currently no approved drug for the treatment of this disease. Iguratimod, as a novel clinical anti-rheumatic drug in China and Japan, has shown remarkable efficacy in improving the symptoms of patients with pSS in clinical studies. In this study we investigated the mechanisms underlying the therapeutic effect of iguratimod in the treatment of pSS. Experimental Sjögren's syndrome (ESS) model was established in female mice by immunizing with salivary gland protein. After immunization, ESS mice were orally treated with iguratimod (10, 30, 100 mg·kg-1·d-1) or hydroxychloroquine (50 mg·kg-1·d-1) for 70 days. We showed that iguratimod administration dose-dependently increased saliva secretion, and ameliorated ESS development by predominantly inhibiting B cells activation and plasma cell differentiation. Iguratimod (30 and 100 mg·kg-1·d-1) was more effective than hydroxychloroquine (50 mg·kg-1·d-1). When the potential target of iguratimod was searched, we found that iguratimod bound to TEC kinase and promoted its degradation through the autophagy-lysosome pathway in BAFF-activated B cells, thereby directly inhibiting TEC-regulated B cells function, suggesting that the action mode of iguratimod on TEC was different from that of conventional kinase inhibitors. In addition, we found a crucial role of TEC overexpression in plasma cells of patients with pSS. Together, we demonstrate that iguratimod effectively ameliorates ESS via its unique suppression of TEC function, which will be helpful for its clinical application. Targeting TEC kinase, a new regulatory factor for B cells, may be a promising therapeutic option.

Immunomodulatory Effects of Yu-Ping-Feng Formula on Primary Sjögren's Syndrome: Interrogating the T Cell Response.

Ethnopharmacological treatments have shown beneficial effects in the clinical practice of autoimmune disorders. However, the underlying mechanism of immunomodulatory effects remains challenging, given the complicate composition of herbal medicines. Here, we developed an immunological approach to interrogate the T helper cell response. Through data mining we hypothesized that Chinese medicine formula, Yu-Ping-Feng (YPF) might be a promising candidate for treating primary Sjögren's syndrome (pSS), a common autoimmune disease manifested by exocrine gland dysfunction. We took advantage of a mouse model of experimental Sjögren's syndrome (ESS) that we previously established for YPF formula treatment. YPF therapy ameliorated the ESS pathology in mice with active disease, showing improved salivary function and decreased serum levels of autoantibodies. Phenotypic analysis suggested that both effector T and B cells were significantly suppressed. Using co-culture assay and adoptive transfer models, we demonstrated that YPF formula directly restrained effector/memory T cell expansion and differentiation into Th17 and T follicular helper (Tfh) cells, the key subsets in ESS pathogenesis. Importantly, we recruited 20 pSS patients and conducted a pilot study of 8-week therapy of YPF formula. YPF treatment effectively improved fatigue symptoms, exocrine gland functions and reduced serum IgG/IgA levels, while effector T and B cell subsets were significantly decreased. There was a trend of reduction on disease activity, but not statistically significant. Together, our findings suggested a novel approach to assess the immunomodulatory effects of YPF formula, which may be favorable for patients with autoimmune disorders.

Iguratimod Alleviates Experimental Sjögren's Syndrome by Inhibiting NLRP3 Inflammasome Activation.

Iguratimod (T-614) is a compound widely used as anti-rheumatic drug. This study investigated the effect and underlying mechanism of T-614 on experimental Sjögren's syndrome (ESS). ESS mice model was established by injection of submandibular gland protein. Mice were randomly divided into control, experimental Sjögren's syndrome (ESS), ESS + T-614 (10 mg/kg), ESS + T-614 (20 mg/kg), and ESS + T-614 (30 mg/kg) groups. Human submandibular gland (HSG) were cultured with 0, 0.5, 5, or 50 μg/ml T-614 in the absence or presence of interferon-α (IFN-α). Haematoxylin and eosin (H&E) and cytokine levels were used to detect immune cells activation in submandibular glands. Apoptosis in submandibular glands tissues and cells was determined by TUNEL and flow cytometry. Apoptosis and NLRP3 inflammasome-related proteins were detected by western blotting. T-614 treatment attenuated submandibular gland damage in ESS mice. T-614 administration inhibited submandibular gland cell apoptosis in ESS mice. Furthermore, T-614 blocked inflammatory factor levels and NLRP3 inflammasome activation in the submandibular glands. In vitro, results corroborated that T-614 could protect HSG cells from IFN-α-induced cell apoptosis and inflammation by inhibiting NLRP3 inflammasome activation. Our results expounded that T-614 alleviated ESS by inhibiting NLRP3 inflammasome activation.

Integrative analysis of transcriptome and proteome in primary Sjögren syndrome

ObjectivePrimary Sjögren's syndrome (pSS) is a intricate autoimmune disease mainly characterized of immune-mediated destruction of exocrine tissues, such as salivary and lacrimal glands, occurring dry mouth and eyes. Although some breakthroughs in understanding pSS have been uncovered, many questions remain about its pathogenesis, especially the internal relations between exocrine glands and secretions. MethodTranscriptomic and proteomic analyses were conducted on salivary tissues and saliva in experimental Sjögren syndrome (ESS). The ESS model was established by immunization with salivary gland protein. The expression of mRNAs and proteins in salivary tissues and saliva were determined by high-throughput sequencing transcriptomic analysis and LC-MS/MS-based proteome, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were used to recognize dysregulated genes and proteins. The association between RNA and protein abundance was investigated to provides a comprehensive understanding of RNA-protein correlations in the pathogenesis of pSS. ResultsAs a result, we successfully established the ESS model. We recognized 3221 differentially expressed genes (DEGs) and 253 differentially expressed proteins (DEPs). The sample analysis showed that 61 proteins overlapped through the integrative analysis of transcriptomics and proteomics data. The enrichment pathway analysis of DEGs and DEPs in samples showed alterations in renin-angiotensin-system (RAS), lysosome, and apoptosis. Notably, we found that some genes, such as AGT, FN1, Klk1b26, Klk1, Klk1b5, Klk1b3 had a consistent trend in the regulation at the RNA and protein levels and might be potential diagnostic biomarkers of pSS. ConclusionHerein, we found critical processes and potential biomakers that may contribute to pSS pathogenesis by analyzing dysregulated genes and pathways. Additionally, the integrative multi-omics datasets provided additional insight into understanding complicated disease mechanisms.

IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental Sjögren's syndrome.

Excessive NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome activation has an important function in the pathogenesis of Sjögren's syndrome (SS). Increased and dysfunctional myeloid-derived suppressor cells (MDSCs) promoted SS. However, NLRP3 inflammasome activation of MDSCs in SS and its regulated components are unclear. Splenic MDSCs were purified by immunomagnetic beads and cultured. Western blot was used to assess NLRP3 inflammasomes. Interleukin-1β (IL-1β) and IL-18 were measured using enzyme-linked immunosorbent assay. Here we showed that the NLRP3 inflammasome was activated in non-obese diabetic (NOD) mice with SS-like manifestations. We found that NLRP3 inflammasome activation was augmented in MDSCs of SS mice and NLRP3 inflammasome activation was suppressed in IL-27-deficient NOD mice. Consistent with findings of SS mice in vivo, we observed that NLRP3 inflammasome activation by adenosine triphosphate and lipopolysaccharide was remarkably intensified in MDSCs with IL-27 treatment in vitro. Collectively, our data highlighted that IL-27 regulates NLRP3 inflammasome activation of MDSCs in experimental SS.

MANF inhibits Sjögren's syndrome salivary gland epithelial cell apoptosis and antigen expression of Ro52/SSA through endoplasmic reticulum stress/autophagy pathway.

Sjögren's syndrome (SS) is a typical autoimmune disease characterized by lymphocyte infiltration accompanied by the production of Ro52/SSA and La/SSB autoantibodies against whole body ribonucleoprotein particles. The release of type I IFN can induce endoplasmic reticulum stress (ERS) in submandibular gland cells. ERS not only produces a large number of Ro52/SSA antigens and changes their location, but also down-regulates autophagy and increases apoptosis. We collected human submandibular gland tissue samples, established an Experimental Sjögren's syndrome (ESS) mouse model, and used submandibular gland cells to test whether Mesencephalic astrocyte-derived neurotrophic factor (MANF) could reverse ERS-induced autophagy downregulation and reduce apoptosis and Ro52/SSA antigen expression. It was found that MANF could reduce lymphocyte infiltration and the proportion of CD4+ T cell subsets in the salivary glands, reduce the phosphorylation of AKT and mTOR proteins and the expression of ERS-related proteins, and increase the expression of autophagy proteins. We also found that MANF can reduce the expression of Ro52/SSA antigen on the cell membrane and reduce apoptosis. In short, we found that MANF can activate autophagy, inhibit apoptosis and reduce the expression of Ro52/SSA by regulating the AKT/mTOR/LC3B signaling pathway. The above results suggest that MANF may be a protective factor against SS.

Extracellular vesicle-encapsulated miR-10a-5p derived from MDSCs restrains germinal center B cells in experimental Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) is a progressive systemic autoimmune disease characterized by chronic inflammation of the exocrine glands, resulting in damage to the salivary and lacrimal glands. Our group and other researchers have reported that myeloid-derived suppressor cell-derived extracellular vesicles (MDSC-EVs) could attenuate the progression of autoimmune disease by impairing T-cell function. However, the effect of MDSC-EVs on B-cell function and the underlying mechanism remains largely unknown. In this study, we found that MDSC-EVs significantly attenuated the progression of experimental Sjögren's syndrome (ESS). Moreover, treatment with MDSC-EVs via intravenous injection markedly reduced the percentage of germinal center (GC) B cells in ESS mice. In vitro, MDSC-EVs could directly suppress the generation of GC B cells and theexpression of B cell lymphoma 6 (Bcl-6) in B cells under GC B-cell-polarizing conditions. Mechanistically, miR-10a-5p carried by MDSC-EVs regulated the differentiation of GC B cells by targeting Bcl-6, and inhibition of miR-10a-5p in MDSC-EVs significantly reversed the effect of MDSC-EVs involved in alleviating the development of ESS. Taken together, our findings demonstrated that miR-10a-5p carried by MDSC-EVs inhibited the generation of B cells by targeting Bcl-6 and eventually alleviated the progression of ESS, which may provide novel therapeutic targets for the treatment of pSS.

Immune infiltration analysis reveals immune cell signatures in salivary gland tissue of primary Sjögren's syndrome.

Mouse models are the basis for primary Sjögren's syndrome (pSS) research. However, the depth of comparisons between mice and humans in salivary gland (SG) immune cells remains limited. The gene expression profiles of SGs from normal subjects and pSS patients were downloaded from the Gene Expression Comprehensive Database. The proportion of infiltrating immune cell subsets was then assessed by cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT). An experimental Sjögren's syndrome (ESS) mouse model was successfully constructed using SG protein. Based on mouse SG tissue RNA-Seq data, the seq-ImmuCC model was used to quantitatively analyze the compositional ratios of 10 immune cells in pSS patients and mouse model SG tissues. Computed and obtained 31 human data samples using the CIBERSORT deconvolution method. The immune cell infiltration results showed that, compared to normal human SG tissue, the content of gamma delta T cells was significantly different from naive CD4+ T cells and significantly increased, while the plasma cell content decreased. Principal component analysis indicated differences in immune cell infiltration between pSS patients and normal subjects. Meanwhile, for ESS model mouse data analysis, we found that the proportion of macrophages increased, while the proportion of CD4+ T cells, B cells, and monocytes decreased. Furthermore, we found that the proportion of monocytes was decreased, while the proportion of macrophages was increased in the SG tissues of pSS patients and model mice. The infiltration of CD4+ T, CD8+ T, and B cells also showed some differences. We comprehensively analyzed SG immune infiltration in pSS patients and model mice. We demonstrated conserved and nonconserved aspects of the immune system in mice and humans at the level of immune cells to help explain the primary regulation of immune mechanisms during the development of Sjögren's syndrome.

Detection of T Follicular Helper Cells and T Follicular Regulatory Cells in Experimental Sjögren's Syndrome.

With the compiling studies on the autoimmune pathogenesis in the developing of Sjögren's syndrome, the functional importance of T follicular helper cells (Tfh) and T follicular regulatory cells (Tfr) was investigated, including our recent findings, among which various techniques for detecting Tfh and Tfr cells in Sjögren's syndrome have been reported. In this chapter, we describe detailed methods for the effective detection of Tfh and Tfr cells in mice with experimental Sjögren's syndrome (ESS), a mouse model with evident salivary hypofunction, increased serum levels of autoantibodies, and histopathological changes in the salivary glands. We provide representative detections of surface markers, cytokines, and transcription factors of Tfh and Tfr cells by flow cytometry and ELISpot assay. Moreover, a detailed protocol for detecting Tfh and Tfr cells in the draining cervical lymph nodes (CLNs) in ESS mice by immunofluorescence microscopy is also described. Together, these techniques of Tfh and Tfr cell detection in ESS mice may facilitate the investigation of autoimmune pathogenesis and enhance the understanding of Tfh and Tfr cell functions in the development of Sjögren's syndrome.

Deficiency of β-arrestin2 alleviates apoptosis through GRP78-ATF6-CHOP signaling pathway in primary Sjögren's syndrome

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no ideal drug for the specific treatment of pSS. β-arrestin2 is a key protein that mediates desensitization and internalization of G protein-coupled receptors (GPCRs) and it participates in inflammatory and immune responses that have been found to mediate apoptosis in autoimmune disease. In this study, we established an experimental Sjögren's syndrome (ESS) mouse model to elucidate the molecular mechanisms of β-arrestin2 in pSS. First, excessive activation of β-arrestin2 and GRP78-ATF6-CHOP apoptosis signaling were detected in specimens from pSS patients. In vivo, we found that inhibition of GRP78-ATF6-CHOP apoptosis signaling improved ESS symptoms, and the targeted deletion of β-arrestin2 significantly increased saliva flow, alleviated salivary gland indices, and improved tissue integrity in the ESS model by downregulating GRP78-ATF6-CHOP apoptosis signaling. In vitro, we used IFNα to stimulate human salivary gland epithelial cells (HSGECs), and the results showed that IFNα activated GRP78-ATF6-CHOP apoptosis signaling, decreased cell viability, and induced apoptosis, which were negatively regulated by the ERS inhibitor 4-PBA. In addition, β-arrestin2 depletion downregulated GRP78-ATF6-CHOP apoptosis signaling to alleviate cell apoptosis, and the effect depended on the interaction between GRP78 and β-arrestin2. In summary, our results suggest that excessive activation of GRP78-ATF6-CHOP apoptosis signaling is involved in the pathogenesis of pSS and that β-arrestin2 encourages inflammation-induced epithelial apoptosis through GRP78-ATF6-CHOP apoptosis signaling. This research further clarified the underlying role of β-arrestin2 and provided an experimental foundation for β-arrestin2 depletion in the treatment of the human autoimmune disorder pSS.

Proteomic profiling of saliva reveals association of complement system with primary Sjögren's syndrome.

IntroductionTo compare the saliva proteomes of experimental Sjögren's syndrome (ESS) model mice and healthy controls to identify potential diagnostic biomarkers for primary Sjögren's syndrome (pSS).MethodsProteins were extracted from the saliva of three ESS and three normal control mice using the data‐independent acquisition technique. R language was used to identify the differentially expressed proteins (DEPs). Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to functionally annotate the DEPs. The protein–protein interaction (PPI) network was constructed and the core proteins were identified with the STRING website and Cytoscape software. The concentrations of Serpin family G member 1 (SERPING1), C3, complement factor H (CFH), fibrinogen alpha (FGA), and fibrinogen gamma (FGG) in saliva were determined by ELISA.ResultsA total of 1722 DEPs were identified in the saliva of the ESS mice relative to the controls, of which 50 showed significantly different expression levels between the two groups. SERPING1, C3, CFH, FGA, and FGG were significantly downregulated, and keratin 4 (Krt4) and transglutaminase 3 (TGM3) were upregulated in the saliva of ESS mice. The PPI network showed that SERPING1, C3, FGG, FGA, TGM3, and hemopexin (HPX) were the core proteins. ELISA results showed that the expression of C3, CFH, FGA, and SERPING1 were significantly downregulated in the saliva of ESS mice. However, the expression of FGG was a little downregulated but with no significant difference. SERPING1, FGG, and FGA may downregulate the complement C3 by inhibiting immune complement system, thereby promoting pSS progression.ConclusionsThe salivary proteome of ESS mice was markedly different from that of healthy controls, suggesting that salivary proteomics is a promising noninvasive diagnostic tool for pSS. SERPING1, C3, CFH, FGA, and FGG are potential biomarkers of pSS.

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6′-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.The therapeutic effects of CP-25 on experimental Sjögren's syndrome has been shown to be associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. The study provides an experimental foundation for CP-25 as a potential drug in the treatment of human autoimmune disorder, Sjögren's syndrome.

IL‐17 drives salivary gland dysfunction via inhibiting TRPC1‐mediated calcium movement in Sjögren’s syndrome

ObjectivesThis study aims to determine a role of interleukin‐17A (IL‐17) in salivary gland (SG) dysfunction and therapeutic effects of targeting IL‐17 in SG for treating autoimmune sialadenitis in primary Sjögren’s syndrome (pSS).MethodsSalivary IL‐17 levels and IL‐17‐secreting cells in labial glands of pSS patients were examined. Kinetic changes of IL‐17‐producing cells in SG from mice with experimental Sjögren’s syndrome (ESS) were analysed. To determine a role of IL‐17 in salivary secretion, IL‐17‐deficient mice and constructed chimeric mice with IL‐17 receptor C (IL‐17RC) deficiency in non‐hematopoietic and hematopoietic cells were examined for saliva flow rates during ESS development. Both human and murine primary SG epithelial cells were treated with IL‐17 for measuring cholinergic activation‐induced calcium movement. Moreover, SG functions were assessed in ESS mice with salivary retrograde cannulation of IL‐17 neutralisation antibodies.ResultsIncreased salivary IL‐17 levels were negatively correlated with saliva flow rates in pSS patients. Both IL‐17‐deficient mice and chimeric mice with non‐hematopoietic cell‐restricted IL‐17RC deficiency exhibited no obvious salivary reduction while chimeric mice with hematopoietic cell‐restricted IL‐17RC deficiency showed significantly decreased saliva secretion during ESS development. In SG epithelial cells, IL‐17 inhibited acetylcholine‐induced calcium movement and downregulated the expression of transient receptor potential canonical 1 via promoting Nfkbiz mRNA stabilisation. Moreover, local IL‐17 neutralisation in SG markedly attenuated hyposalivation and ameliorated tissue inflammation in ESS mice.ConclusionThese findings identify a novel function of IL‐17 in driving salivary dysfunction during pSS development and may provide a new therapeutic strategy for targeting SG dysfunction in pSS patients.

Recent Advances in Mouse Models of Sjögren's Syndrome

Sjögren's syndrome (SS) is a complex rheumatoid disease that mainly affects exocrine glands, resulting in xerostomia (dry mouth) and xerophthalmia (dry eye). SS is characterized by autoantibodies, infiltration into exocrine glands, and ectopic expression of MHC II molecules on glandular epithelial cells. In contrast to the well-characterized clinical and immunological features, the etiology and pathogenesis of SS remain largely unknown. Animal models are powerful research tools for elucidating the pathogenesis of human diseases. To date, many mouse models of SS, including induced models, in which disease is induced in mice, and genetic models, in which mice spontaneously develop SS-like disease, have been established. These mouse models have provided new insight into the pathogenesis of SS. In this review, we aim to provide a comprehensive overview of recent advances in the field of experimental SS.

Bufotalin ameliorates experimental Sjögren's syndrome development by inhibiting Th17 generation.

Chronic inflammatory autoimmune disease Sjögren's syndrome (SS) is characterized by the reduced secretion of exocrine glands, suggesting strategies targeting inflammation to be a potential option for SS therapy. Bufotalin, an active constituent of Bufadienolides, exerts potent antitumor effects with unknown effects on autoimmune diseases including SS. This study aims to investigate whether bufotalin possesses therapeutic potentials to SS and the underlying mechanisms. The experimental Sjögren's syndrome (ESS) murine model was constructed by SG-immunization and murine naïve CD4+ T cells were cultured under Th17 polarization conditions with or without low doses of bufotalin treatment. Saliva flow rate was measured, and flow cytometry was applied to analyze T cell subpopulations. ELISA was conducted to determine the levels of targeted inflammatory cytokines. Bufotalin-treated ESS mice showed higher saliva flow rates, lower serum levels of autoantibodies (anti-M3R and anti-SSA IgG), lower serum levels of pro-inflammatory cytokines, as well as lower Th17 cell population from spleens and cervical lymph nodes. Additionally, in vitro study showed that bufotalin inhibits Th17 polarization and secretion of cytokines IL-17 and IFN-γ. Bufotalin at a low dose significantly ameliorates ESS development, possibly via inhibiting pro-inflammatory Th17 population and secretion of inflammatory cytokines during ESS pathogenesis.

Alleviating effect of paeoniflorin-6′-O-benzene sulfonate in antigen-induced experimental Sjögren’s syndrome by modulating B lymphocyte migration via CXCR5-GRK2-ERK/p38 signaling pathway

Primary Sjögren's syndrome (pSS) is an autoimmune disease of unresolved aetiology that affects the exocrine glands. Clinical symptoms frequently also involve skin, liver, kidney and neurovascular components. The pathogenesis of pSS is still unclear but B cell hyperactivity has been identified as a hallmark of pSS. Currently, a curative therapeutic agent is lacking. In this study, we explored whether paeoniflorin-6′-O-benzene (CP-25) exerted therapeutic effects through regulating B lymphocyte migration via CXCR5-GRK2-MAPK mediated signaling pathways in a mouse model of antigen-induced, experimental Sjögren's syndrome (ESS). We found that CP-25 increased the salivary flow and alleviated the histopathology of ESS. Furthermore, CP-25 reduced the viability of B lymphocyte and limited the target organs index. In the peripheral blood and salivary gland of ESS mice, CP-25 down-regulated the proportion of total B cells, CXCR5+ B cells and PDCA1 + CD19- and limited the presence of phosphorylated (p-) p38 and ERK (p-ERK). Besides, CP-25 increased the percentage of memory B cells in the peripheral blood and reduced it in salivary gland. Furthermore, in vitro, CP-25 down-regulated p-p38, p-ERK, CXCR5 and membrane GRK2, and increased cytoplasm GRK2 in Maver-1 cells, a mantle cell lymphoma cell line, causing a lower migration ability of Maver-1 cells. Thus, we define CP-25 as a novel compound that is a potent therapeutic agent for pSS which modulates B lymphocyte subsets and impacts the migration of B lymphocytes through regulating the CXCR5-GRK2-ERK/p38 signaling pathway.

IL-10-producing regulatory B cells restrain the T follicular helper cell response in primary Sjögren's syndrome.

Increased numbers of T follicular helper (Tfh) cells have been implicated in the development of autoimmune diseases including primary Sjögren's syndrome (pSS), but how the Tfh cell response is regulated during autoimmune pathogenesis remains largely unclear. Here, we first found negative correlations between IL-10+ regulatory B (Breg) cell numbers and Tfh cell responses and disease activity in patients with pSS and mice with experimental Sjögren's syndrome (ESS). Moreover, we detected high expression of IL-10 receptor on Tfh cells and their precursors in both humans and mice. In culture, IL-10 suppressed human and murine Tfh cell differentiation by promoting STAT5 phosphorylation. By using an adoptive transfer approach and two-photon live imaging, we found significantly increased numbers of Tfh cells with enhanced T cell homing into B cell follicles in the draining cervical lymph nodes of RAG-2-/- mice transferred with IL-10-deficient B cells during ESS development compared with those of RAG-2-/- mice transferred with wild-type B cells. In ESS mice, CD19+CD1dhiCD5+ Breg cells with decreased IL-10 production exhibited severely impaired suppressive effects on T cell proliferation. Consistently, CD19+CD24+CD38hi Breg cells from pSS patients showed significantly reduced IL-10 production with defective inhibitory function in the suppression of autologous Tfh cell expansion. Furthermore, the adoptive transfer of IL-10-producing Breg cells markedly suppressed the Tfh cell response and ameliorated ESS progression in ESS mice. Together, these findings demonstrate a critical role for IL-10-producing Breg cells in restraining the effector Tfh cell response during pSS development.

Increased GITRL Impairs the Function of Myeloid-Derived Suppressor Cells and Exacerbates Primary Sjögren Syndrome.

Although the expansion of myeloid-derived suppressor cells (MDSCs) has been reported in autoimmune disorders, it is largely unclear how MDSCs contribute to the development of primary Sjögren syndrome (pSS). In this study, we found significantly increased MDSCs with gradually diminished suppressive capacity during disease development in mice with experimental Sjögren syndrome (ESS). The ligand for glucocorticoid-induced TNFR family-related protein (GITRL) was increased along ESS progression, whereas the increased GITRL was found to attenuate the immunosuppressive function of MDSCs. Moreover, blocking GITR signal in MDSCs significantly restored their immunosuppressive function and alleviated ESS progression in mice. In pSS patients, expanded MDSCs were found to express low levels of arginase. Significantly increased serum GITRL levels were closely correlated with patients with higher Sjögren syndrome disease activity index. Furthermore, treatment with recombinant GITRL markedly reduced the immunosuppressive function of human MDSCs. Together, our studies have demonstrated a critical role of GITRL in modulating the suppressive function of MDSCs, which may facilitate the validation of GITRL as a therapeutic target for the treatment of pSS.

Interleukin-25 Axis Is Involved in the Pathogenesis of Human Primary and Experimental Murine Sjögren's Syndrome.

To investigate the role of the interleukin-25 (IL-25)/IL-17 receptor B (IL-17RB) axis in experimental Sjögren's syndrome (SS) and in patients with primary SS and primary SS-associated lymphoma. Expression of IL-25, IL-17RB, IL-17B, and tumor necrosis factor receptor-associated factor 6 (TRAF6) was analyzed on minor salivary gland (SG) samples from patients with primary SS and on parotid gland samples from patients with primary SS-associated B cell non-Hodgkin's lymphoma (NHL). IL-17RB expression and the frequencies of natural group 2 innate lymphoid cells (ILC2s), inflammatory ILC2s, and M2-polarized macrophages were assessed by flow cytometry in SG mononuclear cells and peripheral blood mononuclear cells (PBMCs). Tissue distribution of ILC2s was studied by confocal microscopy. The role of recombinant IL-25 and of rituximab in modulating IL-25 expression was investigated in in vitro studies. IL-25/IL-17RB and TRAF6 expression and the role of IL-25 inhibition were also studied in the experimental murine model of SS. Activation of the IL-25/IL-17RB/TRAF6 axis correlated with the focus score and was observed in patients with primary SS and in patients with primary SS-associated NHL. A significant increase in the frequency of inflammatory ILC2s was observed both in SG mononuclear cells and in PBMCs. IL-25 stimulation of isolated SG mononuclear cells and PBMCs from patients and controls resulted both in inflammatory ILC2 expansion and in increased autoantibody production. Rituximab modulated expression of inflammatory ILC2s and IL-25 in primary SS. SG protein-immunized mice developed overt SS symptoms with increased IL-25 expression and increased frequency of CD4+IL-17RB+TRAF6+ cells. IL-25 neutralization attenuated disease progression and tissue pathology in mice with experimental SS. IL-25 may promote the inflammatory state in primary SS and may be a potential target for novel disease-modifying therapeutic strategies in patients with primary SS.

Proteasome inhibition suppresses Th17 cell generation and ameliorates autoimmune development in experimental Sjögren's syndrome.

Immunoproteasome activation in immune cells is involved in the modulation of immune responses. Increasing evidence indicates that proteasome inhibitors show beneficial effects in treating autoimmune diseases, but it remains unclear whether proteasome inhibition is an effective approach for suppressing autoimmune development in Sjögren's syndrome (SS). Our previous work has demonstrated a critical role for Th17 cells in the development of experimental SS (ESS) in mice. In this study, we detected high levels of low-molecular-weight protein 7 (LMP7), a key subunit of the immunoproteasome, in Th17 cells from ESS mice. Moreover, treatment with bortezomib (BTZ), a proteasome inhibitor, markedly suppressed Th17 differentiation in both murine and human naive T cells in culture. Furthermore, ESS mice treated with BTZ displayed significantly higher saliva flow rates and a reduction in tissue destruction in the salivary glands compared with vehicle-treated ESS mice. Notably, BTZ-treated ESS mice showed markedly decreased Th17 cells, germinal center B cells and plasma cells in the peripheral lymphoid organs. In addition, adoptively transferred wild type naive CD4+ T cells rapidly differentiated into Th17 cells and induced salivary dysfunction in IL-17-deficient mice immunized for ESS induction. However, BTZ treatment profoundly suppressed the donor T-cell-derived Th17 response and ameliorated the reduction in salivary secretion in IL-17-deficient recipient mice. Taken together, our findings demonstrate that proteasome inhibition can effectively ameliorate ESS by suppressing the Th17 response, which may contribute to the development of a novel therapeutic strategy for the treatment of SS.Cellular &Molecular Immunology advance online publication, 10 July 2017; doi:10.1038/cmi.2017.8.