Angela Bikker

Increased expression of interleukin-7 in labial salivary glands of patients with primary Sjögren's syndrome correlates with increased inflammation

OBJECTIVE To study the expression levels and immunostimulatory capacities of interleukin-7 (IL-7) in primary Sjögrens syndrome. METHODS Labial salivary gland (LSG) IL-7 expression was determined by immunohistochemistry, using a quantitative scoring system, in 30 patients with sicca syndrome: 15 patients with primary Sjögrens syndrome (SS) and 15 patients with non-SS sicca syndrome. The correlation of IL-7 expression in LSGs with parameters of local and peripheral disease was studied, and serum and salivary IL-7 levels were determined. Additionally, the effects of IL-7 on cytokine production by peripheral blood mononuclear cells (PBMCs) from patients with primary SS were determined in vitro by Luminex multicytokine assay and compared with the effects in control subjects. RESULTS The expression of IL-7 in LSGs was higher in patients with primary SS compared with that in patients with non-SS sicca syndrome. IL-7 was observed primarily in the vicinity of lymphocytic infiltrates. Salivary IL-7 levels in patients with primary SS were higher than those in control subjects. In all 30 patients with sicca syndrome, IL-7 expression in LSGs correlated with parameters of both local and peripheral disease. Furthermore, IL-7 stimulated T cell-attracting and T cell-differentiating cytokines (monokine induced by interferon-gamma [IFNgamma], IFNgamma-inducible 10-kd protein, IL-12, and IL-15), as well as Th1 (IFNgamma), Th2 (IL-4), Th17 (IL-17A), proinflammatory (tumor necrosis factor alpha and IL-1alpha), and regulatory (IL-10 and IL-13) cytokine production by PBMCs. All of these cytokines were previously shown to be associated with primary SS. The IL-7-induced increase in IL-10 production in patients with primary SS was reduced compared with that in control subjects. CONCLUSION The correlation between LSG IL-7 expression and (local) disease parameters in primary SS as well as the IL-7-mediated induction of inflammatory cytokines indicate that IL-7 might contribute to the immunopathology of primary SS.

IL-7 drives Th1 and Th17 cytokine production in patients with primary SS despite an increase in CD4 T cells lacking the IL-7Rα

OBJECTIVE To study the phenotypic characteristics of and the balance between systemic IL-7 receptor (IL-7R)α+ and IL-7Rα- Tregs in primary SS (pSS) patients as compared with control subjects and to assess the functional consequences this has for (IL-7-induced) T-cell activation. METHODS The functional properties of IL-7Rα+ and IL-7Rα- (CD25+) CD4 T cells from pSS patients were tested in vitro. Expression of CD25 and FoxP3 by IL-7Rα+ and IL-7Rα- CD4 T cells from pSS patients and healthy controls (HCs) were assessed. Also, the net ex vivo T-cell cytokine production and the capacity of IL-7 to activate total CD4 T cells from pSS patients compared with HCs in vitro was tested. RESULTS IL-7Rα+ T cells from pSS patients strongly proliferated and their numbers were slightly reduced compared with HCs. This reduced number was caused by an increase in both anergic and suppressive IL-7Rα- CD25+ T cells expressing high levels of FoxP3, but also by increases in IL-7Rα- CD25- CD4 T cells that only moderately expressed FoxP3. This altered balance in IL-7Rα+ and IL-7Rα- CD4 T cells was accompanied by unchanged ex vivo Th1, Th2 and Th17 cytokine production of total CD4 T cells. Furthermore, the increased numbers of IL-7Rα- CD25+ T cells did not prevent specific IL-7-induced Th1 and Th17 cytokine production by IL-7Rα+ T cells. CONCLUSION IL-7Rα+ cells are highly proliferating cells that respond strongly to IL-7 despite an increased number of IL-7Rα- T cells that express FoxP3 and CD25. The recent finding that IL-7 and IL-7Rα+ T cells were both found to be increased in exocrine glands of pSS patients indicates that IL-7 could contribute to glandular inflammation by activation of IL-7Rα+ responder T cells despite the increased numbers of Tregs.

Interleukin-7: a key Mediator in T Cell-driven Autoimmunity, Inflammation, and Tissue Destruction

IL-7, expressed by stromal cells in primary lymphoid organs, is known for its critical role in the development and homeostatic expansion of T cells in humans and mice. IL-7 is equally important for B cell development in human and mice, but only in mice seems critical for B cell development and expansion. Recent studies demonstrate that this potent immunostimulatory cytokine is overexpressed in inflamed tissues of patients with (rheumatic) autoimmune diseases and that expression levels correlate with clinical parameters of disease. In inflamed tissues several cell types, including macrophages, dendritic cells, and fibroblasts produce IL-7. IL-7 primarily acts on T cells that abundantly express the IL-7 receptor and that are increased at the inflammatory sites, and predominantly induces Th1 and Th17-associated cytokine secretion. IL-7-mediated T cell-dependent activation of macrophages, dendritic cells and B cells is accompanied by up regulation of T cell differentiating factors, chemokines, adhesion/co-stimulatory molecules and catabolic cytokines and enzymes. Moreover, overexpression of IL-7 is associated with ectopic lymphoid aggregate formation, corresponding with the capacity of IL-7 to induce LTβ and TNFα and to activate innate lymphoid tissue inducer cells. Additionally, IL-7 promotes T cell-driven osteoclastogenesis and fibroblast activation, processes involved in tissue destruction in chronic inflammation. Altogether this suggests that IL-7 is an important proinflammatory mediator in several chronic (rheumatic) inflammatory autoimmune diseases. The substantial amelioration of inflammation and immunopathology in experimental animal models for these diseases by blocking IL-7(receptor) supports this role of IL-7 and demonstrates that IL-7 and its receptor represent novel targets for immunotherapy.

Increased interleukin (IL)-7Rα expression in salivary glands of patients with primary Sjögren's syndrome is restricted to T cells and correlates with IL-7 expression, lymphocyte numbers and activity

Objective To identify interleukin (IL)-7Rα expression in the labial salivary gland (LSG) of patients with primary Sjögrens syndrome (pSS) and non-Sjögrens syndrome sicca (nSS-sicca) and to study its correlation with glandular inflammation and IL-7 expression. Methods The presence of infiltrating immune cells and IL-7Rα cells in inflamed LSG of patients with pSS (n=12) and nSS-sicca controls (n=7) was studied by immunohistochemistry and fluorescence activated cell sorting analysis upon tissue digestion (n=15 and n=13, respectively). Additionally, the correlations of IL-7Rα cells with hallmark disease parameters of pSS, major infiltrating inflammatory cells and IL-7 were assessed. Results In the LSG of patients with pSS increased numbers of IL-7Rα cells were found as compared with nSS-sicca patients. IL7Rα cells strongly correlated with the lymphocytic focus score, IL-7 expression, the decrease in percentage of IgA plasma cells and numbers of CD3 T cells, CD20 B cells, and CD1a and CD208 myeloid dendritic cells. Analysis of isolated cells from the LSG demonstrated strongly increased percentages of IL-7Rα CD3 T cells in pSS as compared with nSS, showing abundant IL-7Rα expression on both CD4 and CD8 T cells. Other CD45 leucocytes and CD45- tissue cells scarcely expressed IL-7Rα. Percentages of IL-7Rα T cells also significantly correlated with glandular inflammation. Conclusions This study shows the presence of increased IL-7Rα T cells in the LSG of patients with pSS and their association with the severity of sialadenitis, disease parameters and IL-7 expression. Considering the immunostimulatory ability of IL-7Rα T cells and IL-7, this suggests that IL-7(R)-dependent T cell-driven immune activation plays an important role in inflammation in pSS.

Interleukin-7 and Toll-like receptor 7 induce synergistic B cell and T cell activation

Objectives To investigate the potential synergy of IL-7-driven T cell-dependent and TLR7-mediated B cell activation and to assess the additive effects of monocyte/macrophages in this respect. Methods Isolated CD19 B cells and CD4 T cells from healthy donors were co-cultured with TLR7 agonist (TLR7A, Gardiquimod), IL-7, or their combination with or without CD14 monocytes/macrophages (T/B/mono; 1 : 1 : 0,1). Proliferation was measured using 3H-thymidine incorporation and Ki67 expression. Activation marker (CD19, HLA-DR, CD25) expression was measured by FACS analysis. Immunoglobulins were measured by ELISA and release of cytokines was measured by Luminex assay. Results TLR7-induced B cell activation was not associated with T cell activation. IL-7-induced T cell activation alone and together with TLR7A synergistically increased numbers of both proliferating (Ki67+) B cells and T cells, which was further increased in the presence of monocytes/macrophages. This was associated by up regulation of activation markers on B cells and T cells. Additive or synergistic induction of production of immunoglobulins by TLR7 and IL-7 was associated by synergistic induction of T cell cytokines (IFNγ, IL-17A, IL-22), which was only evident in the presence of monocytes/macrophages. Conclusions IL-7-induced CD4 T cell activation and TLR7-induced B cell activation synergistically induce T helper cell cytokine and B cell immunoglobulin production, which is critically dependent on monocytes/macrophages. Our results indicate that previously described increased expression of IL-7 and TLR7 together with increased numbers of macrophages at sites of inflammation in autoimmune diseases like RA and pSS significantly contributes to enhanced lymphocyte activation.

Clinical efficacy of leflunomide in primary Sjögren's syndrome is associated with regulation of T-cell activity and upregulation of IL-7 receptor α expression

Objectives To investigate whether the immunomodulatory capacities of leflunomide are associated with clinical efficacy in the treatment of primary Sjögrens syndrome (SS) in a phase II pilot study. Methods Peripheral blood mononuclear cells from 13 primary SS patients were obtained at baseline and after 24 weeks of leflunomide treatment. Ex-vivo production of interleukin (IL) 1β and tumour necrosis factor α (TNFα) and of interferon (IFN), IL-4, as well as TNFα ELISA measured production on T-cell and monocyte stimulation. In addition, the authors investigated the ability of leflunomide to influence systemic levels of inflammatory cytokines, as well as T-cell activation markers and the expression of IL-7 receptor α by flow cytometry. Correlations between changes in cytokine levels and changes in clinical response parameters were studied. Results Ex-vivo production of IL-1β and TNFα was decreased at 24 weeks in the whole patient group, whereas IFN and IL-4 production were not significantly changed. However, a significant decrease in T-cell-stimulated IFN and TNFα production was observed in clinical responders, but not in non-responders. Moreover, significant correlations were found between increased sialometry values and decreased IFN and TNFα production. In addition, leflunomide reduced levels of inflammatory serum cytokines and CD40L expression, whereas it upregulated IL-7Rα expression on CD4 T cells with persistent serum IL-7 concentrations. Conclusions Leflunomide treatment suppressed cytokine release from circulating immune cells. Inhibition of T-helper 1 cell cytokine production was related to clinical efficacy. This suggests that selective T-cell targeting might be a relevant therapeutic strategy in primary SS, possibly enhancing clinical efficacy and safety.

High soluble IL-7 receptor expression in Sjögren's syndrome identifies patients with increased immunopathology and dryness

Increased expression of interleukin (IL)-7 and its receptor is suggested to play a critical role in immunopathology of primary Sjogrens syndrome (pSS).1–3 Data from humans and mice demonstrate that IL-7 drives a range of processes involved in pSS immunopathology, including epithelial cell apoptosis, lymphocyte infiltration and reduction of salivary output.3 The IL-7/IL-7R axis is involved in the formation of (ectopic) lymphoid structures in salivary glands (SGs),3 ,4 which is a predictor for lymphoma development in pSS.5 ,6 IL-7 activity is potentiated by the soluble form of its receptor (sIL-7R), which is produced in inflamed tissues by activated stromal cells.7 As sIL-7R is a possible biomarker for IL-7-driven immune activation and lymphoid neogenesis, we studied the expression of sIL-7R in pSS in relation to markers of inflammation and saliva production. Ninety-five patients with pSS were diagnosed according to the 2002 criteria (table 1).8 sIL-7R was measured in serum of 68 patients with pSS using ELISA as previously described9 and compared with 51 healthy individuals (HC). Labial SG biopsy tissues were taken from 27 patients and, after thorough rinsing, were incubated in 200 µL of sterile saline for 1 hour at room temperature. In these tissue supernatants, sIL-7R was measured …

Decreased expression of thymic stromal lymphopoietin in salivary glands of patients with primary Sjögren's syndrome is associated with increased disease activity.

Abstract Objectives: Thymic Stromal Lymphopoietin (TSLP) is a potent immunomodulatory cytokine involved in Th2- and Th17-mediated immune responses in different autoimmune diseases. TSLP expression in relation to disease activity was studied in salivary glands of primary Sjögren’s syndrome (pSS) patients as compared to non-SS sicca (nSS) controls. Methods: Tissue sections of minor salivary glands from pSS and nSS patients were stained with monoclonal antibodies against human TSLP, CD3, CD19 and cytokeratin high molecular weight (CK HMW) or stained for Alcian blue to detect mucus production. The number of TSLP-expressing cells was quantified and expression was correlated to local and systemic disease parameters. Results: The number of TSLP-expressing cells was significantly lower in pSS patients than in nSS controls and correlated with a range of disease markers. In pSS patients, TSLP was expressed outside of lymphocytic infiltrates at sections that also encompassed high numbers of intact acinar cells. This difference was independent of tissue destruction. Conclusions: Reduced TSLP expression in pSS patients is associated with increased local and systemic inflammatory markers. Loss of TSLP expression may contribute to Th1/Th17-associated immunopathology in pSS, in line with previous studies demonstrating that TSLP promotes a protective Th2 milieu at mucosal sites.

Decreased expression of TSLP in labial salivary glands of patients with primary Sjögren’s syndrome is associated with local- and systemic disease parameters

Background Thymic Stromal Lymphopoietin (TSLP) is a potent immunomodulatory cytokine involved in Th2-mediated immune responses and homeostatic T-cell expansion. Reduced TSLP expression by intestinal epithelial cells was recently shown to lead to reduced Th2 responses and development of Th1-mediated experimental colitis. Additionally, TSLP is described as a proinflammatory factor in autoimmune diseases. TSLP expression was studied in salivary glands of primary Sjogren’s syndrome (pSS) patients as compared to non-SS Sicca (nSS) patients and the relationship to localand systemic disease parameters was examined.

SAT0158 IL-7 and IL-7 receptor blockade selectively inhibit TLR7-induced B cell activation in primary sjögren’s syndrome

Background Toll-like receptors (TLRs) have been implicated in several auto-immune diseases, especially those involved in the recognition of nucleic acids (viral, bacterial, and possibly self). In primary Sjögren’s syndrome (pSS) TLR7-induced B cell activation has been implicated in the immunopathology. An increased expression of TLR7 mRNA has been found in the parotid gland of pSS patients. In addition, TLR7 specifically recognizes ssRNA from viruses, and possibly self-ssRNA, with which Ro- and/or La-proteins form complexes, facilitating anti-SSA/SSB auto-antibody production, one of the hallmark disease parameters of pSS. Interestingly, EBV-transformed B cells have been shown to express the IL-7R and IL-7. Recently, we found increased levels of IL-7 in the minor salivary gland as well. Objectives To investigate the role of IL-7/IL-7 receptor-mediated immune activation in TLR7-induced B cell activation in pSS patients. Methods Isolated CD4 T cells and CD19 B cells from HC (n=7) and pSS patients (n=5) were co-cultured with and without a TLR7 agonist (TLR7A, Gardiquimod) in the presence or absence of CD14 monocytes/macrophages. Additionally, PBMCs (HC n=5, pSS n=8) were cultured with TLR7A with and without soluble human IL-7R (shuIL7R) and fully human anti-human IL-7 mAb. Proliferation of T cells and B cells was measured using 3H-thymidine incorporation and Ki67 expression (FACS analysis). Activation markers (CD19, HLA-DR, CD25) and intracellular IL-7 and IL-7Rα expression by B cells were measured by FACS analysis. Results TLR7A-increased proliferation of T and B cell co-cultures was associated with significant and selective increases in Ki67+ CD19 B cells (HC from 1.2±0.2% to 9.3±1.3%, p<0.01 vs. pSS from 1.2±0.2% to 7.0±2.2%, p<0.05), but not CD4 T cells. Additionally, markers of activation on CD19 B cells (HC: CD25+ from 42.2±4.8% to 80.1±4.4%; CD19 MFI from 26.8±3.3% to 63.4±9.6%; HLA-DR MFI from 214±32 to 649±105) were significantly increased, equally effective in HC and pSS. TLR7-induced B cell activation was further increased in the presence of monocytes (Ki67+ B cells: HC from 0.9±0.1% to 30.2±8.9% vs. pSS from 1.0±0.1% to 11.6±2.9%, both p<0.05). IL-7(R) blockade markedly inhibited proliferation of TLR7A stimulated PBMCs from HC (from 8880±2069 cpm to 2289±624 cpm; p<0.05) and pSS patients (from 8580±2555 cpm to 4802±1526 cpm; p<0.01), associated by a selective inhibition of Ki67-proliferating B cells. The specific role of IL-7R-mediated activation was supported by an increase in intracellular IL-7Rα and IL-7 upon TLR7 triggering in B cells, and confirmed by blockade of TLR7-induced B cell activation with a fully human anti-IL-7 mAb (mean inhibition 50%, p<0.05). Conclusions Our results show that TLR7 triggering activates B cells in pSS, which is facilitated by monocytes. Interestingly, TLR7A-induced B cell activation is potently and selectively inhibited by IL-7/IL-7R blockade. Together with the up regulation of IL-7 and IL-7R upon TLR7 activation, these results suggest that shuIL-7R/anti-IL-7 mAb blocks an autocrine function of IL-7 in B cells. This indicates that IL-7/IL-7R blockade, targeting not only T cells as previously shown, but also B cells, represents an interesting new therapeutic approach in pSS. Disclosure of Interest None Declared