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 block...
Background Thymic stromal lymphopoietin (TSLP) is well known for its potent activation of myeloid dendritic cells (mDCs) resulting in Th2-mediated immune responses. TSLP signals cells via the IL-7 receptor-alpha chain (IL-7Rα), shared with IL-7, together with the TSLP receptor (TSLPR) subunit. Recently, we have demonstrated that prevention of TSLPR signalling strongly reduces Th17-driven experimental arthritis and immunopathology. Furthermore, we have shown that administration of TSLP enhances severity of inflammation and joint destruction in collagen induced arthritis. Objectives To determine the levels of TSLP and numbers of TSLPR-expressing mDCs in joints of rheumatoid arthritis (RA) patients as compared to peripheral blood (PB) and the capacity of TSLP to induce mDC-dependent T-cell activation. Methods TSLP was measured in synovial fluid (SF) of patients with RA (n=50) and osteoarthritis (OA, n=24) by ELISA. CD1c+ mDC numbers and TSLPR expression on these cells were assessed by FACS analysis in paired samples of SF and PB from RA patients (n=9). CD1c+ mDCs, isolated from PB as well as SF of RA patients (n=6), were stimulated with TSLP for 20 hours and cytokine production was measured by multiplex immunoassay (measuring 51 cytokines). Washed TSLP-activated CD1c+ mDCs from PB (n=11) and SF (n=5) were added to autologous CD4 T cells in the absence of additional stimuli, cultured for 6 days and subsequently proliferation was measured. Additionally, T-cell cytokine production was measured (by ELISA) upon restimulation with ionomycin/PMA. Results TSLP levels in SF of RA patients were significantly increased compared to OA patients (mean 297 vs. 80 pg/ml, resp., p<0.01). mDCs numbers from SF were significantly increased compared to PB (5.0% vs. 0.6%, resp., p<0.01) and expressed increased levels of TSLPR (MFI 24 vs. 15, resp., p<0.01). TSLP significantly stimulated production of chemokines TARC and MIP1α by mDCs from PB and SF (TARC: PB from 1 to 42 pg/ml, p<0.05 and SF from 26 to 186 pg/ml, p<0.05; MIP1α: PB from 1268 to 5486 pg/ml, p<0.05 and SF from 2776 to 3733 pg/ml, p<0.05). Upon incubation with TSLP, TSLPR-expressing mDCs from PB potently stimulated proliferation of autologous CD4 T cells as compared to unstimulated mDCs (ratio T cell:DC 5:1, from 1503 to 16036 cpm, p<0.01). However, TSLP-mDCs from SF had a strongly increased capacity to activate CD4 T cells (ratio T cell:DC 5:1, from 26395 to 57387 cpm, p<0.05). Enhanced proliferation was associated with increased production of IFNγ (ratio T cell:DC 5:1, PB from 179 to 655 pg/ml, p<0.01 and SF from 601 to 1867 pg/ml, p<0.05), IL-17 (PB from 39 to 353 pg/ml, p<0.05 and SF from 363 to 1382 pg/ml, p<0.05), and IL-4 (PB from 17 to 246 pg/ml, p<0.01 and SF from 193 to 775 pg/ml, n.s.). Conclusions Our data indicate that increased intra-articular TSLP concentrations in RA potently activate TSLPR-expressing mDCs from SF to secrete enhanced levels of proinflammatory mediators causing T cell chemotaxis and to potently increase arthritogenic T cell activation. This...
Background IL-7 is a potent T cell activating cytokine that has been shown to cause proliferation, survival and differentiation of T cells as well as T cell-dependent activation of myeloid cells in numerous conditions. In inflamed tissues of patients with several autoimmune diseases (RA, pSS, psoriasis) increased IL-7 production by tissue cells and immune cells has been documented. Although reduced serum immunoglobulin levels in IL-7R-deficient individuals suggested that IL-7 might play a role in activation of mature human B cells, direct evidence for this is lacking. Previously, it has been demonstrated that EBV, one of the suggested triggers of pSS, induces TLR7-dependent B cell activation and that EBV-transformed B cells express significant levels of the IL-7R as well as IL-7. Furthermore, we have shown that both intracellular IL-7R and IL-7 expression is up regulated in vitro upon TLR7 triggering of B cells. 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 HC (n=7) were co-cultured (1:1) with and without IL-7, TLR7 agonist (TLR7A, Gardiquimod) or the combination of IL-7/TLR7A with and without CD14 monocytes/macrophages (T/B/mono; 1:1:0,1). Proliferation of T and B cells was measured using 3H-thymidine incorporation and by Ki67 expression (FACS analysis). Activation markers (CD19, HLA-DR, CD25) expression were measured by FACS analysis. Results Exogenously added IL-7 did not activate B cells directly, in line with the absence of surface IL-7R. However, in the presence of T cells, IL-7 activated both T and B cells (Ki67+ CD4 cells from 1.1±0.2% to 14.4±3.7%, p<0.01 and Ki67+ B cells from 1.9±0.3% to 4.1±0.9%, p<0.05). TLR7A induced B cell activation, as measured by increased proliferation (%Ki67 from 1.2±0.2% to 9.3±1.4%) and up regulation of activation markers on B cells, which was facilitated in the presence of monocytes. TLR7-induced B cell activation in T/B or T/B/monocyte co-cultures was not associated with T cell activation. IL-7 added to TLR7A synergistically increased both B cell (TLR7A vs. IL-7/TLR7A; 9.3±1.4% vs. 33.4±7.3%) and T cell proliferation (IL-7 vs. IL-7/TLR7A; 0.8±0.1% vs. 29.2±5.2%), which for B cells again was further increased by monocytes (TLR7A vs. IL-7/TLR7A; 30.2±8.9% vs. 63.0±8.0%). Similar results were observed for activation marker expression on B cells (CD19, HLA-DR CD25) and on T cells (HLA-DR, CD25). Conclusions IL-7-induced T cell activation and TLR7A-induced B cell activation synergistically activate B cells, which is markedly enhanced by the presence of monocytes. Our results indicate that previously described increased local expression of IL-7 and TLR7 and increased numbers of macrophages could contribute to enhanced lymphocyte activation in patients with pSS. Disclosure of Interest None Declared
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