M Cristina Lebre

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: A role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte‐derived DC can be differentiated into Th1‐, Th2‐ or Th1/Th2‐promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte‐derived mature Th1/Th2‐, Th1‐ or Th2‐promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1‐ and Th1/Th2‐, but not Th2‐promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP‐1, CCL3/MIP‐1α, CCL4/MIP‐1β and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP‐3β. CCL21/6Ckine is preferentially expressed by Th2‐promoting DC. Production of the Th1‐attracting chemokines, CXCL9/Mig, CXCL10/IP‐10 and CXCL11/I‐TAC, is restricted to Th1‐promoting DC. In contrast, expression of Th2‐associated chemokines does not strictly correlate with the Th2‐promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2‐promoting DC. Because inflammatory chemokines and Th1‐associated chemokines are constitutively expressed by mature Th1‐promoting DC and CCL22/MDC is constitutively expressed by mature Th2‐promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.

Rheumatoid Arthritis Synovium Contains Two Subsets of CD83−DC-LAMP− Dendritic Cells with Distinct Cytokine Profiles

Dendritic cells (DCs) have been proposed to play a pivotal role in the initiation and perpetuation of rheumatoid arthritis (RA) by presentation of arthritogenic antigens to T cells. We investigated the in vivo characteristics of two major DC subsets, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs), in RA synovial tissue (ST) by measuring their frequency, phenotype, distribution, and cytokine expression. ST was obtained by arthroscopy from 20 RA, 8 psoriatic arthritis, and 10 inflammatory osteoarthritis patients. Levels of CD1c(+) mDCs and CD304(+) pDCs present in ST were quantified by digital image analysis, and their distribution was assessed by double immunolabeling with antibodies against CD3 and CD8. The maturation status and cytokine profile of mDCs and pDCs were quantified by double-immunofluorescence microscopy. In RA patients, the number of CD304(+) pDCs exceeded that of CD1c(+) mDCs, with the majority of infiltrating DCs being CD83(-) or DC-LAMP(-). Synovial pDC numbers were especially increased in RA patients who were positive for rheumatoid factor and anti-citrullinated peptide antibody. mDCs and pDCs were localized adjacent to lymphocyte aggregates. In ST from RA patients, both mDCs and pDCs expressed interleukin (IL)-15. IL-18 and interferon (IFN)-alpha/beta were mainly expressed by pDCs whereas IL-12p70 and IL-23p19 expression was predominant in mDCs. These data characterize the phenotypes of mDCs and pDCs in inflammatory synovitis and define for the first time the cytokine expression profile of these DC subsets.

Aberrant function of peripheral blood myeloid and plasmacytoid dendritic cells in atopic dermatitis patients

BACKGROUND Dendritic cells (DCs) can act both as innate cells in host defense and as antigen-presenting cells for naive T cells in adaptive immunity. These functions, among others, are determined by the level of production of particular cytokines. Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by an initial phase predominated by T(H)2 cytokines that switches into a second, more chronic T(H)1-dominated eczematous phase. OBJECTIVE To assess to what extent the AD phenotype is associated with an aberrant phenotype and function of DCs. METHODS Classic CD1c(+)/blood DC antigen (BDCA)-1(+) myeloid (m) DCs and CD304(+)/BDCA4(+) plasmacytoid (p) DCs, the natural IFN-producing cells, were isolated from peripheral blood of patients with AD and healthy controls and analyzed for their phenotype and function. RESULTS Purified CD1c(+)/BDCA1(+) mDCs from patients with AD showed a selective and dramatic reduction of IL-12p70 and TNF-alpha release. IL-12p70 reduction was attributed to a defective expression of both IL-12p35 and IL-12p40 subunits. Accordingly, mature CD1c(+)/BDCA1(+) mDCs from patients with AD induced considerably less IFN-gamma-producing and more IL-4-producing T(H) cells compared with mDCs from healthy controls. In addition, CD304(+)/BDCA4(+) pDCs from patients with AD produced significantly lower levels of IFN-alpha compared with healthy controls. CONCLUSION Myeloid DCs and pDCs from patients with AD show defective IL-12, TNF-alpha, and IFN-alpha production, which may contribute to increased susceptibility to infection and to the maintenance of the T(H)2 cell-mediated allergic state in patients with AD.

A8.9 Rheumatoid arthritis synovial IL-21 + CD4+ t cells specifically induce matrix metalloproteinase production by fibroblast-like synoviocytes

Background/Purpose IL-21 is a cytokine produced by activated CD4+ T cells and T follicular helper cells (TFh) that has been implicated in several autoimmune diseases including rheumatoid arthritis (RA). IL-21 regulates antibody production by B cells and induces osteoclastogenesis, mechanisms that contribute to rheumatoid arthritis (RA) pathology. Importantly, IL-21R blockade ameliorates arthritis in mice. Here we investigated the functional characteristics of synovial CD4+ IL-21 + T cells in RA. Methods Matched peripheral blood (PB) and synovial fluid (SF) from 13 RA and 6 psoriatic arthritis (PsA) patients, and PB of 17 healthy control (HC) subjects were stimulated with PMA/Ionomycin/brefeldin A and intracellular cytokine production assessed by FACS. STAT3-dependent IL-21 production by SF CD4+ T cells was investigated by using a STAT3 specific inhibitor (WP1066). The effects of IL-21 were evaluated on cytokine and matrix metalloproteinase (MMP) release by RA synovial biopsies. In addition, the capacity of sorted RA SF IL-21 + or IL-21-CD4+ T cells in mediator release by fibroblast-like synoviocytes (FLS) was evaluated in co-cultures. IL-21, IL-6 and MMP-1 and MMP-3 concentrations were assessed by ELISA. Results The frequency of both SF IL-21 + CD4+ or IL-21 + TNF-a + CD4+ T cells in RA was significantly higher compared to PsA (p = 0.0140 and p = 0.0038, respectively). STAT3-specific inhibitor blocked significantly the production of IL-21 by SF CD4+ T cells. Synovial IL-21 + CD4+ T cells did not phenotypically fit the TFh cell paradigm in that they did not co-express CXCR5 and ICOS. The levels of SF IL-21 were associated with CRP, MMP-1 and MMP-3. Related to this, IL-21 selectively induced MMP-1 and MMP-3 secretion by RA synovial biopsies. Sorted SF IL-21 + CD4+ T cells induced specifically the release of MMP-1 and MMP-3 by FLS compared to medium (both p < 0.0001) while IL-21-CD4+ T cells were not able to induce these MMPs. In addition, the capacity of IL-21 + and IL-21- CD4+ T cells to induce IL-6 production by FLS was similar. Conclusion The results of this study support the notion that RA IL-21-producing CD4 T cells are involved in promoting joint destruction by inducing MMP release. Therefore IL-21 might be a therapeutic target in RA.

CD40 signaling instructs chronic lymphocytic leukemia cells to attract monocytes via the CCR2 axis

Chronic lymphocytic leukemia (CLL) cells are provided with essential survival and proliferative signals in the lymph node microenvironment. Here, CLL cells engage in various interactions with bystander cells such as T cells and macrophages. Phenotypically distinct types of tumor infiltrating macrophages can either be tumor supportive (M2) or play a role in tumor immune surveillance (M1). Although recent in vitro findings suggest a protective role for macrophages in CLL, the actual balance between these macrophage subsets in CLL lymphoid tissue is still unclear. Furthermore, the mechanism of recruitment of monocytes towards the CLL lymph node is currently unknown. Both questions are addressed in this paper. Immunofluorescence staining of lymph node samples showed macrophage skewing towards an M2 tumor-promoting phenotype. This polarization likely results from CLL-secreted soluble factors, as both patient serum and CLL-conditioned medium recapitulated the skewing effect. Considering that CLL cell cytokine secretion is affected by adjacent T cells, we next studied CLL-mediated monocyte recruitment in the presence or absence of T-cell signals. While unstimulated CLL cells were inactive, T cell-stimulated CLL cells actively recruited monocytes. This correlated with secretion of various chemokines such as C-C-motif-ligand-2,3,4,5,7,24, C-X-C-motif-ligand-5,10, and Interleukin-10. We also identified CD40L as the responsible T-cell factor that mediated recruitment, and showed that recruitment critically depended on the C-C-motif-chemokine-receptor-2 axis. These studies show that the shaping of a tumor supportive microenvironment depends on cytokinome alterations (including C-C-motif-ligand-2) that occur after interactions between CLL, T cells and monocytes. Therefore, targeted inhibition of CD40L or C-C-motif-chemokine-receptor-2 may be relevant therapeutic options.

Indoor pollutant hexabromocyclododecane enhances house dust mite-induced activation of human monocyte-derived dendritic cells

Abstract The indoor pollutant hexabromocyclododecane (HBCD) has been added as flame retardant to many consumer products but detaches and accumulates in house dust. Inhalation of house dust leads to exposure to house dust mite (HDM) allergens in the presence of HBCD. Activation of dendritic cells is crucial in the sensitization to HDM allergens. The current study examined whether exposure to HBCD affected activation/maturation of HDM-exposed human dendritic cells (DC). Human monocyte-derived DC (moDC) were exposed simultaneously to HDM and a concentration range of HBCD (0.1–20 μM) in vitro. HDM exposure of moDC induced expression of co-stimulatory molecule CD80 and production of pro-inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. However, simultaneous exposure of moDC to HBCD and HDM enhanced the expression of antigen presenting molecule HLA-DR, co-stimulatory molecule CD86 and pro-inflammatory cytokine IL-8 depending on the dose of HBCD. Our results indicate that simultaneous exposure of HDM and HBCD can enhance the antigen presentation and maturation/activation of DC.

A3.16 Specific Overexpression of Synovial Interleukin-21+ CD4+ T Cells Co-Expressing Tumour Necrosis Factor in Rheumatoid Arthritis: Role in Joint Destruction?

Background and Objectives IL-21 is a cytokine produced by activated CD4+ T cells and T follicular helper cells (TFh) that has been implicated in several autoimmune diseases. IL-21 regulates antibody production by B cells and induces osteoclastogenesis, mechanisms that contribute to rheumatoid arthritis (RA) pathology. Importantly, IL-21R blockade ameliorates arthritis in mice. Here we investigated the functional characteristics of CD4+IL-21+ T cells in RA. Materials and Methods We evaluated the expression of surface markers and cytokine production in matched peripheral blood (PB) and synovial fluid (SF) from 13 RA and 6 psoriatic arthritis (PsA) patients, and PB of 17 healthy control (HC) subjects by flow cytometry following PMA/Ionomycin stimulation ex-vivo. IL-21 concentrations were assessed by ELISA in cell-free SF samples of RA (n = 15), PsA (n = 14) and OA (n = 6) patients and in synovial biopsy culture supernatants (6 days) of RA (n = 6) and ankylosing spondylitis (AS; n = 5) patients. The effects of IL-21 were evaluated on cytokine and matrix metalloproteinase (MMP) release by RA synovial biopsies. Results The frequency of IL-21+CD4+ T cells in RA and PsA SF was significantly higher compared to PB (P < 0.0001 and P < 0.0001, respectively). Moreover, the frequency of both total IL-21+CD4+ (P = 0.0140) and IL-21+TNF+CD4+ T cells (P = 0.0038) in RA SF were significantly increased compared to PsA patients. The frequency of IL-21+CD4+ T cells in RA PB was positively correlated with DAS28 (r = 0.592, P = 0.033), serum anti-cyclic citrullinated peptide (anti-CCP) antibodies (r = 0.788, P = 0.001) and IgM-rheumatoid factor (IgM-RF; r = 0.691, P = 0.009). In addition, the percentage of IL-21+CD4+ T cells in anti-CCP+ or IgM-RF+ patients was significantly higher compared to anti-CCP- (P = 0.03) and IgM-RF- (P = 0.01) patients respectively. In addition to PB and SF, we found IL-21+CD4+ T cells present in RA synovial tissue. Significant higher levels of IL-21 in RA SF compared to OA SF (P = 0.04) were observed confirming previous observations. RA synovial biopsies released significantly higher levels of IL-21 compared to biopsies from AS patients. The levels of IL-21 in SF were associated with CRP and with the MMP-1 and MMP-3. Related to this, IL-21 selectively induced MMP-1 and MMP-3 secretion by RA synovial biopsies. Synovial IL-21+CD4+ T cells did not phenotypically fit the TFh cell paradigm in that they did not express CXCR5 and ICOS. Conclusions The results of this study support the notion that IL-21-producing CD4 T cells are involved in promoting synovial inflammation (TNF) and joint destruction (MMP) in RA and might be a therapeutic target in this disease.

Mice lacking Flt3L are protected from collagen-induced arthritis: impaired T cell function?

Backgroundand objectives Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. The induction and maintenance of T cell tolerance to tissue antigens is essential to prevent autoimmunity. Combinations of central and peripheral mechanisms act in parallel to inactivate, eliminate or control autoreactive T cells. A key requirement for tolerance is the presentation of antigens in a correct context. Dendritic cells (DCs) are the central antigen-presenting cells (APCs) for the initiation of T cell responses. In this context, stimulation of the Flt3 via Flt3L is known to drive expansion and differentiation of DCs. Traditional approaches to treatment of autoimmune diseases through immunosuppression have focused on direct inhibition of T cells. In the present study, the authors examined the targeted inhibition of APCs as a mean to downregulate /prevent autoimmune disease in a mouse model for rheumatoid arthritis. Materials and methods Collagen-induced arthritis (CIA) was induced in mice lacking Flt3L (Flt3L−/−) and WTlittermates (C57/BL6 background, 9–10 weeks old). The severity of the arthritis was assessed using an established semiquantitative scoring system (0–4). After 60 days (chronic phase) phenotypical and functional analysis of spleen and lymph nodes was performed: T and B cell markers, FoxP3 expression, activation markers, co-stimulatory markers and cytokine production. Results In steady-state, Flt3L−/− mice show reduced celularity in both spleen (p=0.007) and lymph nodes (p=0.01) and reduced T and B cell numbers compared with WT. In CIA abrogation of Flt3 signaling led to decreased disease incidence and severity. As shown for steady-state conditions, in CIA Flt3L-/- mice showed reduced spleen and lymph node cellularity (p<0.0001) but also reduced percentage of CD4+CD25+ T cells compared with WT (p=0.03). Flt3L−/− CD4+ T cells also produce significantly less IL-17 (p=0.016) and tumour necrosis factor α (p=0.010), and CD8+ T cells less interferon (IFNγ) (p=0.029) compared to WT. Conclusions Mice lacking Flt3L are protected from collagen-induced arthritis. The authors observed that Flt3L deletion influences the magnitude (cell numbers) and quality (CD25 expression) of T cell responses. Stimulation of lymphocytes by different types of DC, DC at different stages of maturity and producing and responding to different growth factors might contribute for this change in T cell numbers and/or effector functions in Flt3L−/− mice. Targeting this signalling pathway might be considered as a good therapeutic strategy in RA.

Flt3 inhibition leads to decreased production of IL-6 by synovial biopsies and fibroblast-like synoviocytes by reducing NFKb activity

Background Rheumatoid arthritis (RA) is characterized by inflammation and hyperplasia of the synovial membrane which ultimately results in erosion of cartilage and bone. Fibroblast-like synoviocytes (FLS) play ac ritical role in this destructive process by producing inflammatory cytokines. Interleukin 6 (IL-6) is a key cytokine in the pathobiology of RA and biological therapies targeting the IL-6 receptor have shown clinical benefit. The Fmslike tyrosine kinase 3 (Flt3) is a membrane bound tyrosine kinase receptor which has a crucial role in hematopoiesis, regulating cellular differentiation, proliferation and apoptosis. Both Flt3L and its receptor are strongly expressed in RA and it has been shown that this axis exerts both pro-inflammatory and tissue destructive properties once in the joint cavity. Therefore we examined the effect(s) of a specific Flt3 inhibitor on IL-6 production by RA synovial biopsies and FLS. Methods

TNFα and chemerin cross-talk in rheumatoid arthritis

Background and objectives Rheumatoid arthritis (RA) synovium is characterised by a dense infiltrate, consisting of macrophages, T and B cells, plasma cells and dendritic cells (DC). Inflammatory chemokines present in RA synovium may contribute to the accumulation of these immune cells. The authors have recently shown that plasmacytoid DC (pDC) are enriched in RA synovial tissue (ST) compared to CD1c myeloid DC. In line with these observations, the authors have shown that chemerin (and its receptor ChemR23) expression is upregulated in RA ST compared to non-RA arthritis patients. Moreover, in RA ST ChemR23 was specifically expressed by CD68 macrophages and pDC, while chemerin expression was confined to endothelial cells (CD31 and von Willebrand factor positive). Therefore the authors aimed at investigating the regulation of chemerin expression in an ex vivo model of human RA. Materials and methods Arthroscopic ST biopsies were obtained from patients with active RA and cultured in medium or in the presence of recombinant (r)-tumour necrosis factor α (TNFα) or r-chemerin. After 6 days, cell-free supernatants were harvested and the levels of TNFα or chemerin were analysed by Luminex or ELISA, respectively. When indicated, antichemerin or anti-ChemR23 neutralising antibodies were added to TNFα-stimulated cultures. Results RA synovial biopsies released chemerin spontaneously. Interestingly, TNFα stimulation induced significantly higher levels of chemerin compared to medium control. In addition, RA synovial biopsies released TNFα spontaneously and addition of chemerin to the cultures strongly induced TNFα release, suggesting a vicious cycle. Of importance, spontaneous and TNFα-induced chemerin could be blocked by the addition of neutralising antibodies against chemerin. Moreover, spontaneous TNFα could also be blocked by the addition of neutralising antibodies against chemerin. Conclusions These findings suggest that elevated levels of chemerin in RA ST might regulate local TNFα release and vice-versa in a positive feedback loop. The reciprocal interplay between chemerin and TNFα is novel and might represent an attractive candidate for future drug development by blocking the chemerin/ChemR23 system to disrupt disease perpetuation.