Saida Aarrass

Transcriptional Profiling Reveals Novel Markers of Liver Fibrogenesis GREMLIN AND INSULIN-LIKE GROWTH FACTOR-BINDING PROTEINS

Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression, representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. Serial analysis of gene expression was performed on RNA isolated from quiescent, activated, and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor-binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice, whereas it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs it could be a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver.

Heterogeneous expression pattern of interleukin 17A (IL-17A), IL-17F and their receptors in synovium of rheumatoid arthritis, psoriatic arthritis and osteoarthritis: possible explanation for nonresponse to anti-IL-17 therapy?

IntroductionAccumulating evidence suggests an important role for interleukin 17 (IL-17) in the pathogenesis of several inflammatory diseases, including rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Accordingly, clinical trials aimed at blocking IL-17 have been initiated, but clinical results between patients and across different diseases have been highly variable. The objective was to determine the variability in expression of IL-17A, IL-17F and their receptors IL-17RA and IL-17RC in the synovia of patients with arthritis.MethodsSynovial biopsies were obtained from patients with RA (n = 11), PsA (n = 15) and inflammatory osteoarthritis (OA, n = 14). For comparison, synovia from noninflamed knee joints (n = 7) obtained from controls were included. Frozen sections were stained for IL-17A, IL-17F, IL-17RA and IL-17RC and evaluated by digital image analysis. We used confocal microscopy to determine which cells in the synovium express IL-17A and IL-17F, double-staining with CD4, CD8, CD15, CD68, CD163, CD31, von Willebrand factor, peripheral lymph node address in, lymphatic vessel endothelial hyaluronan receptor 1, mast cell tryptase and retinoic acid receptor-related orphan receptor γt (RORγt).ResultsIL-17A, IL-17F, IL-17RA and IL-17RC were abundantly expressed in synovial tissues of all patient groups. Whereas IL-17RA was present mostly in the synovial sublining, IL-17RC was abundantly expressed in the intimal lining layer. Digital image analysis showed a significant (P < 0.05) increase of only IL-17A in arthritis patients compared to noninflamed control tissues. The expression of IL-17A, IL-17F and their receptors was similar in the different patient groups, but highly variable between individual patients. CD4+ and CD8+ cells coexpressed IL-17A, and few cells coexpressed IL-17F. IL-17A and IL-17F were not expressed by CD15+ neutrophils. Mast cells were only occasionally positive for IL-17A or IL-17F. Interestingly, IL-17A and IL-17F staining was also observed in macrophages, as well as in blood vessels and lymphatics. This staining probably reflects receptor-bound cytokine staining. Many infiltrated cells were positive for the transcription factor RORγt. Colocalisation between RORγt and IL-17A and IL-17F indicates local IL-17 production.ConclusionsIncreased expression of IL-17A is not restricted to synovial tissues of RA and PsA patients; it is also observed in inflammatory OA. The heterogeneous expression levels may explain nonresponse to anti-IL-17 therapy in subsets of patients.

FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis in rheumatoid arthritis

IntroductionThe FMS-related tyrosine kinase 3 ligand (Flt3L)/CD135 axis plays a fundamental role in proliferation and differentiation of dendritic cells (DCs). As DCs play an important role in rheumatoid arthritis (RA) immunopathology we studied in detail the Flt3L/CD135 axis in RA patients.MethodsThe levels of Flt3L in (paired) serum and synovial fluid (SF) were quantified by enzyme-link immunosorbent assay (ELISA). Expression of Flt3L and CD135 in paired peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) was quantified by fluorescence-activated cell sorting (FACS). The expression of Flt3L, CD135 and TNF-Converting Enzyme (TACE) in synovial tissues (STs) and in vitro polarized macrophages and monocyte-derived DCs (Mo-DCs) was assessed by quantitative PCR (qPCR). CD135 ST expression was evaluated by immunohistochemistry and TACE ST expression was assessed by immunofluorescence. Flt3L serum levels were assessed in RA patients treated with oral prednisolone or adalimumab.ResultsFlt3L levels in RA serum, SF and ST were significantly elevated compared to gout patients and healthy individuals (HI). RA SF monocytes, natural killer cells and DCs expressed high levels of Flt3L and CD135 compared to HI. RA ST CD68+ and CD163+ macrophages, CD55+ fibroblast-like synoviocytes (FLS), CD31+ endothelial cells or infiltrating monocytes and CD19+ B cells co-expressed TACE. IFN-γ-differentiated macrophages expressed higher levels of Flt3L compared to other polarized macrophages. Importantly, Flt3L serum levels were reduced by effective therapy.ConclusionsThe Flt3L/CD135 axis is active in RA patients and is responsive to both prednisolone and adalimumab treatment. Conceivably, this ligand receptor pair represents a novel therapeutic target.

Synovial IL-21/TNF-producing CD4+ T cells induce joint destruction in rheumatoid arthritis by inducing matrix metalloproteinase production by fibroblast-like synoviocytes

Bone and cartilage destruction is one of the key manifestations of rheumatoid arthritis (RA). Although the role of T helper (Th)17 cells in these processes is clear, the role of IL‐21–producing cells T cells has been neglected. We sought to investigate the role of IL‐21 in RA by focusing on the functional characteristics of the main producers of this cytokine, synovial CD4+IL‐21+ T cells. We show that the frequency of both synovial fluid (SF) CD4+IL‐21+ or CD4+IL‐21+TNF+ T cells in patients with RA was significantly higher compared with patients with psoriatic arthritis (PsA). The frequency of peripheral blood (PB) IL‐21+CD4+ T cells in patients with RA positively correlated with disease activity score 28 (DAS28), serum anticyclic citrullinated peptide (anti‐CCP) antibodies and IgM‐rheumatoid factor (IgM‐RF). IL‐21 levels in RA SF were associated with matrix metalloproteinase (MMP)‐1 and MMP‐3. Related to this, IL‐21 induced significantly the secretion of MMP‐1 and MMP‐3 in RA synovial biopsies. Sorted SF CD4+IL‐21+ T cells significantly induced the release of MMP‐1 and MMP‐3 by fibroblast‐like synoviocytes (FLS) compared with medium or CD4+IL‐21− T cells in a coculture system. Neutralization of both IL‐21 and TNF resulted in significantly less production of MMP by FLS. The results of this study indicate a new role for synovial CD4+IL‐21+TNF+ T cells in promoting synovial inflammation/joint destruction in patients with RA. Importantly, IL‐21 blockade in combination with anti‐TNF might be an effective therapy in patients with RA by inhibiting MMP‐induced inflammation/joint destruction.

Absence of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling protects against collagen-induced arthritis

Objective Comprehending the mechanisms that regulate activation of autoreactive T cells and B cell antibody production is fundamental for understanding the breakdown in self-tolerance and development of autoimmunity. Here we studied the role of Fms-like tyrosine kinase 3 ligand (Flt3L) signalling in the pathogenesis of collagen-induced arthritis (CIA). Methods CIA was induced in mice lacking Flt3L (Flt3L−/−) and wild-type (WT) littermates (C57/BL6, 8–10 weeks old). Mice were killed in the initial phase (acute phase: experiment 1) and late phase (chronic phase: experiment 2) of the disease. Arthritis severity was assessed using a semiquantitative scoring system (0–4), and histological analysis of cellular infiltration, cartilage destruction and peptidoglycan loss was performed. Phenotypic and functional analysis of T and B cells, FoxP3 expression, activation and lymphocyte costimulatory markers, and cytokine production were performed ex vivo by flow cytometry in lymph nodes. Serum collagen type II (CII)-specific antibodies were measured by ELISA. Results Flt3L−/− mice showed a marked decrease in clinical arthritis scores and incidence of arthritis in both acute and chronic phases of CIA compared with WT mice. Moreover, decreased synovial inflammation and joint destruction was observed. Both the magnitude and quality of T cell responses were altered in Flt3L−/−. In the acute phase, the amount of CII-specific IgG2a antibodies was lower in Flt3L−/− than WT mice. Conclusions These results strongly suggest a role for Flt3L signalling in the development of arthritis.

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.

A8.19 Non-lymphoid CD103+ dendritic cells are required for the initiation of collagen-induced arthritis

Background and Objectives Dendritic cells (DCs) are essential for the initiation of synovial inflammation, but it is unclear which subset of DCs performs this task. We have shown that FMS-like tyrosine kinase 3 ligand (Flt3L)-dependent DCs are important for disease development in a mouse model for RA. However since Flt3L-/- mice have reductions in all conventional (c)DCs it remained unclear which subset is required for disease induction. Materials and Methods CIA was induced in Flt3L-/-, Batf3-/- (mice lacking both CD103+ and CD8a+DCs) and WT C57/BL6 littermates. In vitro and in vivo uptake and migration was performed using bone marrow (BM)-DCs and dermal DCs. Antigen presentation was studied in vivo by adoptive transfer of CFSE-labeled OT-I or OT-II T cells + OVA in Flt3L-/- and WT mice and in vitro by culturing BM-DCs with OT-I and OT-II cells. T cell proliferation was analysed by CFSE dilution. To study BM-DC function qPCR array (Dendritic and Antigen Presenting Cell PCR Array- Qiagen) for 84 genes was performed. To test the potential of a Flt3 inhibitor (CEP701) in the prevention of CIA, an in vivo study was performed injecting CEP701 before the onset of disease in DBA-1 mice. Results Flt3L-/- mice were susceptible to innate K/BxN arthritic model and arthritic T cell transfer. Batf3-/- mice lacking both CD103 + and CD8a + DCs were resistant to CIA, demonstrating that CD11b + and monocyte-derived DCs (moDCs) were not sufficient to induce CIA. The amount of DCs carrying antigen reaching the LN in Flt3L-/- mice was reduced compared with WT. Uptake and migratory capacity was similar in Flt3L-/- BM-DCs compared to WT BM-DCs. CEP701 (a Flt3L inhibitor) treatment prevented CIA induction, and reduced dramatically CD103 + DCs in the lymph nodes and synovium. Human CD141hi DCs, homologues of mouse CD103 + DCs, were present and increased in inflamed rheumatoid arthritis (RA) synovium. Conclusions Antigen presentation in Flt3L-/- mice is impaired. As CD103 + DCs are absent in Flt3L-/- mice and are important in (cross)-presenting antigens our data reveals a crucial role for CD103 + DCs in the induction of CIA. As a consequence of CD103 + DC absence Flt3L-/- mice showed a reduction in T cell activation in particularly reduced CD8 T cell responses. Hence this study identified non-lymphoid CD103 + DCs as the main subset orchestrating the initiation of cell-mediated immunity in arthritis. Targeting this DC subset might be of interest in individuals at risk of developing autoimmune disorders.

A9.4 Flt3L-Dependent CD103+ DC are Crucial for the Initiation and Maintenance of Collagen-Induced Arthritis

Background and Objectives Dendritic cells (DCs) are a heterogeneous population of antigen-sensing and-presenting cells that play an important role for the initiation of protective and pathogenic immunity. Flt3L is a growth factor that can drive DC development from bone marrow (BM) progenitors and it is crucial for steady-state DC maintenance. Collagen-induced arthritis (CIA) induction requires dermal DC uptake, processing, migration and presentation of antigen to lymph node (LN) T cells. The objective of this study was to find the mechanism of protection in CIA observed in mice lacking Flt3L (Flt3L-/-). Materials and Methods CIA was induced in Flt3L-/- and WT littermates. The severity of the arthritis was assessed using an established semiquantitative scoring system (0–4). In vitro uptake and migration was assessed by using BM-DC (differentiated with GM-CSF for 9 days). OVA-FITC was injected intradermally in Flt3L-/- and WT animals, after 36 h LNs were collected and the number of FITC positive cells was determined. Antigen presentation was studied by adoptive transfer of CFSE-labelled OT-I or OT-II T cells + OVA (i.d.) into Flt3L-/- and WT animals. After 3 days LNs were collected and T cell proliferation was analysed by CFSE dilution. Results Flt3L-/- mice (steady-state) have severe reductions in DC populations in lymphoid and non-lymphoid organs. Flt3L-/- mice are protected from CIA showing impaired T and B cell responses. Flt3L-/- mice have reduced dermal CD11b+ DCs and CD103+ DCs are almost absent. In addition, the amount of DCs carrying antigen reaching the LN in Flt3L-/- mice was reduced compared with WT. In vitro and in vivo experiments using BMDCs demonstrated that Flt3L-/- BMDCs have the same capacity to uptake and migrate as WT BMDCs. Moreover, adoptive transfer of OT-I and OT-II T cells + OVA in Flt3L-/- mice resulted in a dramatic reduction of total cell proliferation and more importantly less divisions compared with WT animals. FACS analysis of WT and Flt3L-/- mice synovium at the acute phase of CIA showed that CD11b+ DC are present and increased in arthritic animals compared with immunised but not arthritic animals. CD103+ DC are only present in WT animals, and increased in animals with a high clinical score. Conclusions Our data shows that antigen presentation in Flt3L-/- mice is impaired. As CD103+ DC are important in presenting and cross-presenting antigens our data reveals an important role for CD103+ DC in both induction and maintenance of CIA. Specifically targeting CD103+ DC could provide a novel antirheumatic strategy.

Expansions of interleukin 21-secreting CD4 T helper cells in inflammatory arthritides

Rheumatoid arthritis (RA) is a chronic inflammatory disease affecting synovial tissue in multiple joints. The inflammatory process in RA is regulated by several cytokines, especially tumour necrosis factor (TNF), which is produced not only by macrophages and dendritic cells but also by activated antigen-specific CD4 T helper (Th) cells. An increasing number of inflammatory molecules are being identified that may contribute to RA pathology. Therefore the authors investigated the expression of interleukin 17A (IL17A), IL21 …

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.