B. Walgreen

Shift from toll-like receptor 2 (TLR-2) toward TLR-4 dependency in the erosive stage of chronic streptococcal cell wall arthritis coincident with TLR-4-mediated interleukin-17 production.

OBJECTIVEnToll-like receptors (TLRs) may activate innate and adaptive immune responses in rheumatoid arthritis (RA) through recognition of microbial as well as endogenous ligands that have repeatedly been found in arthritic joints. The objective of this study was to investigate the involvement of TLR-2 and TLR-4 in the development of chronic destructive streptococcal cell wall (SCW)-induced arthritis, in which interleukin-1 (IL-1)/IL-17-dependent T cell-driven pathologic changes replace the macrophage-driven acute phase.nnnMETHODSnChronic SCW arthritis was induced by 4 repeated intraarticular injections of SCW fragments in wild-type, TLR-2(-/-), and TLR-4(-/-) mice. Clinical, histopathologic, and immunologic parameters of arthritis were evaluated.nnnRESULTSnThe TLR-2 dependency of joint swelling during the acute phase was shifted to TLR-4 dependency during the chronic phase. Persistent joint inflammation in the latter phase of the model was significantly suppressed in TLR-4(-/-) mice. In the chronic phase, TLR-4 actively contributed to matrix metalloproteinase (MMP)-mediated cartilage destruction and to osteoclast formation, since the expression of the MMP-specific aggrecan neoepitope VDIPEN and the osteoclast marker cathepsin K was significantly reduced in TLR-4(-/-) mice. Furthermore, TLR-4(-/-) mice expressed less IL-1beta, tumor necrosis factor alpha, IL-6, and IL-23, cytokines that are implicated in IL-17 production. Accordingly, SCW-specific IL-17 production was found to be dependent on TLR-4 activation, since T cells from arthritic TLR-4(-/-) mice produced markedly less IL-17 upon SCW stimulation, whereas interferon-gamma production remained unaffected.nnnCONCLUSIONnThese data indicate the involvement of TLR-4 in the chronicity and erosive character of arthritis coincident with the antigen-specific IL-17 response. The high position of TLR-4 in the hierarchy of erosive arthritis provides an interesting therapeutic target for RA.

Periodontal pathogens directly promote autoimmune experimental arthritis by inducing a TLR2- and IL-1-driven Th17 response

Increasing epidemiologic evidence supports a link between periodontitis and rheumatoid arthritis. The actual involvement of periodontitis in the pathogenesis of rheumatoid arthritis and the underlying mechanisms remain, however, poorly understood. We investigated the influence of concomitant periodontitis on clinical and histopathologic characteristics of T cell–mediated experimental arthritis and evaluated modulation of type II collagen (CII)–reactive Th cell phenotype as a potential mechanism. Repeated oral inoculations of periodontal pathogens Porphyromonas gingivalis and Prevotella nigrescens induced periodontitis in mice, as evidenced by alveolar bone resorption. Interestingly, concurrent periodontitis induced by both bacteria significantly aggravated the severity of collagen-induced arthritis. Exacerbation of arthritis was characterized by increased arthritic bone erosion, whereas cartilage damage remained unaffected. Both P. gingivalis and P. nigrescens skewed the CII-specific T cell response in lymph nodes draining arthritic joints toward the Th17 phenotype without affecting Th1. Importantly, the levels of IL-17 induced by periodontal pathogens in CII-specific T cells directly correlated with the intensity of arthritic bone erosion, suggesting relevance in pathology. Furthermore, IL-17 production was significantly correlated with periodontal disease–induced IL-6 in lymph node cell cultures. The effects of the two bacteria diverged in that P. nigrescens, in contrast to P. gingivalis, suppressed the joint-protective type 2 cytokines, including IL-4. Further in vitro studies showed that the Th17 induction strongly depended on TLR2 expression on APCs and was highly promoted by IL-1. Our data provide evidence of the involvement of periodontitis in the pathogenesis of T cell–driven arthritis through induction of Ag-specific Th17 response.

Destructive role of myeloid differentiation factor 88 and protective role of TRIF in interleukin-17-dependent arthritis in mice.

OBJECTIVEnIncreasing evidence indicates the involvement of Toll-like receptors (TLRs) in the progression of arthritis; however, the contribution of the two signaling pathways used by TLRs, which are mediated by myeloid differentiation factor 88 (MyD88) and TRIF, remains unclear. The objective of this study was to investigate the specific roles of MyD88 and TRIF in chronic experimental arthritis and the accompanying adaptive immune responses.nnnMETHODSnChronic arthritis was induced in wild-type, MyD88(-/-) , and Trif(lps2) (TRIF(-/-) ) mice by repetitive intraarticular injections of streptococcal cell wall (SCW) fragments. SCW-specific T cell and B cell responses, joint swelling, and histopathologic changes were analyzed during chronic arthritis.nnnRESULTSnBoth MyD88 and TRIF pathways contributed to antigen-specific T cell proliferation and antibody production, with the MyD88 pathway playing the dominant role. The severity of joint swelling and synovial inflammation, as well as the histopathologic damage to cartilage and bone, was strongly dependent on MyD88 signaling, whereas TRIF was redundant. MyD88 signaling was critical for the development of pathogenic T cell response (i.e., interleukin-17 [IL-17] production) in response to SCW antigen. Interestingly, when the T cell-dependent phase was prolonged, TRIF signaling appeared to down-regulate bone erosion, an effect accompanied by an inhibitory effect on IL-17 production.nnnCONCLUSIONnThis study reveals a central role of MyD88 and a counterregulatory function of TRIF in T cell-driven arthritis. The findings provide a rationale for a pathway-specific interference in order to block the pathogenic features and to preserve or stimulate the beneficial aspects of TLR signaling.

Different amplifying mechanisms of interleukin-17 and interferon-gamma in Fcgamma receptor-mediated cartilage destruction in murine immune complex-mediated arthritis.

OBJECTIVEnPreviously, we reported that interferon-gamma (IFNgamma) aggravates cartilage destruction in immune complex (IC)-mediated arthritis via up-regulation of activating Fcgamma receptors (FcgammaR). Recently, we found that interleukin-17 (IL-17) also aggravates cartilage destruction in arthritis models in which ICs are involved, but the underlying mechanism remains unknown. This study was undertaken to determine the role of IL-17 in FcgammaR-mediated cartilage destruction in IC-mediated arthritis and to compare its effect with that of IFNgamma.nnnMETHODSnIC-mediated arthritis was passively induced in gamma-chain(-/-) mice, which lack functional activating FcgammaR, and in wild-type controls. AdIL-17 or a control vector was injected into the knee joints 1 day prior to induction of IC-mediated arthritis. Knee joints were isolated for histologic analysis, and synovium samples were obtained for reverse transcriptase-polymerase chain reaction (RT-PCR). Macrophage (RAW 264.7) cell lines and polymorphonuclear cell (PMN; 32Dcl3) lines were stimulated with IFNgamma or IL-17 for analysis of FcgammaR expression using RT-PCR and fluorescence-activated cell sorting.nnnRESULTSnIL-17 overexpression prior to induction of IC-mediated arthritis significantly aggravated cartilage destruction and inflammation, characterized by a massive influx of PMNs, which adhered to the cartilage surface. Although IL-17 overexpression increased FcgammaR messenger RNA levels in the synovium, in vitro stimulation of macrophages and PMNs revealed that, in contrast to IFNgamma, IL-17 did not directly regulate FcgammaR expression. Despite similar inflammation in AdIL-17-enhanced IC-mediated arthritis in gamma-chain(-/-) mice and wild-type controls, severe cartilage destruction and PMN adherence were completely absent in gamma-chain(-/-) mice.nnnCONCLUSIONnOur findings indicate that IL-17-mediated aggravation of cartilage destruction in IC-mediated arthritis is FcgammaR dependent. However, in contrast to IFNgamma, which directly up-regulates FcgammaR expression on macrophages and PMNs, IL-17 enhances cartilage destruction by increasing the local amount of FcgammaR-bearing neutrophils.

Toll-like receptor 2 controls acute immune complex-driven arthritis in mice by regulating the inhibitory Fcγ receptor IIB.

OBJECTIVEnPrevious studies have demonstrated a protective role of Toll-like receptor 2 (TLR-2) and a proinflammatory function of TLR-4 during chronic T cell-driven arthritis. The involvement of TLRs in T cell-independent arthritic processes, however, remains unclear. This study was undertaken to determine the functional significance of TLR-2 and TLR-4 in T cell-independent immune complex-driven arthritis.nnnMETHODSnSerum-transfer arthritis was induced in wild-type and TLR-deficient mice by intraperitoneal injections of arthritogenic K/BxN mouse serum. Arthritis was assessed macroscopically and by histologic analysis. The influence of TLR-2 on macrophage cytokine profile, Fcγ receptor (FcγR) expression, and response to immune complexes was determined.nnnRESULTSnWhile TLR-4, unexpectedly, did not play any significant role, TLR-2 deficiency accelerated the onset and markedly increased the severity of acute immune complex-driven arthritis in mice. TLR-2 deficiency resulted in a substantial increase in joint inflammation, bone erosion, and cartilage pathology, indicating a protective function of TLR-2 in passive FcγR-driven disease. Ex vivo study of the macrophage inflammatory phenotype revealed increased production of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) despite similar levels of IL-10, along with a significant increase in FcγR-specific response, in TLR-2-/- mouse macrophages early in the disease. Although distinct FcγR messenger RNA expression was not affected, cell surface protein expression of the inhibitory FcγRIIB in TLR-2-/- naive primary macrophages was specifically diminished, resulting in a higher proinflammatory response. Accordingly, TLR-2 stimulation specifically up-regulated FcγRIIB, but not the activating FcγR, on macrophages.nnnCONCLUSIONnTLR-2 regulates acute immune complex-driven arthritis by controlling macrophage FcγR response. Our findings indicate that the protective role of TLR-2 is extended beyond its previously described role in promoting Treg cells during T cell-mediated arthritis.

Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis

Th17 cells and their cytokines are linked to the pathogenesis of rheumatoid arthritis, a chronic autoimmune disease characterized by joint inflammation. Th17 development is initiated by combined signaling of TGF-β and IL-6 or IL-21, and can be reduced in the absence of either IL-6 or IL-21. The aim of this study was to assess whether combinatorial IL-6/IL-21 blockade would more potently inhibit Th17 development, and be more efficacious in treating arthritis than targeting either cytokine. We assessed in vitro Th17 differentiation efficacy in the absence of IL-6 and/or IL-21. To investigate in vivo effects of IL-6/IL-21 blockade on Th17 and arthritis development, antigen-induced arthritis (AIA) was induced in IL-6-/- x IL-21R-/- mice. The therapeutic potential of this combined blocking strategy was assessed by treating mice with collagen-induced arthritis (CIA) with anti-IL-6R antibodies and soluble (s)IL-21R.Fc. We demonstrated that combined IL-6/IL-21 blocking synergistically reduced in vitro Th17 differentiation. In mice with AIA, absence of IL-6 and IL-21 signaling more strongly reduced Th17 levels and resulted in stronger suppression of arthritis than the absence of either cytokine. Additionally, anti-IL-6/anti-IL-21 treatment of CIA mice during the arthritis induction phase reduced disease development more potent than IL-6 or IL-21 inhibition alone, as effective as anti-TNF treatment. Collectively, these results suggest dual IL-6/IL-21 inhibition may be a more efficacious therapeutic strategy compared to single cytokine blockade to suppress arthritis development.

A1.14 Synovial macrophages promote TGF-β signalling but protect against influx of S100A8/S100A9-producing cells after intra-articular injections of oxidised low-density lipoproteins

Background and objectives LDL in inflamed synovium is oxidised and taken-up by macrophages, leading to an activated macrophage phenotype. In this study, we investigate whether injection of oxLDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Materials and Methods Synovium was obtained from end-stage OA patients. Murine knee joints were injected five consecutive days with oxLDL, LDL, or vehicle (PBS). This procedure was repeated in mice depleted of synovial lining macrophages by intraarticular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry, flow cytometry (FCM) and synovial RNA expression and protein production. Results Synovial macrophages and fibroblast of OA patients showed extensive accumulation apolipoprotein B, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs, but did not induce catabolic or inflammatory processes. In contrast, repeated injections of specifically oxLDL in macrophage-depleted knee joints led to increased synovial thickening. Furthermore, protein and RNA levels of CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections and FCM-analyses revealed increased presence of monocytes and neutrophils in the synovium, which was confirmed by immunohistochemistry. Also protein levels of S100A8/A9 were significantly increased in synovial wash-outs of oxLDL-injected joints, as was expression of aggrecanase-induced neo-epitopes. Interestingly, no raise in active TGF-β was measured in macrophage-depleted joints. Conclusions Synovial macrophages promote anabolic processes after oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity in combination with increased influx of monocytes and neutrophils.

OP0210 Apolipoprotein E Aggravates Inflammation and Joint Destruction in Murine Antigen-Induced Arthritis

Background Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by severe bone destruction which has been associated with altered lipid metabolism. Apolipoprotein E (Apo E) is a lipoprotein crucial in lipid metabolism, mainly produced by macrophages. Moreover ApoE has been recently described as an important anti-inflammatory mediator regulating innate immunity and bone turnover. Objectives In the present study we investigated the role of Apo E in inflammation and bone destruction during antigen-induced arthritis (AIA). Methods Experimental arthritis (AIA) was induced by injection of 60 μg mBSA into the right knee joint of ApoE–/– and wild type (WT) control mice previously immunized with mBSA/CFA. Joint swelling was measured by uptake of 99mTechnecium (99mTc) and expressed as a ratio of the uptake in right (injected) and the left (non injected) knee joint. Humoral immunity (mBSA antibody titer) was measured by ELISA. Joint inflammation and bone erosion were measured by histological analysis using an arbitrary scale from 0 to 3. TRAP+ cells were determined using immunohistochemistry. Results ApoE–/– mice showed significantly less joint swelling at day 1, 3 and 7 after AIA induction compared to WT controls (21%, 17%, 18% lower, respectively). Serum mBSA antibody levels (total IgG, IgG1, IgG2a and IgG2b) are comparable between the two immunized mouse strains. At day 21, histology of the knee joints showed less infiltration of inflammatory cells (25% lower) and decreased bone erosion in the ApoE–/– mice compared to WT controls (25% lower from 1.5±0.2 to 1.1 ±0.1). In line with that, ApoE–/– mice showed a reduction of the number of osteoclasts present at the area of resorption within the arthritic knee joints (36% lower from 20±4 osteoclasts/section in WT mice to 12±5 in ApoE–/– mice), as measured by image analysis of TRAP staining. Conclusions ApoE aggravates bone destruction within the knee joints during AIA by increasing influx of inflammatory cells within the synovium and elevating the number of resorbing osteoclasts on the bone surface. Disclosure of Interest None declared

FRI0039 Synovial Macrophages Promote TGF-Beta Signaling but Protect against Influx of S100a8/s100a9-Producing Cells Following Intra-Articular Injections of Oxidized Low-Density Lipoproteins

Background In previous studies we found that synovial macrophages regulate joint pathology during experimental osteoarthritis (OA) and, more recently, we found that high systemic levels of low-density lipoproteins (LDL) aggravate joint pathology during experimental OA with synovitis. LDL in inflamed synovium is oxidized and taken-up by macrophages, leading to an activated macrophage phenotype. Objectives In this study, we investigate whether injection of oxidized LDL directly into a murine knee joint induces joint pathology and elucidate the role of synovial macrophages in that process. Methods Synovium was obtained from end-stage OA patients and stained for apolipoprotein B (APOB). Murine knee joints were injected five consecutive days with oxLDL, LDL, or an equal volume of vehicle (PBS). This procedure was repeated in mice depleted of synovial macrophages by intra-articular injection of clodronate liposomes seven days prior to the consecutive injections. Joint pathology was investigated by immunohistochemistry and flow cytometry (FCM) analysis, and RNA expression and protein production by synovium were determined using RT-PCR and luminex, respectively. Aggrecanase activity was measured using NITEGE-staining and active TGF-β was measured using a functional CAGA-luciferase assay. Data are depicted as mean ± standard deviation. Results Synovial macrophages and fibroblasts of end-stage OA patients showed extensive accumulation of APOB, the main protein present in LDL and oxLDL. Multiple injections of oxLDL in murine knee joints significantly increased TGF-β activity in synovial wash-outs by 28% [from 16562 ± 2326 relative light units (RLU) to 21151 ± 3823 RLU; P<0.05], but did not induce catabolic or inflammatory processes. In contrast, repeated injections of oxLDL in macrophage-depleted knee joints led to a 3.1 fold increase of synovial thickening, compared with injection of PBS (P<0.01), while LDL injections did not alter synovial thickening. Protein and RNA levels of chemokines CCL2 and CCL3 were significantly upregulated in macrophage-depleted joints after oxLDL injections (6.7 fold and 4.6 fold, respectively; P<0.01). Furthermore, FCM-analysis revealed increased presence of monocytes (from 1422 ± 1105 to 3029 ± 1644 cells) and neutrophils (from 4014 ± 3511 to 12708 ± 7829 cells) in the synovium of macrophage-depleted joints after injection of oxLDL (P<0.05), which was confirmed by immunohistochemical staining. Also protein levels of S100A8/A9, markers for inflammation, were significantly increased in synovial wash-outs of oxLDL-injected joints compared with LDL (fold increase 5.6; P<0.05) or PBS (fold increase 8.3; P<0.01) injection, as was NITEGE expression (fold increase 1.92; P<0.05). Interestingly, no raise in active TGF-β was measured in these macrophage-depleted joints. Conclusions Synovial macrophages promote anabolic processes after intra-articular oxLDL injections. In absence of synovial macrophages, however, oxLDL induces production of pro-inflammatory mediators and aggrecanase activity, in combination with increased influx of monocytes and neutrophils. Disclosure of Interest None declared

FRI0005 Combination Blocking of IL-22 and IL-17 During Experimental Arthritis Potently Reduces TH17-Driven Disease Progression

Background Rheumatoid arthritis (RA) patients show elevated levels of IL-22 and IL-22-producing T helper cells that correlate to erosive disease, suggesting a role for this cytokine in the pathogenesis of RA. Interestingly, IL-22 is a dual cytokine with pro- and anti-inflammatory properties, and its effects might be regulated by cooperation and crosstalk with IL-17. Objectives The purpose of this study was to elucidate the role of IL-22 in the development of a spontaneous model of experimental arthritis by using IL-1Ra knockout mice. Additionally, we aimed to investigate the therapeutic potential of combined IL-22/IL-17 blocking during experimental arthritis. Methods IL-1Ra-deficient mice develop spontaneous arthritis due to excess IL-1 signaling, and we previously demonstrated the importance of IL-17 and Th17 cells in this model1. To investigate the role of IL-22 in this arthritis model, we compared IL-1Ra-/- x IL-22+/+ mice to IL-1Ra-/- mice lacking IL-22 expression. Paw joint swelling was scored weekly, and mice were sacrificed at the age of fifteen weeks. In addition, arthritic IL-1Ra-/- x IL-22-/- mice were treated with anti-IL-17 antibodies to determine the therapeutic potency of this combined blocking strategy during experimental arthritis. Results IL-1Ra-/- mice that also lack IL-22 showed strongly reduced arthritis development, reaching a disease incidence of only 54% at the age of 15 weeks compared to 93% in IL-1Ra-/- x IL-22+/+ mice. In addition, arthritis severity of the mice that did develop arthritis was significantly reduced by 30.6% in the absence of IL-22. Interestingly, combined blocking of IL-22 and IL-17 using IL-1Ra-/- x IL-22-/- mice treated with neutralizing anti-IL-17 antibodies after the onset of arthritis demonstrated clear additive effects compared to blocking these single cytokines alone, thereby potently reducing progression of this Th17-driven arthritis model. Conclusions These findings suggest that IL-22 plays an important role both in the initiation and augmentation of Th17-dependent experimental arthritis, and that targeting IL-22, especially in combination with IL-17 therefore seems an interesting, potent strategy in RA treatment. References Koenders MI, Devesa I, Marijnissen RJ, Abdollahi-Roodsaz S, Boots AM, Walgreen B, di Padova FE, Nicklin MJ, Joosten LA, van den Berg WB. Interleukin-1 drives pathogenic Th17 cells during spontaneous arthritis in interleuking-1 receptor antagonist-deficient mice. Arthritis Rheum. 58(11): 3461-70, 2008. Disclosure of Interest None declared