Liduine van den Bersselaar

IL-4 gene therapy for collagen arthritis suppresses synovial IL-17 and osteoprotegerin ligand and prevents bone erosion

Bone destruction is the most difficult target in the treatment of rheumatoid arthritis (RA). Here, we report that local overexpression of IL-4, introduced by a recombinant human type 5 adenovirus vector (Ad5E1mIL-4) prevents joint damage and bone erosion in the knees of mice with collagen arthritis (CIA). No difference was noted in the course of CIA in the injected knee joints between Ad5E1mIL-4 and the control vector, but radiographic analysis revealed impressive reduction of joint erosion and more compact bone structure in the Ad5E1mIL-4 group. Although severe inflammation persisted in treated mice, Ad5E1mIL-4 prevented bone erosion and diminished tartrate-resistant acid phosphatase (TRAP) activity, indicating that local IL-4 inhibits the formation of osteoclast-like cells. Messenger RNA levels of IL-17, IL-12, and cathepsin K in the synovial tissue were suppressed, as were IL-6 and IL-12 protein production. Osteoprotegerin ligand (OPGL) expression was markedly suppressed by local IL-4, but no loss of OPG expression was noted with Ad5E1mIL-4 treatment. Finally, in in vitro studies, bone samples of patients with arthritis revealed consistent suppression by IL-4 of type I collagen breakdown. IL-4 also enhanced synthesis of type I procollagen, suggesting that it promoted tissue repair. These findings may have significant implications for the prevention of bone erosion in arthritis.

IL-17 promotes bone erosion in murine collagen-induced arthritis through loss of the receptor activator of NF-kappa B ligand/osteoprotegerin balance.

IL-17 is a T cell-derived proinflammatory cytokine in experimental arthritis and is a stimulator of osteoclastogenesis in vitro. In this study, we report the effects of IL-17 overexpression (AdIL-17) in the knee joint of type II collagen-immunized mice on bone erosion and synovial receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB/osteoprotegerin (OPG) expression. Local IL-17 promoted osteoclastic bone destruction, which was accompanied with marked tartrate-resistant acid phosphatase activity at sites of bone erosion in cortical, subchondral, and trabecular bone. Accelerated expression of RANKL and its receptor, receptor activator of NF-κB, was found in the synovial infiltrate and at sites of focal bone erosion, using specific immunohistochemistry. Interestingly, AdIL-17 not only enhanced RANKL expression but also strongly up-regulated the RANKL/OPG ratio in the synovium. Comparison of arthritic mice from the AdIL-17 collagen-induced arthritis group with full-blown collagen-arthritic mice having similar clinical scores for joint inflammation revealed lower RANKL/OPG ratio and tartrate-resistant acid phosphatase activity in the latter group. Interestingly, systemic OPG treatment prevented joint damage induced by local AdIL-17 gene transfer in type II collagen-immunized mice. These findings suggest T cell IL-17 to be an important inducer of RANKL expression leading to loss of the RANKL/OPG balance, stimulating osteoclastogenesis and bone erosion in arthritis.

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Interleukin-17 acts independently of TNF-alpha under arthritic conditions.

The proinflammatory T cell cytokine IL-17 is a potent inducer of other cytokines such as IL-1 and TNF-α. The contribution of TNF in IL-17-induced joint inflammation is unclear. In this work we demonstrate using TNF-α-deficient mice that TNF-α is required in IL-17-induced joint pathology under naive conditions in vivo. However, overexpression of IL-17 aggravated K/B×N serum transfer arthritis to a similar degree in TNF-α-deficient mice and their wild-type counterparts, indicating that the TNF dependency of IL-17-induced pathology is lost under arthritic conditions. Also, during the course of the streptococcal cell wall-induced arthritis model, IL-17 was able to enhance inflammation and cartilage damage in the absence of TNF. Additional blocking of IL-1 during IL-17-enhanced streptococcal cell wall-induced arthritis did not reduce joint pathology in TNF-deficient mice, indicating that IL-1 is not responsible for this loss of TNF dependency. These data provide further understanding of the cytokine interplay during inflammation and demonstrate that, despite a strong TNF dependency under naive conditions, IL-17 acts independently of TNF under arthritic conditions.

Reduction of interleukin-17-induced inhibition of chondrocyte proteoglycan synthesis in intact murine articular cartilage by interleukin-4.

OBJECTIVE To investigate the role of interleukin-4 (IL-4) and IL-10 in basal and IL-1- and IL-17-mediated inhibition of chondrocyte metabolism. METHODS Cartilage explants of patellae from naive mice were incubated with IL-17 and/or IL-1 alone or were pretreated with IL-4 and IL-10. In addition, knee joints of naive mice were injected intraarticularly with IL-4 and IL-10 alone or were coinjected with IL-1. Chondrocyte proteoglycan (PG) synthesis was measured in intact murine articular cartilage. Levels of nitric oxide (NO) were measured using the Griess reagent. RESULTS IL-17, a novel cytokine secreted by CD4+ activated memory T cells, inhibited chondrocyte PG synthesis in intact murine articular cartilage, although the suppressive effect was less potent than that of IL-1. The suppressive effect of IL-17 was completely abolished in the presence of L-NIO (L-NS-[1-iminoethyl]ornithine), an inhibitor of NO metabolism, and IL-17 only slightly induced inhibition of PG synthesis in cartilage explants of patellae from iNOS (inducible NO synthase) knockout mice. This indicates that the suppressive effect of IL-17 was mediated by NO. Pretreatment with IL-4, but not IL-10, significantly reduced the inhibition of chondrocyte PG synthesis induced by IL-1 or IL-17. The IL-4-induced reduction in the inhibitory effects of IL-1 and IL-17 on chondrocyte PG synthesis was accompanied by decreased NO formation in the culture supernatants. CONCLUSION IL-17 plays a role in the inhibition of chondrocyte PG synthesis. IL-4 and IL-10 have no effect on basal chondrocyte metabolism. However, IL-4-pretreated cartilage is less sensitive to the suppressive effect of IL-1 as well as IL-17. This suggests that IL-4 is protective in T cell-driven cartilage disturbances, probably through reduction of iNOS.

Interleukin-18 Promotes Joint Inflammation and Induces Interleukin-1-Driven Cartilage Destruction

Interleukin (IL)-18 is a member of the IL-1 family of proteins that exerts proinflammatory effects and is a pivotal cytokine for the development of Th1 responses. The goal of the present study was to investigate whether IL-18 induces joint inflammation and joint destruction directly or via induction of other cytokines such as IL-1 and tumor necrosis factor (TNF). To this end we performed both in vitro and in vivo kinetic studies. For in vivo IL-18 exposure studies C57BL/6, TNF-deficient, and IL-1-deficient mice were injected intra-articularly with 1.10(7) pfu mIL-18 adenovirus followed by histopathological examination. Local overexpression of IL-18 resulted in pronounced joint inflammation and cartilage proteoglycan loss in control mice. Of high interest, IL-18 gene transfer in IL-1-deficient mice did not show cartilage damage, although joint inflammation was similar to that in wild-type animals. Overexpression of IL-18 in TNF-deficient mice showed that TNF was partly involved in IL-18-induced joint swelling and influx of inflammatory cells, but cartilage proteoglycan loss occurred independent of TNF. In vitro cartilage degradation by IL-18 was found after a 72-hour culture period. Blocking of IL-1 with IL-1Ra or an ICE-inhibitor resulted in complete protection against IL-18-mediated cartilage degradation. The present study demonstrated that IL-18 induces joint inflammation independently of IL-1. In addition, we showed that IL-1beta generation, because of IL-18 exposure, was essential for marked cartilage degradation both in vitro and in vivo. These findings implicate that IL-18, in contrast to TNF, contributes through separate pathways to joint inflammation and cartilage destruction.

Role of PMN in early cartilage destruction

PMN are the dominant cells in the joint cavity in the early phase of experimental models like antigen-induced arthritis (AIA) [1] or immune complex arthritis (ICA) [2]. Early cartilage damage is evident in those models. In vitro, PMN are able to degrade the cartilage matrix and to inhibit chondrocyte proteoglycan synthesis whereas the neutral protease elastase appears the dominant destructive mediator [3]. However, it is still a matter of debate whether PMN are actively involved in cartilage damage in vivo. Recent data suggests that in AIA, there is the involvement of other mediators, like IL-1, and that PMN are of minor importance. We investigated whether the same holds true for ICA. We developed a new model of ICA in the mouse. The highly cationic protein lysozyme was coupled to poly-L-lysine and injected intra-articularly (30 lag) into the knee joint, shortly after systemic administration of specific antibodies (0.2 ml). The latter were raised in rabbits. The cationic protein binds electrostatically to the cartilage and high amounts of immune complexes were formed at the surface layer. A vehement joint inflammation developed and within two days marked cartilage degradation was observed quantitatively by loss of 35-sulphate proteoglycan from the patellar cartilage and qualitatively by safranin-o staining on whole knee joint sections. We studied the in vivo role of PMN in early cartilage degradation in two ways. First by depleting PMN from arthritic joints and second by studying beige mice which possess PM N which are deficient in elastase. Results and discussion

Alteration of the intestinal microbiome characterizes preclinical inflammatory arthritis in mice and its modulation attenuates established arthritis

Perturbations of the intestinal microbiome have been observed in patients with new-onset and chronic autoimmune inflammatory arthritis. However, it is currently unknown whether these alterations precede the development of arthritis or are rather a consequence of disease. Modulation of intestinal microbiota by oral antibiotics or germ-free condition can prevent arthritis in mice. Yet, the therapeutic potential of modulation of the microbiota after the onset of arthritis is not well characterized. We here show that the intestinal microbial community undergoes marked changes in the preclinical phase of collagen induced arthritis (CIA). The abundance of the phylum Bacteroidetes, specifically families S24-7 and Bacteroidaceae was reduced, whereas Firmicutes and Proteobacteria, such as Ruminococcaceae, Lachnospiraceae and Desulfovibrinocaceae, were expanded during the immune-priming phase of arthritis. In addition, we found that the abundance of lamina propria Th17, but not Th1, cells is highly correlated with the severity of arthritis. Elimination of the intestinal microbiota during established arthritis specifically reduced intestinal Th17 cells and attenuated arthritis. These effects were associated with reduced serum amyloid A expression in ileum and synovial tissue. Our observations suggest that intestinal microbiota perturbations precede arthritis, and that modulation of the intestinal microbiota after the onset of arthritis may offer therapeutic opportunities.

1.56 Synergism between GM-CSF and IL-17 causes enhanced joint pathology via the production of IL-6 and IL-23

Background and Objective T helper-17 (Th17) cells are important mediators of inflammatory diseases, and are the main pathogenic cell type in many animal models of autoimmunity. Recent studies highlight a surprising role for T-cell derived granulocyte-macrophage colony stimulating factor (GM-CSF) in the pathogenicity of Th17 cells. We examined the mechanism by which interleukin 17 (IL-17) and GM-CSF contribute to cartilage- and bone damage of synovial joints during experimental arthritis, and provide a rationale for combination therapy in auto-inflammatory conditions. Materials and Methods Collagen-induced arthritis (CIA) was elicited in DBA/1J mice. Neutralizing antibodies to IL-17 and/or GM-CSF were administered after onset of disease for 14 days. Arthritis progression was followed by macroscopic scoring of the paws (maximum score of 12 per mouse). In addition, the effect of local over-expression of IL-17 and/or GM-CSF was studied by adenoviral transfection in naïve knee joints. Results Combined therapeutic treatment of mice early after the onset of CIA ameliorated disease progression. Macroscopic joint inflammation was significantly reduced, from a total score of 5.6 ± 0.4 for mice treated with isotype control antibodies to 2 ± 0.6 for mice treated with combination therapy. Treatment with anti-IL-17 or anti-GM-CSF alone resulted in scores of 3.4 ± 0.5 and 3.5 ± 0.4, respectively. Simultaneous blocking of GM-CSF and IL-17 was also the most effective treatment in the prevention of radiological bone damage and histological cartilage destruction. To provide further insight in local additive or synergistic effects of IL-17 and GM-CSF, overexpression of IL-17, GM-CSF or the combination was achieved with adenoviral vectors. Inflammatory infiltrate and cartilage- and bone damage developed in all groups from day 1 after adenoviral transfer, with the most severe effect observed in the combination group. On day 7, partial destruction of joint architecture was apparent in knee joints after combined overexpression of IL-17 and GM-CSF. Overexpression of GM-CSF alone induced IL-1β, which production was elevated by IL-17. Interestingly, overexpression of IL-17 alone caused a clear increase in synovial IL-6 production (179 ± 63pg/ml), which was dramatically enhanced in the co-presence of GM-CSF (1.9 ± 0.4 ng/ml). In addition, a strong synergistic effect of combined overexpression was seen on the Th17 differentiation factor IL-23. Conclusions We show that IL-17 and GM-CSF cause joint damage through synergistic effects on inflammatory mediators in synovial joints. In view of the moderate success of therapeutic IL-17 or GM-CSF blockade in rheumatoid arthritis, combined inhibition of IL-17 and GM-CSF might be an interesting option for patients that do not fully respond to inhibition of the separate cytokines. Abstract 1.56 Figure 1 (A) Macroscopic disease scores followed over time from day 21 of collagen-induced arthritis (day 0 of treatment). (B) End-point X-ray analysis of ankle joints after CIA with four different antibody treatments.

07.04 Partial elimination of intestinal microbiota dampens t helper 17 cell differentiation and established collagen-induced arthritis in mice

Background/objectives Both germ-free condition and administration of oral antibiotics before the onset of arthritis modulate T cell differentiation and attenuate arthritis in mice. However, it is not known whether and how the modulation of intestinal microbiota after the onset of arthritis may influence the disease. Here, we investigated the involvement of commensal intestinal microbiota in the progression of established arthritis in both T cell-dependent and -independent mouse models. Materials and methods Mice with established collagen-induced arthritis (CIA) as well as mice with K/BxN serum-transfer arthritis were treated orally with broad-spectrum antibiotics for one week to partially eliminate intestinal microbiota. 16S rRNA gene high-throughput sequencing was performed to assess the effect of arthritis and antibiotic treatment on the gut microbiota composition. Arthritis was assessed macroscopically and by histology. Differentiation of Th1, Th17 and regulatory T (Treg) cells and production of their prototypic cytokines in intestinal lamina propria and joint-draining lymph nodes were assessed by flow cytometry and Luminex cytokine array. Results Induction of arthritis as such resulted in significant alterations in the gut microbiota. Antibiotic treatment of CIA mice eliminated the majority of the gut microbiota, while the microbiota composition of control mice remained relatively stable. Partial elimination of intestinal microbiota during ongoing CIA specifically suppressed intestinal Th17 cell differentiation without affecting Th1 and Treg cells. Accordingly, production of IL-17, but not IFNγ, IL-4 and IL-10, by lamina propria lymphocytes was significantly diminished in antibiotic-treated mice. Importantly, elimination of intestinal microbiota resulted in suppressed Th17 cell differentiation in joint-draining lymph nodes and reduced the severity of established CIA. In contrast, antibiotic treatment did not influence disease severity in the T cell-independent K/BxN serum-transfer arthritis. Intriguingly, the abundance of intestinal Th17 cells strongly correlated with the severity of arthritis in the CIA mice. However, elimination of intestinal microbiota after disease onset did not affect the development of anti-collagen type II auto-antibodies. Conclusion These observations suggest that modulation of commensal intestinal microbiota during established arthritis specifically suppresses Th17 differentiation and dampens T cell-mediated arthritic processes. Our study supports the notion that inflammatory signals provided by the gut microbiota continue to promote arthritis after its onset.