Antonia Puchner

Essential role of microRNA-155 in the pathogenesis of autoimmune arthritis in mice

OBJECTIVE MicroRNAs (miRNA) are a new class of regulatory elements. Altered expression of miRNA has been demonstrated in the inflamed joints of patients with rheumatoid arthritis (RA). The aim of this study was to examine the role of miRNA in the pathogenesis of autoimmune arthritis, using 2 murine models. METHODS Collagen-induced arthritis (CIA) and K/BxN serum-transfer arthritis were induced in wild-type (WT) and miR-155-deficient (miR-155(-/-) ) mice. The severity of arthritis was determined clinically and histologically. Anticollagen antibodies and cytokines were measured by enzyme-linked immunosorbent assay. The cellular composition of the draining lymph nodes after induction of CIA was measured by flow cytometry. RESULTS The miR-155(-/-) mice did not develop CIA. Deficiency in miR-155 prevented the generation of pathogenic autoreactive B and T cells, since anticollagen antibodies and the expression levels of antigen-specific T cells were strongly reduced in miR-155(-/-) mice. Moreover, Th17 polarization of miR-155(-/-) mouse T cells was impaired, as shown by a significant decrease in the levels of interleukin-17 (IL-17) and IL-22. In the K/BxN serum-transfer arthritis model, which only depends on innate effector mechanisms, miR-155(-/-) mice showed significantly reduced local bone destruction, attributed to reduced generation of osteoclasts, although the severity of joint inflammation was similar to that in WT mice. CONCLUSION These results demonstrate that miR-155 is essentially involved in the adaptive and innate immune reactions leading to autoimmune arthritis, and therefore miR-155 might provide a novel target for the treatment of patients with RA.

Tumor necrosis factor-inhibiting therapy preferentially targets bone destruction but not synovial inflammation in a tumor necrosis factor-driven model of rheumatoid arthritis.

OBJECTIVE To investigate how tumor necrosis factor (TNF)-inhibiting therapy affects bone destruction and inflammation in a TNF-driven mouse model of rheumatoid arthritis. METHODS In order to evaluate the influence of TNF on osteoclastogenesis in vitro, different concentrations of TNF were added to spleen cell-derived monocytes in the absence or presence of different concentrations of RANKL. In addition, the effects of TNF inhibition on osteoclast precursors as well as local bone destruction in vivo were assessed by treating TNF-transgenic mice with different doses of adalimumab. RESULTS TNF stimulated osteoclastogenesis mainly by increasing the number of osteoclast precursor cells in vitro. This TNF effect was independent of the presence of RANKL. In the hTNF-transgenic mouse model of destructive arthritis, low-dose TNF-inhibiting therapy with adalimumab had no effect on synovial inflammation but significantly inhibited local bone destruction and the generation of osteoclasts. This inhibition was accompanied by a reduction in the number of c-Fms-positive osteoclast precursor cells in the bone marrow and a reduction of the osteoclast precursor pools in the blood and inflamed synovial membrane of hTNF-transgenic mice. CONCLUSION Low-dose TNF-inhibiting therapy significantly reduces bone erosions by reducing the number of circulating and joint-invading osteoclast precursors. This effect is uncoupled from its antiinflammatory action.

Loss of phosphatase and tensin homolog (PTEN) in myeloid cells controls inflammatory bone destruction by regulating the osteoclastogenic potential of myeloid cells

Objective Local bone destruction in rheumatic diseases, which often leads to disability and severely reduced quality of life, is almost exclusively mediated by osteoclasts. Therefore, it is important to understand pathways regulating the generation of osteoclasts. Here, we analysed the impact of the Phosphoinositide-3-Kinase (PI3K)/Phosphatase and tensin homolog (PTEN) axis on osteoclast generation and bone biology under basal and inflammatory conditions. Methods We analysed osteoclastogenesis of wildtype (wt) and PTEN−/− cells in vitro and in vivo, pit resorption and qPCR of osteoclasts in vitro. Mice with a myeloid cell-specific deletion of PTEN and wt littermate mice were investigated by bone histomorphometry and clinical and histological assessment in the human tumour necrosis factor (TNF)-transgenic (hTNFtg) arthritis model. Results We show that myeloid-specific PTEN−/− mice display increased osteoclastogenesis in vitro and in vivo compared to wt mice. Loss of PTEN did not affect the generation or survival of osteoclast precursor cells. However, PTEN deficiency greatly enhanced receptor activator of nuclear factor κ-B ligand (RANKL)-induced expression of the master transcription factor of osteoclastogenesis, nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), resulting in markedly increased terminal differentiation of osteoclasts in vitro. We also observed increased osteoclastogenesis under inflammatory conditions in the hTNFtg mouse model of arthritis, where hTNFtg/myeloid-specific PTEN−/− mice displayed enhanced local bone destruction as well as osteoclast formation in the inflamed joints. The extent of synovial inflammation, however, as well as recruitment of osteoclast precursor cells was not different between wt and myeloid-specific PTEN−/− mice. Conclusions These data demonstrate that loss of PTEN and, therefore, sustained PI3-Kinase signalling in myeloid cells especially, elevates the osteoclastogenic potential of myeloid cells, leading to enhanced inflammatory local bone destruction. Therefore, although our study allows no direct translational conclusion since we used a conditional knockout approach, the therapeutic targeting of the PI3-Kinase pathway may be of benefit in preventing structural joint damage.

Interface Management between General Practitioners and Rheumatologists—Results of a Survey Defining a Concept for Future Joint Recommendations

Objective To measure the views of general practitioners (GPs) and rheumatologists in a nationwide evaluation, so as to optimise their cooperation in managing patients with inflammatory rheumatic diseases. Methods A questionnaire covering aspects of collaboration was sent, both by mail and/or by email, to all GPs and rheumatologists in Austria. Topics covered were (i) examinations and interventions to be performed before referral, (ii) the spectrum of diseases to be referred, and (iii) the role of GPs in follow-up and continuous management of patients. Results 1,229 GPs of the 4,016 GPs (31%) and 110 of the 180 rheumatologists (61%) responded to the questionnaire. In cases of suspected arthritis, 99% of the GPs and 92% of the rheumatologists recommended specific laboratory tests, and 92% and 70%, respectively, recommended X-rays of affected joints before referral. Rheumatoid arthritis and spondyloarthritis, psoriatic arthritis and connective tissue disease were unanimously seen as indications for referral to a rheumatologist. Only 12% of rheumatologists felt responsible for the treatment of hand osteoarthritis and fibromyalgia. 80% of GPs and 85% of rheumatologists were of the opinion that treatment with disease-modifying drugs should be initiated by a specialist. Subsequent drug prescription and administration by GPs was supported by a majority of GPs and rheumatologists, with a concomitant rheumatologist follow-up every three to six months. Conclusion The considerable consensus between the two professional groups constitutes a solid base for future joint recommendations, with the aim to accelerate the diagnostic process and the initiation of adequate therapy.

MicroRNA 155-deficiency leads to decreased autoantibody levels and reduced severity of nephritis and pneumonitis in pristane-induced lupus

Objective We herein examine the role of endogenous miR155 in the development of systemic manifestations in pristane induced lupus. Materials and methods Systemic lupus in miR155-deficient and wild type mice was induced upon injection of pristane and analyzed after 8 months, PBS-injected mice served as controls. Glomerulonephritis and pneumonitis were quantified using the kidney biopsy score and a newly adapted histomorphometric image analysis system; lung tissue was further analyzed by tissue cytometry. Serum levels of anti-dsDNA, anti-histone and anti-chromatin antibodies were measured by ELISA. Frequencies of B cells, activated and regulatory CD4+ T cells as well as Th1, Th2, Th17 cells were measured by flow cytometry. RT-qPCR was used to measure expression levels of interferon-signature and T-cell subset related as well as miR155-associated genes. Results After induction of lupus, miR155-deficient mice had significant less pulmonary involvement (perivascular inflammatory area in mm2/mm2 lung area 0.00092±0.00015 vs. 0.0027±0.00075, p = 0.0347) and renal disease (glomerular activity score 1.95±0.19 vs 3±0.26, p = 0.0029) compared to wild types. MiR155-deficient mice had significantly lower serum levels of disease-associated auto-antibodies and decreased frequencies of activated CD4+CD25+ (Foxp3-) cells. Upon restimulation, CD4+ cells showed a less pronounced Th2 and Th17 and a slightly decreased Th1 response in mir155-deficient mice. Pristane-treated wild types showed significantly up-regulated expression of genes related to the INF-signature (MX1, IP10, IRF7, ISG15). Conclusions MiR155-deficient mice had less severe organ involvement, lower serum auto-antibody levels, a less prominent T cell response and lower expressions of genes jointly responsible for disease development. Thus, antagonizing miR155 might be a future approach in treating SLE.

Therapeutic Modulation of Plasmacytoid Dendritic Cells in Experimental Arthritis

The role of plasmacytoid dendritic cells (PDCs) and type I interferons (IFNs) in rheumatoid arthritis (RA) remains a subject of controversy. This study was undertaken to explore the contribution of PDCs and type I IFNs to RA pathogenesis using various animal models of PDC depletion and to monitor the effect of localized PDC recruitment and activation on joint inflammation and bone damage.

Effects of 18β-Glycyrrhetinic acid in hTNFtg mice - a model of rheumatoid arthritis.

ZusammenfassungEINLEITUNG: Die rheumatoide Arthritis ist eine chronische, entzündliche Autoimmunerkrankung, die aufgrund einer ätiologisch ungeklärten Entzündungsreaktion zu einer zunehmenden Zerstörung der Gelenke führt. Mit derzeitigen Therapien ist nur ungefähr die Hälfte der Patienten in eine klinische Remission zu bringen. Daher werden neue Therapien benötigt um die Gelenkszerstörung vollständig zu verhindern. In den letzten Jahren konnte der Einfluss, der 18β-Glycyrrhetinsäure die aus Süßholz gewonnen wird auf zahlreiche Entzündungsmediatoren gezeigt werden. Außerdem wurde berichtet, dass Süßholz die Entzündung und Gelenkszerstörung in der Collagen induzierten Arthritis vermindert. Ziel dieser Studie war, den Effekt der 18β-Glycyrrhetinsäure in einem Mausmodel der rheumatoiden Arthritis zu studieren. MATERIAL UND METHODEN: Humane Tumornekrosefaktor transgene (hTNFtg) Mäuse wurden mit 18β-Glycyrrhetinsäure durch subkutane Injektion behandelt. Untersucht wurde, ob die Behandlung eine Besserung der Arthritis bringt. Als Negativkontrollen wurden hTNFtg Mäuse in gleicher Weise mit der Trägerlösung (Olivenöl) behandelt. Als Positivkontrolle diente die Hemmung von Tumornekrosefaktor mit dem anti-Tumornekrosefaktor Antikörper Infliximab. RESULTATE: 18β-Glycyrrhetinsäure behandelte hTNFtg Mäuse zeigten während der Studie denselben Krankheitsverlauf wie die mit der Trägerlösung behandelten Mäuse. Im Gegensatz dazu bewirkte eine TNF hemmende Therapie mit Infliximab eine signifikante Verbesserung der klinischen Arthritiszeichen (Pfotenschwellung und Griffstärkenabnahme) im Vergleich zur Kontrollgruppe. In Übereinstimmung mit den klinischen Daten führte die Therapie mit der 18β-Glycyrrhetinsäure auch zu keinem Rückgang der Inflammation, Erosion und der Zahl der Osteoklasten im Gelenkschnitt, während die Gabe von Infliximab auch zu einer signifikanten Besserung der histologischen Arthritiszeichen führte.SummaryINTRODUCTION: Rheumatoid arthritis is a chronic autoimmune disease characterised by inflammation of joints with cartilage and bone destruction leading to progressive disability. While the cause of rheumatoid arthritis is not known and the disease cannot be cured, conventional disease modifying antirheumatic drugs and biologicals are effective treatments for many patients. However, new therapies are needed in order to achieve better relief from rheumatoid arthritis symptoms than currently possible and to fully prevent joint damage. 18β-Glycyrrhetinic acid is not only used frequently in traditional Chinese medicine, but has been reported to target some of the inflammatory mediators involved in the pathogenesis of rheumatoid arthritis. Moreover, it has been reported that liquorice, which contains high levels of 18β-Glycyrrhetinic acid, reduces inflammation and articular damage in collagen induced arthritis. Therefore, we studied the effects of 18β-Glycyrrhetinic acid in a Tumor necrosis factor (TNF) dependent mouse model of rheumatoid arthritis. MATERIAL AND METHODS: HTNFtg mice were treated with 18ß-Glycyrrhetinic acid from day 28 after birth every second or third day for 2 weeks, or 3 times a week for six weeks. TNF inhibitor treated animals served as positive control. RESULTS: Clinical scores of arthritis were not altered in animals treated with 18β-Glycyrrhetinic acid compared to placebo treated animals. Histological data also indicate no effects of 18β-Glycyrrhetinic acid on inflammatory joint destruction. TNF inhibitors, however markedly reduced not only clinical signs of TNF triggered joint inflammation but also histological signs of erosive disease. Therefore, in contrast to previous reports our data indicate that 18β-Glycyrrhetinic acid does not provide a new therapeutic option for treating patients with rheumatoid arthritis.

02.06 Ccr6 modulates severity of arthritis in T cell dependent manner

Backgrounds and objectives Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, driven by the infiltration of various cell types. Evidence suggests that chemokines are involved in a variety of immunological and inflammatory responses. Increased production of MIP-3-α and accumulation of CCR6 expressing mononuclear cells could be found in joints of RA patients. CCR6 is also expressed in human osteoblasts. However, CCR6 expression has also been reported in CD4+ T cells, in particular regulatory as well as Th17 cells. Recent data suggest that CD25-Foxp3+ T cells express CCR6 and have osteoclastogenic potential. Therefore we investigated which CCR6 expressing cell type is involved in the pathogenesis of arthritis using different arthritis models. Materials and methods Clinical as well as histological signs of arthritis were investigated in the collagen-induced arthritis (CIA), K/BxN serum transfer arthritis and in the human tumour necrosis factor (hTNFtg) arthritis model, comparing wt and CCR6-/- mice. We analysed the phenotype of lymph node cells by flow cytometry and cytokine concentrations in serum. Anti-collagen antibodies and cytokines were measured by enzyme-linked immunosorbent assay. Results Since CCR6 is an important component of the innate immune system we compared the development of arthritis in CCR6-/- mice in 2 different arthritis models, known to be T cell independent. Contrary to expectations, no difference could be detected in clinical signs of inflammation as well as in joint histomorphology. In line with this, we could not see differences in bone density between wt and CCR6-/- mice. To investigate the role of CCR6 as part of the adaptive immune system in the development of arthritis we induced CIA in wt and CCR6-/- mice. Surprisingly CCR6-/- mice were completely protected from arthritis. Analyses of different T cell subsets by flow cytomtry revealed a significant reduction of CD25-Foxp3+ cells. Conclusions These data suggest that CCR6 expression on T cells is a major driver of arthritis and confirm the pathologic importance of CCR6 expression in T cells. CCR6 as part of the adaptive immune system outcompetes its importance as part of the innate immune system.

A1.02 Important role of microrna-146A in inflammatory arthritis by controlling local bone destruction

Background MicroRNA (MiR-) 146a is a key regulator of the innate immune response and has also been shown to suppress cancer development in myeloid cells., Although in late stages of arthritis elevated expression of miR-146a in synovial tissue of rheumatoid arthritis patients was detected, the level of this miRNA was found to be down regulated in early disease, but its role in the development of inflammatory arthritis is yet unknown. Materials and Methods We induced K/BxN serum transfer arthritis in wild type and miR-146a-/- mice. As a second inflammatory arthritis disease model we crossed miR-146adeficient into hTNFtg mice. Disease severity was assessed clinically and histologically in both arthritis models. Blood of arthritis animals was analysed by flow cytometry. Serum cytokine levels were measured by Elisa. RNA expression levels were measured by qPCR. Results Absence of miR-146a leads to increased clinical signs of the induced serum transfer arthritis. In line, higher serum levels of the proinflammatory cytokines IL-12 and IL-6 were measured in miR146a deficient compared to wt mice. When we crossed miR-146a-/- into hTNFtg mice, histological examination revealed a significant increase in synovial inflammation and even more striking a more than twofold increase in local bone destruction due to increased generation of osteoclasts in the tarsal joints in miR-146a-/-/hTNFtg mice compared to hTNFtg mice. Interestingly, systemic bone loss was comparable in hTNFtg compared to miR-146a-/-/hTNFtg mice, suggesting an important local role of miR-146a. Indeed, we detected increased levels of IL-1β and RANKL and decreased expression of OPG locally in the paws of miR-146a-/-/hTNFtg compared to hTNFtg mice. Analysing the content of myeloid cells in the blood of arthritis diseased mice, revealed significantly increased numbers of circulating CD11b+ as well as CD11c+ cells in mice lacking miR-146a. Bone marrow transplants demonstrated a pivotal role for miR-146a in mesenchymal cells in controlling local osteoclast generation and bone destruction. Conclusion These data demonstrate an important mitigating role of miR-146a in inflammatory arthritis, most importantly in local bone destruction, by controlling mesenchymal expression of osteoclastogenic factors. This shows a crucial anti-inflammatory role of miR-146a, which might possibly be exploited for therapeutic purposes.

A1.82 Limited role of microrna 155 innate immunity dependent arthritis

Background MicroRNA 155 (miR155) has been demonstrated to be essential for the development of collagen induced arthritis by controlling the generation of autoreactive T and B cells. However, the contribution of miR155 in innate immune cells is not known. Materials and Methods We analysed activation and cytokine production of macrophages and dendritic cells (DCs) in vitro and in vivo. We analysed T-cells stimulatory capacity of DCs. We crossed miR155 deficient mice into hTNFtg mice and analysed arthritis development clinically as well as histologically. Results MiR155 deficiency did not alter the differentiation of BMDC from bone marrow cells or the expression of costimulatory molecules or MHCII expression after stimulation of macrophages and DCs in vitro and in vivo. Proinflammatory cytokine production (IL-12p40, IL-6) after LPS challenge in vivo was not different in serum of wt mice and miR155-/-mice. We also Facs-sorted DCs after stimulation with LPS in vivo and determined the production of proinflammatory cytokines such as IL-23, IL-6 as well as TNF. We did not detect differences between wt and miR155-/- mice. In addition, the T cell stimulatory capacity of wt and miR155-/-was identical in vitro and in vivo. When we analysed miR155-/-mice compared to wt mice in the hTNFtg model of inflammatory arthritis, which is independent of the adaptive immune system, we did not detect differences in the clinical signs and symptoms of arthritis. Histologically, we even found slightly increased synovial inflammation in hTNFtg/ miR155-/-mice compared to wt mice. Conclusion In contrast to the pivotal role of miR155 in autoimmunity requiring the adaptive immune system, the role of miR155 in innate immunity seems to be limited. This is emphasised by the fact that miR155 hardly influences the course of TNF-driven arthritis, which is mainly dependent on components of the innate immune system.