Collagen-induced Arthritis Disease Research Articles

Activation of β2-adrenergic Receptor Alleviates Collagen-induced Arthritis by Ameliorating Th17/Treg Imbalance.

Recent research in our laboratory shows that CD4+ T cells express the β2 adrenergic receptor (β2-AR), and the sympathetic neurotransmitter norepinephrine regulates the function of T cells via β2-AR signaling. However, the immunoregulatory effect of β2-AR and its related mechanisms on rheumatoid arthritis is unknown. To explore the effects of β2-AR in collagen-induced arthritis (CIA) on the imbalance of T helper (Th) 17/ regulatory T (Treg) cells. In DBA1/J mice, collagen type II was injected intradermally at the tail base to prepare the CIA model. The specific β2-AR agonist, terbutaline (TBL), was administered intraperitoneally beginning on day 31 and continuing until day 47 after primary vaccination, twice a day. Magnetic beads were used to sort CD3+ T cells subsets from spleen tissues. In vivo, β2-AR agonist TBL alleviated arthritis symptoms in the CIA mice including histopathology of the ankle joints, four limbs' arthritis score, the thickness of ankle joints, and rear paws. After TBL treatment, in the ankle joints, the levels of proinflammatory factors (IL-17/22) notably decreased and the levels of immunosuppressive factors (IL-10/TGF-β) significantly increased. In vitro, ROR-γt protein expression, Th17 cell number, mRNA expression and the releasing of IL-17/22 from CD3+ T cells reduced following TBL administration. Moreover, TBL enhanced the anti-inflammatory responses of Treg cells. These results suggest that β2-AR activation exerts anti-inflammatory effects through the amelioration of Th17/Treg imbalance in the CIA disease.

Suppression of inflammatory arthritis by the parasitic worm product ES-62 is associated with epigenetic changes in synovial fibroblasts.

ES-62 is the major secreted protein of the parasitic filarial nematode, Acanthocheilonema viteae. The molecule exists as a large tetramer (MW, ~240kD), which possesses immunomodulatory properties by virtue of multiple phosphorylcholine (PC) moieties attached to N-type glycans. By suppressing inflammatory immune responses, ES-62 can prevent disease development in certain mouse models of allergic and autoimmune conditions, including joint pathology in collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Such protection is associated with functional suppression of “pathogenic” hyper-responsive synovial fibroblasts (SFs), which exhibit an aggressive inflammatory and bone-damaging phenotype induced by their epigenetic rewiring in response to the inflammatory microenvironment of the arthritic joint. Critically, exposure to ES-62 in vivo induces a stably-imprinted CIA-SF phenotype that exhibits functional responses more typical of healthy, Naïve-SFs. Consistent with this, ES-62 “rewiring” of SFs away from the hyper-responsive phenotype is associated with suppression of ERK activation, STAT3 activation and miR-155 upregulation, signals widely associated with SF pathogenesis. Surprisingly however, DNA methylome analysis of Naïve-, CIA- and ES-62-CIA-SF cohorts reveals that rather than simply preventing pathogenic rewiring of SFs, ES-62 induces further changes in DNA methylation under the inflammatory conditions pertaining in the inflamed joint, including targeting genes associated with ciliogenesis, to programme a novel “resolving” CIA-SF phenotype. In addition to introducing a previously unsuspected aspect of ES-62’s mechanism of action, such unique behaviour signposts the potential for developing DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA. Pertinent to these translational aspects of ES-62-behavior, small molecule analogues (SMAs) based on ES-62’s active PC-moieties mimic the rewiring of SFs as well as the protection against joint disease in CIA afforded by the parasitic worm product.

Microstructure and mechanical behaviors of tibia for collagen-induced arthritic mice treated with gingiva-derived mesenchymal stem cells

Rheumatoid arthritis (RA) is a systemic polyarticular arthritis that primarily affects the small joints but also causes bone erosion in large joints. None of the currently existing treatment approaches is curable. In this study, the effects of human gingiva-derived mesenchymal stem cells (GMSCs) on collagen-induced arthritis (CIA) mice are examined by experimentally assessing the microstructure and mechanical behaviors of tibia. Bone morphology and mineral density of mouse tibiae were assessed using micro-X-ray computed tomography (micro-CT). Compression testing was performed on mouse tibia to access its stiffness. The deformation and strain localized inside proximal tibia were mapped using mechanical testing coupled with micro-CT and digital volume correlation of micro-CT images. The results show that CIA disease caused bone erosion in epiphyseal cortical bone, which manifested into the adjacent epiphyseal trabecular bone, and also affected the metaphyseal cortical bone. CIA disease also weakened the load-bearing function of proximal tibia. GMSC treatment interfered with the progress of CIA, attenuated the bone erosion in epiphyseal and metaphyseal trabecular bone and resulted in improved load-bearing function of proximal tibia. GMSCs provide a promising potential treatment of autoimmune arthritis.

Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damage. Regulatory T cells (Tregs) are defective in RA patients while adoptive transfer of expanded autologous Tregs efficiently reverse disease in collagen-induced arthritis, an animal model of RA. Moreover, providing an antigen (Ag) specificity to Tregs render them more potent to suppress abnormal inflammation by increasing specific activity and promoting selective migration to the Ag-expressing tissue, reducing the risk of pan-immunosuppression in the meantime. These studies strongly suggest that Treg therapy may be effective in the treatment of RA patients by reducing joint inflammation and inducing immune tolerance and may be improved by using Ag-specific Tregs. Therefore, we aimed to engineer Ag-specific Tregs from polyclonal Tregs using a chimeric antigen receptor (CAR) specifically targeting an Ag present in the joint of RA patients. We generated a CAR directed against a post-translationally modified intermediate filament protein, citrullinated vimentin (CV), an Ag found, abundantly and almost exclusively, in the extracellular matrix of the inflamed synovial tissue of the RA patients. CV-specific CAR was transduced into human Tregs that were efficiently activated, expanding and suppressing following CAR-mediated stimulation. Importantly, CV-CAR Tregs were shown to react with CV expressed in RA patient synovial fluid. Studies are underway to demonstrate the functional activity of these CV-CAR Tregs in in vivo mouse models, a validation step in the development of a promising therapeutic tool to treat RA and potentially other autoimmune disorders.

MIF and MIF2 have distinct but synergistic roles in CIA pathogenesis

Abstract Rheumatoid arthritis (RA) is a relapsing autoimmune disease with progressive joint destruction. High expression alleles of the cytokine macrophage migration inhibitory factor (MIF) are associated with severe erosive RA. MIF and MIF family member D-dopachrome tautomerase (MIF2) signal through the receptor CD74. Previously we reported on the presence of CD74 expressing effector memory T cells in the collagen-induced arthritis (CIA) mouse model and identified CD74+ T cells in human RA synovium fluid. We studied MIF and MIF2 signaling on CD74+ T cells and inflammatory CD74high MΦ in the CIA model. We observed infiltration of CD74+ T cells (1.7 fold increase, p<0.001) and inflammatory CD74high MΦ (2 fold increase, p<0.001) in the joints of WT mice on day 28 post CIA induction. CD74+ T cell infiltration into paws positively correlated with CIA disease score (r=0.731, p=0.003). CD74+ T cells and CD74high MΦ failed to expand or infiltrate in the paws of Mif−/− mice in CIA. Interestingly, CIA induction in Mif2−/− mice expand CD74+ T cells in lymph nodes (0.5 fold increase, p=0.024), but fail to expand in other secondary lymphoid organs or infiltrate joints. Macrophage precursor Gr1high CD74+ monocytes expand in the spleen (3 fold, p<0.001) and blood (1 fold, p<0.001) of Mif2−/− mice, no change in CD74high MΦ levels is observed. The MIF inhibitor MIF098 ameliorates joint inflammation but did not inhibit the expansion or joint infiltration of CD74+ T cells (2 fold, p<0.001), Gr1high CD74+ monocytes, and CD74high MΦ (0.7 fold, p<0.01). Our findings provide evidence for a pathogenic role of novel MIF responsive CD74+ T cell in RA and suggest separate but crucial roles for both MIF and MIF2 in the development and localization for CD74+ T cells and CD74high MΦ in CIA.

The parasitic worm product ES-62 normalises the gut microbiota bone marrow axis in inflammatory arthritis

The human immune system has evolved in the context of our colonisation by bacteria, viruses, fungi and parasitic helminths. Reflecting this, the rapid eradication of pathogens appears to have resulted in reduced microbiome diversity and generation of chronically activated immune systems, presaging the recent rise of allergic, autoimmune and metabolic disorders. Certainly, gastrointestinal helminths can protect against gut and lung mucosa inflammatory conditions by modulating the microbiome and suppressing the chronic inflammation associated with dysbiosis. Here, we employ ES-62, an immunomodulator secreted by tissue-dwelling Acanthocheilonema viteae to show that helminth-modulation of the gut microbiome does not require live infection with gastrointestinal-based worms nor is protection restricted to mucosal diseases. Specifically, subcutaneous administration of this defined immunomodulator affords protection against joint disease in collagen-induced arthritis, a mouse model of rheumatoid arthritis, which is associated with normalisation of gut microbiota and prevention of loss of intestinal barrier integrity.

The number of regulatory B cells is increased in mice with collagen-induced arthritis

The aim of this study is to investigate changes in regulatory B cells (Bregs) and the expression of related cytokines such as interleukin-10 (IL-10) and transforming growth factor (TGF)-β in a mouse model of collagen-induced arthritis (CIA). A total 20 DBA/1 mice (6-8 weeks old) were randomly divided into control and CIA disease groups. For the CIA disease group, animals were injected intradermally with chicken collagen type II and complete Freund's adjuvant. The calculated arthritis index score of the CIA group was significantly higher than that in control group. Hematoxylin and eosin staining showed tumid synovial cells with irregular arrangement and obvious hyperplasia, with a high degree of inflammatory cell infiltration in CIA model group. Cytometric bead array technology and quantitative RT-PCR indicated that the levels of IL-10 and TGF-β in serum, and synovial cells were significantly increased in the CIA group. The proportion of Bregs in the spleen of the CIA group was significantly increased compared to the control group. In conclusion, our findings demonstrate that the number of Bregs and the expression of TGF-β and IL-10 are enhanced in mice with CIA.

The Influence of LPS-Induced Maternal Inflammation on Postnatal Collagen-Induced Arthritis.

Maternal health and nutritional status influence offspring health and the diseases that may develop in them. The effects of maternal inflammation on offspring from the perspective of the inflammatory response and immune changes are not fully understood. We hypothesized that maternal inflammation modulates immune and metabolic functions, affecting the pathophysiology of inflammatory diseases in offspring. This study investigated whether maternal inflammation affects the onset of collagen-induced arthritis (CIA), a murine model of human rheumatoid arthritis. Female DBA/1J mice received a single intraperitoneal injection of lipopolysaccharide (LPS) 5days before conception. Male offspring of LPS-treated dams were placed in the maternal LPS group (MLG). To induce CIA, type II collagen (CII) was emulsified with Freund's complete adjuvant and injected twice into each mouse, at 13 and 16weeks. The offspring were sacrificed at 26weeks to analyze immunological and metabolic parameters. The degree of joint swelling at an early stage of CIA was lower in the MLG than in the control group. From histological analysis, the severity of joint destruction (severity of arthritis score) and CII-specific IgG titer were significantly lower in the MLG. However, at 26weeks, serum interleukin (IL)-6 levels, an index of CIA disease activity, were significantly higher in the MLG. Moreover, serum leptin levels were lower in the MLG, and a negative correlation between leptin and serum IL-6 was observed. In conclusion, maternal inflammation does not merely suppress inflammation; it may delay CIA in offspring. The analysis of inflammatory cytokines and leptin concentrations at 26weeks suggests that the pathophysiology of arthritis was worsening. This study also suggests that maternal inflammation modulates postnatal inflammatory response patterns in offspring.

Development of citrullinated-vimentin-specific CAR for targeting Tregs to treat autoimmune rheumatoid arthritis

Abstract Rheumatoid arthritis (RA) is one of the most common chronic autoimmune diseases that is characterized by an aberrant inflammation of the synovial membrane that causes irreversible joint and bone damage. Regulatory T cells (Tregs) are defective in RA patients while adoptive transfer of expanded autologous Tregs efficiently reversed disease in collagen-induced arthritis (CIA), an animal model of RA. These studies strongly suggest that Treg therapy may be effective in the treatment of RA patients by reducing joint inflammation and inducing immune tolerance. However, clinical treatment with polyclonal Tregs may lead to general immunosuppression. Thus, the development of precise RA Ag-specific Tregs would increase specific activity, promote selective migration to the site of the abnormal inflammation and enhance their function. Thus, we engineered chimeric antigen receptor (CAR)-expressing Tregs specifically targeting an antigen present in the joint of RA patients to induce a localized and effective immunosuppressive response. We developed a single chain Fv directed against a posttranslational modified intermediate filament protein, citrullinated vimentin (CV), an antigen found, almost exclusively, in the extracellular matrix of the synovial tissue of the RA patients and implicated in the pathogenesis of the disease. The scFv was grafted into a functional CAR construct, transduced into human Tregs and shown to react with CV expressed in RA patient synovial fluid and CV-expressing cells. Studies are underway to demonstrate the functional activity of these CAR-expressing Tregs in in vivo mouse models a validation step in the development of a promising therapeutic tool to treat RA and potentially other autoimmune disorders.

Understanding how immune checkpoint modulators alter pre-existing rheumatologic disorders in cancer patients

Abstract The aim of cancer immunotherapy is to reinvigorate an exhausted immune system. Checkpoint modulators PD1/PDL1 and CTLA4 antagonists “release the breaks” on ineffective T cells and subsequently bolster tumor-targeted T cells. These checkpoint modulators are increasingly available for numerous cancers and therefore the number of patients on immunotherapy are ever increasing. In contrast, patients with autoimmune diseases are prescribed immune therapies that dampen the immune system. Of note, immune suppressive therapies increase the incidence of various cancers. The interesting question that has not been fully addressed- what is happening immunologically in patients who have pre-existing autoimmune diseases and are treated with checkpoint modulators for cancer? We have been evaluating the immune correlates in patients with pre-existing rheumatoid arthritis (RA; often being treated with TNF-α blocker) who later receive checkpoint therapy when diagnosed with cancer. Following checkpoint therapy, we saw an increase in central memory T cells, a decrease in PD1 and CTLA4 expression, and a decrease in Th1 and Th17 cells in the peripheral blood. Additionally, we have been working on a dual disease animal model- collagen-induced arthritis (CIA) and lung cancer models. This will has allowed us to study the direct immunological consequences of giving treatments that either dampen or boost the immune system on both cancer and CIA disease states. Outcomes from this work will continue to expand our understanding of the immune system in patients with dual diseases and thus be better equipped to evaluate immunological correlates for exacerbated autoimmune disease and to productively treat patients with both autoimmune and cancer diagnoses.

In-vivo quantitative assessment of the therapeutic response in a mouse model of collagen-induced arthritis using 18 F-fluorodeoxyglucose positron emission tomography.

Mouse collagen-induced arthritis (CIA) is the most commonly used animal model to investigate underlying pathogenesis of autoimmune arthritis and to demonstrate the therapeutic efficacy of novel drugs in autoimmune arthritis. The conventional read-outs of CIA are clinical score and histopathology, which have several limitations, including (i) subjected to observer bias; and (ii) longitudinal therapeutic efficacy of a new drug cannot be determined. Thus, a robust, non-invasive, in-vivo drug screening tool is currently an unmet need. Here we have assessed the utility of 18 F-fluorodeoxyglucose positron emission tomography (18 F-FDG) as an in-vivo screening tool for anti-inflammatory drugs using the mouse CIA model. The radiotracer 18 F-FDG and a PET scanner were employed to monitor CIA disease activity before and after murine anti-tumour necrosis factor (TNF)-α antibody (CNTO5048) therapy in the mouse CIA model. Radiotracer concentration was derived from PET images for individual limb joints and on a per-limb basis, and Spearman's correlation coefficient (ρ) was determined with clinical score and histology of the affected limbs. CNTO5048 improved arthritis efficiently, as evidenced by clinical score and histopathology. PET showed an increased uptake of 18 F-FDG with the progression of the disease and a significant decrease in the post-treatment group. 18 F-FDG uptake patterns showed a strong correlation with clinical score (ρ=0·71, P < 0·05) and histopathology (ρ=0·76, P < 0·05). This study demonstrates the potential of 18 F-FDG PET as a tool for in-vivo drug screening for inflammatory arthritis and to monitor the therapeutic effects in a longitudinal setting.

Feeding transgenic plants that express a tolerogenic fusion protein effectively protects against arthritis.

Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)-COL-DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H-2(q) -restricted DBA/1 mice to assess the preventive effect on collagen-induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4(+) T-cell responses were reduced. Concomitant with a reduced effector T-cell activity with lower IFNγ, IL-13 and IL-17A production, we observed an increase in IL-10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up-regulated IL-10 and regulatory T-cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T-cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis.

Myeloid-derived suppressor cells ameliorate the pathogenesis of autoimmune arthritis (P1058)

Abstract Myeloid-derived suppressor cells (MDSCs) are subtypes of monocytes and granulocytes that suppress T cell activity in diseases like cancer and multiple sclerosis. Our previous studies suggested a subset of monocytes may act as MDSCs in autoimmune arthritis. Using the collagen-induced arthritis (CIA) model we identified an increase in the Ly6Chigh subset of monocytes in the periphery of WT mice with CIA compared to naïve mice. This subset was absent from the periphery of CCR2-deficient mice and these mice developed more severe CIA disease. Ly6Chigh monocytes isolated from immunized mice inhibited collagen-activated CD4+T cell proliferation as well as reduced the overall T cell numbers. In the presence of Ly6Chigh monocytes we observed high levels of nitric oxide in the supernatants and an increase in Annexin V positive CD4+T cells. To examine the biological role of these monocytes, we performed a cell-based treatment assay. Compared to control mice, in vivo transfer of Ly6Chigh monocytes to collagen-immunized mice resulted in delayed onset of disease, reduced joint swelling, decreased IL-6, IL-1β, and IFNγ expression in the joints and decreased IL-6 and IL-17 levels in the serum. In summary, Ly6Chigh monocytes function as MDSCs to inhibit CD4+T cell proliferation and survival. In vivo transfer of Ly6Chigh monocytes ameliorated autoimmune arthritis in the CIA model suggesting these cells could potentially be used in cell-based therapies for treatment of rheumatoid arthritis.

Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis

IntroductionThe immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA).MethodsThe ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo.ResultsAll MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1β was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs.ConclusionsThese data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.

Oral GABA treatment downregulates inflammatory responses in a mouse model of rheumatoid arthritis

Current treatments for rheumatoid arthritis (RA) have long-term side effects such that new treatments are needed that can safely help manage the disease. There is a growing appreciation that GABA receptors (GABA-Rs) on immune cells provide new targets that can be used to modulate immune cell activity. Here, we show for the first time that activation of peripheral GABA-Rs can inhibit the development of disease in the collagen-induced arthritis (CIA) mouse model of RA. Mice that received oral GABA had a reduced incidence of CIA, and those mice that did develop CIA had milder symptoms. T cells from GABA-treated mice displayed reduced proliferative responses to collagen and their APC had a reduced ability to promote the proliferation of collagen-reactive T cells. Thus, GABA downregulated both T-cell autoimmunity and APC activity. Collagen-reactive T cells from GABA-treated mice displayed reduced recall responses in the presence of GABA ex vivo, indicating that GABA consumption did not desensitize these cells to GABA. GABA-treated mice had reduced collagen-reactive IgG2a, but not IgG1 antibodies, consistent with reduced Th1 help. The levels of serum anti-collagen IgG2a antibodies were correlated significantly with the CIA disease scores of individual mice. Our results suggest that activation of peripheral GABA-Rs may provide a new modality to modulate T cell, B cell, and APC activity and help ameliorate RA and other inflammatory diseases.

Maintenance of T cell Rap1 signalling protects mice against collagen-induced arthritis

Activation of the small GTPase Rap1 in T lymphocytes regulates cell adhesion, cytokine production and reactive oxygen species production. The authors have recently identified blocked Rap1 activation as one of...

Fluoxetine and citalopram exhibit potent antiinflammatory activity in human and murine models of rheumatoid arthritis and inhibit toll‐like receptors

Selective serotonin reuptake inhibitors (SSRIs), in addition to their antidepressant effects, have been reported to have antiinflammatory effects. The aim of this study was to assess the antiarthritic potential of 2 SSRIs, fluoxetine and citalopram, in murine collagen-induced arthritis (CIA) and in a human ex vivo disease model of rheumatoid arthritis (RA). Following therapeutic administration of SSRIs, paw swelling was assessed and clinical scores were determined daily in DBA/1 mice with CIA. Joint architecture was examined histologically at the end of the treatment period. Cultures of human RA synovial membranes were treated with SSRIs, and cytokine production was measured. Toll-like receptor (TLR) function was examined in murine and human macrophages, human B cells, and human fibroblast-like synovial cells treated with SSRIs. Both SSRIs significantly inhibited disease progression in mice with CIA, with fluoxetine showing the greatest degree of efficacy at the clinical and histologic levels. In addition, both drugs significantly inhibited the spontaneous production of tumor necrosis factor, interleukin-6, and interferon-gamma-inducible protein 10 in human RA synovial membrane cultures. Fluoxetine and citalopram treatment also inhibited the signaling of TLRs 3, 7, 8, and 9, providing a potential mechanism for their antiinflammatory action. Fluoxetine and citalopram treatment selectively inhibit endosomal TLR signaling, ameliorate disease in CIA, and suppress inflammatory cytokine production in human RA tissue. These data highlight the antiarthritic potential of the SSRI drug family and provide further evidence of the involvement of TLRs in the pathogenesis of RA. The SSRIs may provide a template for potential antiarthritic drug development.

Treatment with Cl-amidine, a peptidyl arginine deiminase (PAD) inhibitor, significantly reduces collagen-induced arthritis (CIA) (50.6)

Abstract Antibodies to citrullinated protein antigens (ACPA) are specific for rheumatoid arthritis (RA). Post-translational citrullination of epitopes to which ACPA are directed is catalyzed by PADs. We have previously shown that ACPA develop in CIA and contribute to joint damage. In this study we used Cl-amidine, a small molecule that specifically inhibits all PAD enzymes, to determine whether limiting PAD activity can modify CIA. CIA and collagen antibody-induced arthritis (CAIA, a model of the antibody-mediated effector pathway of CIA) were induced in DBA/1j mice using standard methods. Mice received either no treatment, vehicle alone (PBS), or 1, 10 or 50 mg/kg/day Cl-amidine by daily IP injection. Cl-amidine reduced CIA disease activity scores by 42%, 53% and 55% in the respective treatment groups (1, 10, 50 mg/kg/day) as well as C3 deposition, IgG1 and IgG2a anti-mouse CII Abs and epitope spreading to citrullinated epitopes. Conversely, Cl-amidine treatment had no effect on the development of CAIA. These results suggest that PADs are essential components in the development of inflammatory arthritis. This work was funded by an ACR REF WOR Grant.

Combination therapies that inhibit cyclooxygenase-2 and leukotriene synthesis prevent disease in murine collagen induced arthritis

To determine the effect of combinations of cyclooxygenase (COX) inhibitors and inhibitors of leukotriene (LT) syntheses on collagen induced arthritis (CIA) in mice. The CIA model was evaluated for the presence of eicosanoids in the paw tissue. Several selective cyclooxygenase 2 (COX-2) inhibitors or non-selective non-steroidal anti inflammatory drugs (NSAIDs) were evaluated alone or in combination with leukotriene (LT) synthesis inhibitors in the CIA model. Arthritic paw tissue showed increased levels of prostaglandins and leukotrienes in comparison to normal paws. Analysis of mRNA levels indicated the inducible form of the COX-2 enzyme to be the source of prostaglandins. NSAIDs, COX-2 or leukotriene synthesis inhibitors administered alone in CIA decreased severity but had little effect on disease incidence. However, the combination of selective COX-2 inhibitors with leukotriene synthesis inhibitors produced significant decreases in both incidence and severity, suggesting an additive or synergistic effect. This effect was reversible with removal of drug. Little decrease in incidence was observed with the NSAID/5-LO inhibitor combinations. These results suggest that the induction of the disease in CIA is mediated by products of the COX-2 enzyme and LTB4 production, and that blockade of both pathways is required to prevent CIA.

Long-term anti-arthritic and anti-osteoporotic effects of raloxifene in established experimental postmenopausal polyarthritis

Both oestrogen deficiency and the inflammatory disease contribute to the generalized bone loss seen in postmenopausal rheumatoid arthritis (RA). Oestradiol and the selective oestrogen receptor modulator raloxifene have been shown to ameliorate the disease in collagen-induced arthritis (CIA), a well-established animal model for human RA. The aim of this study was to investigate whether raloxifene-treatment would be beneficial in long-term treatment of established CIA, both regarding anti-arthritic and anti-osteoporotic properties. Female dilute brown agouti mice were ovariectomized and CIA was induced. Raloxifene or vehicle treatment was administered 5 days per week, and the clinical arthritis score was evaluated continuously. At termination, bone mineral density was analysed, paws were collected for histological examination and sera were analysed for markers of bone and cartilage turnover, as well as antibodies to type II collagen and levels of interleukin (IL)-6. Treatment with raloxifene is beneficial in long-term treatment of established CIA. It hampers the disease severity and frequency, protects the joints from destruction and protects against the development of osteoporosis. The proinflammatory cytokine IL-6 was down-regulated in raloxifene-treated mice compared with controls. The serum levels of antibodies to collagen were not affected by raloxifene-treatment. Long-term treatment with raloxifene has both anti-arthritic and anti-osteoporotic effects in established experimental postmenopausal polyarthritis.