Stéphanie Put

SPECT Imaging of Joint Inflammation with Nanobodies Targeting the Macrophage Mannose Receptor in a Mouse Model for Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease occurring in approximately 1% of the worldwide population. The disease primarily affects the joints, where inflammatory cells, such as macrophages, invade the synovium and cause cartilage and bone destruction. Currently, it is difficult to efficiently diagnose and monitor early-stage RA. In this study, we investigated whether SPECT/micro-CT imaging with 99mTc-labeled Nanobodies directed against the macrophage mannose receptor (MMR) is a useful tool for monitoring and quantifying joint inflammation in collagen-induced arthritis (CIA), a mouse model for RA. The expression of MMR was analyzed on macrophages and osteoclasts generated in vitro and in cells obtained from various organs from mice with CIA. Methods: CIA was induced in DBA/1 mice by injection of collagen type II in complete Freund adjuvant, and cell suspensions from the inflamed joints and other organs were obtained. Macrophages and osteoclasts were generated in vitro from bone marrow cells. Expression of MMR was quantified by quantitative polymerase chain reaction and flow cytometry with specific Nanobodies and conventional antibodies. SPECT/micro-CT imaging was performed with 99mTc-labeled MMR and control Nanobodies. Results: MMR was highly expressed on macrophages and to a lesser extent on osteoclasts generated in vitro. In mice with CIA, MMR expression was detected on cells from the bone marrow, lymph nodes, and spleen. In synovial fluid of arthritic joints, MMR was expressed on CD11b+F4/80+ macrophages. On in vivo SPECT/micro-CT imaging with consecutive injections of MMR and control Nanobodies, a strong MMR signal was seen in the knees, ankles, and toes of arthritic mice. Quantification of the SPECT imaging confirmed the specificity of the MMR signal in inflamed joints as compared with the control Nanobody. Dissection of the paws revealed an additional significant MMR signal in nonarthritic paws of affected mice (i.e., mice displaying symptoms of arthritis in other paws). Conclusion: Our data show that MMR is expressed on macrophages in vitro and in vivo in synovial fluid of inflamed paws, whereas expression is relatively low in other tissues. The use of Nanobodies against MMR in SPECT/micro-CT imaging generates the possibility to track inflammatory cells in vivo in arthritic joints.

Systemic juvenile idiopathic arthritis-like syndrome in mice following stimulation of the immune system with freund's complete adjuvant: Regulation by interferon-γ

Systemic juvenile idiopathic arthritis (JIA) is unique among the rheumatic diseases of childhood, given its distinctive systemic inflammatory character. Inappropriate control of innate immune responses following an initially harmless trigger is thought to account for the excessive inflammatory reaction. The aim of this study was to generate a similar systemic inflammatory syndrome in mice by injecting a relatively innocuous, yet persistent, immune system trigger: Freunds complete adjuvant (CFA), containing heat‐killed mycobacteria.

Molecular Imaging with Macrophage CRIg-Targeting Nanobodies for Early and Preclinical Diagnosis in a Mouse Model of Rheumatoid Arthritis

An accurate and noninvasive tracer able to detect molecular events underlying the development of rheumatoid arthritis (RA) would be useful for RA diagnosis and drug efficacy assessment. A complement receptor of the Ig superfamily (CRIg) is expressed on synovial macrophages of RA patients, making it an interesting target for molecular imaging of RA. We aim to develop a radiotracer for the visualization of CRIg in a mouse model for RA using radiolabeled single-domain variable antibody VHH fragments (Nanobodies). Methods: Quantitative polymerase chain reaction was used to locate CRIg expression in mice with collagen-induced arthritis (CIA). A Nanobody, NbV4m119, was generated to specifically target CRIg. Flow cytometry, phosphorimaging, and confocal microscopy were used to confirm NbVm119 binding to CRIg-positive cells. SPECT (SPECT/CT) was used to image arthritic lesions in the inflamed paws of 29 mice using 99mTc-NbV4m119 Nanobody. Results: CRIg is constitutively expressed in the liver and was found to be upregulated in synovial tissues of CIA mice. SPECT/CT imaging revealed that 99mTc-NbV4m119 specifically targeted CRIg-positive liver macrophages in naïve wild-type but not in CRIg−/− (CRIg knockout) mice. In CIA mice, 99mTc-NbV4m119 accumulation in arthritic lesions increased according to the severity of the inflammation. In the knees of mice with CIA, 99mTc-NbV4m119 was found to accumulate even before the onset of macroscopic clinical symptoms. Conclusion: SPECT/CT imaging with 99mTc-NbV4m119 visualizes joint inflammation in CIA. Furthermore, imaging could predict which mice will develop clinical symptoms during CIA. Consequently, imaging of joint inflammation with CRIg-specific Nanobodies offers perspectives for clinical applications in RA patients.

Molecular imaging of rheumatoid arthritis: emerging markers, tools, and techniques.

Early diagnosis and effective monitoring of rheumatoid arthritis (RA) are important for a positive outcome. Instant treatment often results in faster reduction of inflammation and, as a consequence, less structural damage. Anatomical imaging techniques have been in use for a long time, facilitating diagnosis and monitoring of RA. However, mere imaging of anatomical structures provides little information on the processes preceding changes in synovial tissue, cartilage, and bone. Molecular imaging might facilitate more effective diagnosis and monitoring in addition to providing new information on the disease pathogenesis. A limiting factor in the development of new molecular imaging techniques is the availability of suitable probes. Here, we review which cells and molecules can be targeted in the RA joint and discuss the advances that have been made in imaging of arthritis with a focus on such molecular targets as folate receptor, F4/80, macrophage mannose receptor, E-selectin, intercellular adhesion molecule-1, phosphatidylserine, and matrix metalloproteinases. In addition, we discuss a new tool that is being introduced in the field, namely the use of nanobodies as tracers. Finally, we describe additional molecules displaying specific features in joint inflammation and propose these as potential new molecular imaging targets, more specifically receptor activator of nuclear factor κB and its ligand, chemokine receptors, vascular cell adhesion molecule-1, αVβ3 integrin, P2X7 receptor, suppression of tumorigenicity 2, dendritic cell-specific transmembrane protein, and osteoclast-stimulatory transmembrane protein.

Macrophages have no lineage history of Foxp3 expression

To the editor: Forkhead box P3 (Foxp3) is a transcription factor critical for the differentiation of regulatory T cells (Tregs) and for the prevention of autoimmune disease. Although expression of Foxp3 was initially found to be restricted to CD4+CD25+ T cells,[1][1] some research groups have

Pulmonary inflammation in mice with collagen-induced arthritis is conditioned by complete Freund's adjuvant and regulated by endogenous IFN-γ

Following immunization with collagen II (CII) in complete Freunds adjuvant (CFA), DBA/1 mice develop arthritis of major joints. This collagen‐induced arthritis (CIA) is used as a model for rheumatoid arthritis (RA) in man. Inflammatory changes in lung tissue commonly occur in RA. However, evidence for pulmonary inflammation in CIA is scarce and ambiguous. Here, we demonstrate pulmonary inflammation accompanying CIA in wild‐type DBA/1 mice. In IFN‐γ receptor‐deficient (IFN‐γR KO) mice, inflammation was more frequent and more severe. Injection of CFA only (without CII) proved to be as efficient in eliciting pulmonary inflammation as immunization with CFA + CII, though being less effective in causing arthritis. Significant correlation in severity between joint and pulmonary involvement could not be demonstrated. Macroscopic, microscopic, and functional characteristics of pulmonary inflammation in the mice resembled those seen in human RA. Increased inflammation in IFN‐γR KO mice was accompanied by augmented expression of various cytokines and chemokines, as measured by RT‐PCR on affected tissue. Treatment with a TNF‐α inhibitor ameliorated lung pathology. We conclude that CIA in DBA/1 mice is accompanied by pulmonary inflammation. Although both disease processes are kept in check by endogenous IFN‐γ, lack of strict parallelism indicates that overlap in their pathogeneses is partial.

OP0054 Complete freund’s adjuvant induces, in interferon-gamma-deficient mice, a chronic inflammatory disease reminiscent of systemic juvenile idiopathic arthritis

Background Systemic juvenile idiopathic arthritis (sJIA) is a disease characterized by arthritis and systemic features such as fever, rash, lymphadenopathy and leukocytosis. The underlying pathogenic mechanism of sJIA is not understood but prolonged stimulation of immune cells and excessive production of cytokines are considered to be important in the pathogenesis of the disease. Objectives The lack of a suitable animal model is a major drawback in the investigation of the pathogenesis of sJIA. We examined whether complete Freund’s adjuvant (CFA), a widely used reagent for chronic stimulation of the immune system, can elicit sJIA-like features in mice. Since interferon-gamma (IFN-γ) exerts key functions in activating and regulating innate and adaptive immune responses and given the disputable role of IFN-γ in sJIA patients, we examined our hypothesis in wild type as well as IFN-γ-deficient mice. Methods IFN-γ-deficient (IFN-γ KO) and wild type mice were injected s.c. with CFA. Body weight, clinical signs of arthritis, complete blood cell counts and peripheral blood biochemistry were monitored. Inflammatory cytokine levels were measured by qPCR and ELISA. Flow cytometry and histology were performed on spleen, liver, bone marrow and lymph nodes. Results Consistent with the immune stimulating properties of CFA, both IFN-γ KO and wild type mice showed splenomegaly and lymphadenopathy from 10 days after CFA challenge onward. Both substrains also developed weight loss; however, while this was transient in wild type animals, it was prolonged and more severe in IFN-γ KO mice. Furthermore, a proportion of the IFN-γ KO, but not wild type mice developed skin rashes as well as redness and swelling in the joints, histologically consistent with prominent synovitis. Complete blood counts demonstrated an increase in platelets and neutrophils in both substrains of mice, the increase being more pronounced in IFN-γ KO mice. IL-6 serum levels were significantly elevated in CFA-challenged IFN-γ KO mice only. Interestingly, between 11 to 40 days post CFA injection, approximately 30% of IFN-γ KO mice developed cytopenia and anemia, as evident from their significantly decreased lymphocyte and red blood cell counts, and decreases in hemoglobin and hematocrit levels. Intriguingly, in CFA-challenged IFN-γ KO mice, bone marrow, spleen, liver and blood showed a prominent infiltration of macrophages, including increased numbers of hemophagocytic macrophages. Conclusions Upon challenge with CFA, IFN-γ KO mice developed clinical, biological and pathological features similar to those seen in patients with sJIA. A proportion of these mice also developed severe inflammation with wasting, anemia and hemophagocytosis, features that are seen in a subset of sJIA patients, diagnosed with macrophage activation syndrome (MAS). To our knowledge, this is the first animal model of sJIA showing an association with MAS. Our data demonstrate that IFN-γ is not required for chronic anemia and hemophagocytosis and challenge the concept that IFN-γ is the critical driver of MAS. Disclosure of Interest None Declared

SAT0075 The use of macrophage mannose receptor-targeting nanobodies and spect imaging to study joint inflammation in mice with collagen-induced arthritis

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that occurs in 0.5-1.0% of the population worldwide. The primary affected organ is the small diarthrodial joint, where the synovial membrane, cartilage and bone tissue will be damaged, ultimately leading to joint deformity and disability of the patient. In the pathogenesis of RA, the synovial membrane becomes hyperplastic and will be infiltrated with T cells, B cells, neutrophils and macrophages. A hallmark of RA is the progressive destruction of bone tissue caused by an elevated bone resorption by osteoclasts, multinuclear cells derived from the monocyte/macrophage lineage. Objectives Our goal was to provide a method to visualize and quantify joint inflammation by the use of an animal model of RA, namely collagen-induced arthritis (CIA). We focused on the macrophage mannose receptor (MMR), since this protein is a well described marker for macrophages, which are numerously present in inflamed tissues. Methods CIA was induced in DBA/1 mice by the injection of collagen type II in Complete Freund’s adjuvant. Flow cytometry and qPCR were used to study the expression of MMR in vitro in macrophages and osteoclasts and in vivo in CIA. SPECT/CT imaging with 99mTc-labeled nanobodies generated against MMR was performed to visualize and quantify MMR expression in the joints of mice. Results MMR expression was shown to be highly upregulated in cultures of bone marrow-derived macrophages and osteoclasts by qPCR and by flow cytometry using MMR-targeting nanobodies. Ex vivo, we identified MMR in lymph nodes, spleen and bone marrow of naïve and arthritic mice. Interestingly, we detected expression of MMR in the synovial fluid, and to a lesser extent in synovium, of mice with CIA. More specifically, MMR was present on CD11b+F4/80+ macrophages isolated from the synovial fluid of the inflamed joints. SPECT/CT imaging was used to detect MMR in vivo in mice with CIA. Therefore, nanobodies against MMR were radioactively labeled with 99mTc, while nanobodies targeting a bacterial enzyme were used as controls. We observed high signals of MMR in lymph nodes, spleen and liver in naïve conditions as well as after immunization. Importantly, the joints of arthritic mice displayed high retention of MMR nanobody. The signal from SPECT imaging was significantly higher in mice with arthritic symptoms compared to naïve animals or immunized mice without clinical symptoms. Conclusions The use of MMR nanobodies in SPECT/CT imaging generates the possibility to track and quantify inflammatory macrophages in vivo in arthritic joints. In vivo quantification of joint inflammation by non-invasive techniques would be a great help in diagnosis and monitoring of disease processes as well as testing the efficiency of (new) drugs. Disclosure of Interest None Declared