Anneleen Avau

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.

Inflammatory gene expression profile and defective IFN‐γ and granzyme K in natural killer cells of systemic juvenile idiopathic arthritis patients

Systemic juvenile idiopathic arthritis (JIA) is an immunoinflammatory disease characterized by arthritis and systemic manifestations. The role of natural killer (NK) cells in the pathogenesis of systemic JIA remains unclear. The purpose of this study was to perform a comprehensive analysis of NK cell phenotype and functionality in patients with systemic JIA.

Mouse Cytomegalovirus Infection in BALB/c Mice Resembles Virus-Associated Secondary Hemophagocytic Lymphohistiocytosis and Shows a Pathogenesis Distinct from Primary Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immunological disorder that is characterized by systemic inflammation, widespread organ damage, and hypercytokinemia. Primary HLH is caused by mutations in granule-mediated cytotoxicity, whereas secondary HLH occurs, without a known genetic background, in a context of infections, malignancies, or autoimmune and autoinflammatory disorders. Clinical manifestations of both HLH subtypes are often precipitated by a viral infection, predominantly with Herpesviridae. Exploiting this knowledge, we established an animal model of virus-associated secondary HLH by infecting immunocompetent wild-type mice with the β-herpesvirus murine CMV. C57BL/6 mice developed a mild inflammatory phenotype, whereas BALB/c mice displayed the clinicopathologic features of HLH, as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia, and elevated serum levels of soluble CD25. BALB/c mice also developed lymphadenopathy, liver dysfunction, and decreased NK cell numbers. Lymphoid and myeloid cells were in a hyperactivated state. Nonetheless, depletion of CD8+ T cells could not inhibit or cure the HLH-like syndrome, highlighting a first dissimilarity from mouse models of primary HLH. Immune cell hyperactivation in BALB/c mice was accompanied by a cytokine storm. Notably, plasma levels of IFN-γ, a key pathogenic cytokine in models of primary HLH, were the highest. Nevertheless, murine CMV–infected IFN-γ–deficient mice still developed the aforementioned HLH-like symptoms. In fact, IFN-γ–deficient mice displayed a more complete spectrum of HLH, including splenomegaly, coagulopathy, and decreased NK cell cytotoxicity, indicating a regulatory role for IFN-γ in the pathogenesis of virus-associated secondary HLH as opposed to its central pathogenic role in primary HLH.

Cytokine balance and cytokine-driven natural killer cell dysfunction in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) is a severe inflammatory childhood disorder, characterized by a specific pattern of systemic features and a typical cytokine profile. Patients are at risk to develop macrophage activation syndrome (MAS), an acute life-threatening condition defined by excessive proliferation and activation of macrophages and T cells. Defects of unknown cause in the natural killer (NK) cell cytotoxic capacity are presumed to underlie the pathogenesis of MAS and have been detected in sJIA patients. Here, we provide an overview of the cytokine profiles in sJIA and related mouse models. We discuss the influence of cytokines on NK cell function, and hypothesize that NK cell dysfunction in sJIA is caused by altered cytokine profiles.

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

Therapeutic Potential of Interferon-γ and Its Antagonists in Autoinflammation: Lessons from Murine Models of Systemic Juvenile Idiopathic Arthritis and Macrophage Activation Syndrome

Interferon-γ (IFN-γ) affects immune responses in a complex fashion. Its immunostimulatory actions, such as macrophage activation and induction of T helper 1-type responsiveness, are widely acknowledged, however, as documented by a large body of literature, IFN-γ has also the potential to temper inflammatory processes via other pathways. In autoimmune and autoinflammatory disorders, IFN-γ can either play a disease-enforcing role or act as protective agent, depending on the nature of the disease. In animal models of any particular autoimmune disease, certain changes in the induction procedure can reverse the net outcome of introduction or ablation of IFN-γ. Here, we review the role of endogenous IFN-γ in inflammatory disorders and related murine models, with a focus on systemic juvenile idiopathic arthritis (sJIA) and macrophage activation syndrome (MAS). In particular, we discuss our recent findings in a mouse model of sJIA, in which endogenous IFN-γ acts as a regulatory agent, and compare with results from mouse models of MAS. Also, we elaborate on the complexity in the activity of IFN-γ and the resulting difficulty of predicting its value or that of its antagonists as treatment option.

IDO1 Deficiency Does Not Affect Disease in Mouse Models of Systemic Juvenile Idiopathic Arthritis and Secondary Hemophagocytic Lymphohistiocytosis

Objectives Indoleamine 2,3-dioxygenase-1 (IDO1) is an immune-modulatory enzyme that catalyzes the degradation of tryptophan (Trp) to kynurenine (Kyn) and is strongly induced by interferon (IFN)-γ. We previously reported highly increased levels of IFN-γ and corresponding IDO activity in patients with hemophagocytic lymphohistiocytosis (HLH), a hyper-inflammatory syndrome. On the other hand, IFN-γ and IDO were low in patients with systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory syndrome. As HLH can occur as a complication of sJIA, the opposing levels of both IFN-γ and IDO are remarkable. In animal models for sJIA and HLH, the role of IFN-γ differs from being protective to pathogenic. In this study, we aimed to unravel the role of IDO1 in the pathogenesis of sJIA and HLH. Methods Wild-type and IDO1-knockout (IDO1-KO) mice were used in 3 models of sJIA or HLH: complete Freund’s adjuvant (CFA)-injected mice developed an sJIA-like syndrome and secondary HLH (sHLH) was evoked by either repeated injection of unmethylated CpG oligonucleotide or by primary infection with mouse cytomegalovirus (MCMV). An anti-CD3-induced cytokine release syndrome was used as a non-sJIA/HLH control model. Results No differences were found in clinical, laboratory and hematological features of sJIA/HLH between wild-type and IDO1-KO mice. As IDO modulates the immune response via induction of regulatory T cells and inhibition of T cell proliferation, we investigated both features in a T cell-triggered cytokine release syndrome. Again, no differences were observed in serum cytokine levels, percentages of regulatory T cells, nor of proliferating or apoptotic thymocytes and lymph node cells. Conclusions Our data demonstrate that IDO1 deficiency does not affect inflammation in sJIA, sHLH and a T cell-triggered cytokine release model. We hypothesize that other tryptophan-catabolizing enzymes like IDO2 and tryptophan 2,3-dioxygenase (TDO) might compensate for the lack of IDO1.

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