Systemic overexpression of interleukin-22 induces the negative immune-regulator SOCS3 and potently reduces experimental arthritis in mice.

OBJECTIVE: High levels of IL-22 are present in serum and synovial fluid of patients with RA. As both pro- and anti-inflammatory roles for IL-22 have been described in studies using animal models of RA, its exact function in arthritis remains poorly defined. With this study we aimed to further unravel the mechanism by which IL-22 exerts its effects and to decipher its therapeutic potential by overexpression of IL-22 either locally or systemically during experimental arthritis. METHODS: CIA was induced in DBA-1 mice by immunization and booster injection with type II collagen (col II). Before arthritis onset, IL-22 was overexpressed either locally by intra-articular injection or systemically by i.v. injection using an adenoviral vector and clinical arthritis was scored for a period of 10 days. Subsequently, joints were isolated for histological analysis of arthritis severity and mRNA and protein expression of various inflammatory mediators was determined in the synovium, spleen and serum. RESULTS: Local IL-22 overexpression did not alter arthritis pathology, whereas systemic overexpression of IL-22 potently reduced disease incidence, severity and pathology during CIA. Mice systemically overexpressing IL-22 showed strongly reduced serum cytokine levels of TNF-α and macrophage inflammatory protein 1α that correlated significantly with the enhanced expression of the negative immune regulator SOCS3 in the spleen. CONCLUSION: With this study, we revealed clear anti-inflammatory effects of systemic IL-22 overexpression during CIA. Additionally, we are the first to show that the protective effect of systemic IL-22 during experimental arthritis is likely orchestrated via upregulation of the negative regulator SOCS3.

Microbiota-Dependent Involvement of Th17 Cells in Murine Models of Inflammatory Arthritis.

OBJECTIVE: Intestinal microbiota are associated with the development of inflammatory arthritis. The aim of this study was to dissect intestinal mucosal immune responses in the preclinical phase of arthritis and determine whether the presence of Th17 cells, beyond involvement of the cytokine interleukin-17 (IL-17), is required for arthritis development, and whether the involvement of Th17 cells in arthritis depends on the composition of the host microbiota. METHODS: Mucosal T cell production of IL-17, interferon-γ, tumor necrosis factor α (TNFα), IL-22, and granulocyte-macrophage colony-stimulating factor (GM-CSF) was analyzed by flow cytometry and Luminex assay before arthritis onset in mice immunized to develop collagen-induced arthritis (CIA). Pathogenic features of arthritis in mice with CIA and mice with antigen-induced arthritis were compared between Th17 cell-deficient (CD4-Cre+ Rorcflox/flox ) and Th17 cell-sufficient (CD4-Cre- Rorcflox/flox ) mice. In addition, the impact of intestinal microbiota on the Th17 cell dependence of CIA was assessed. RESULTS: Lamina propria CD4 T cells were activated before the onset of arthritis in mice with CIA, with marked up-regulation of several cytokines, including IL-17A, TNFα, and GM-CSF. CD4-Cre+ Rorcflox/flox  mice showed a specific reduction in intestinal mucosal levels of Th17 cells and partially reduced levels of IL-17-producing CD8 T cells. However, total levels of IL-17A, mostly produced by γδ T cells and neutrophils, were unaffected. The severity of arthritis was significantly reduced in Th17 cell-deficient mice, suggesting that Th17 cells have additional, IL-17A-independent roles in inflammatory arthritis. Accordingly, antigen-stimulated T cells from Th17 cell-deficient mice produced less IL-17A, IL-17F, and GM-CSF. Importantly, the dependence of CIA on the involvement of Th17 cells was mitigated in the presence of an alternative microbiome. CONCLUSION: These data from murine models suggest that activation of mucosal immunity precedes the development of arthritis, and also that Th17 cells have a microbiota-dependent role in arthritis. Therefore, a microbiome-guided stratification of patients might improve the efficacy of Th17-targeted therapies.

Imaging fibroblast activation protein to monitor therapeutic effects of neutralizing interleukin-22 in collagen-induced arthritis.

Objectives: RA is a chronic autoimmune disease leading to progressive destruction of cartilage and bone. RA patients show elevated IL-22 levels and the amount of IL-22-producing Th cells positively correlates with the extent of erosive disease, suggesting a role for this cytokine in RA pathogenesis. The purpose of this study was to determine the feasibility of SPECT/CT imaging with 111In-labelled anti-fibroblast activation protein antibody (28H1) to monitor the therapeutic effect of neutralizing IL-22 in experimental arthritis. Methods: Mice (six mice/group) with CIA received anti-IL-22 or isotype control antibodies. To monitor therapeutic effects after treatment, SPECT/CT images were acquired 24 h after injection of 111In-28H1. Imaging results were compared with macroscopic, histologic and radiographic arthritis scores. Results: Neutralizing IL-22 before CIA onset effectively prevented arthritis development, reaching a disease incidence of only 50%, vs 100% in the control group. SPECT imaging showed significantly lower joint tracer uptake in mice treated early with anti-IL-22 antibodies compared with the control-treated group. Reduction of disease activity in those mice was confirmed by macroscopic, histological and radiographic pathology scores. However, when treatment was initiated in a later phase of CIA, progression of joint pathology could not be prevented. Conclusion: These findings suggest that IL-22 plays an important role in CIA development, and neutralizing this cytokine seems an attractive new strategy in RA treatment. Most importantly, SPECT/CT imaging with 111In-28H1 can be used to specifically monitor therapy responses, and is potentially more sensitive in disease monitoring than the gold standard method of macroscopic arthritis scoring.

Higher efficacy of anti-IL-6/IL-21 combination therapy compared to monotherapy in the induction phase of Th17-driven experimental arthritis.

Th17 cells and their cytokines are linked to the pathogenesis of rheumatoid arthritis, a chronic autoimmune disease characterized by joint inflammation. Th17 development is initiated by combined signaling of TGF-ß and IL-6 or IL-21, and can be reduced in the absence of either IL-6 or IL-21. The aim of this study was to assess whether combinatorial IL-6/IL-21 blockade would more potently inhibit Th17 development, and be more efficacious in treating arthritis than targeting either cytokine. We assessed in vitro Th17 differentiation efficacy in the absence of IL-6 and/or IL-21. To investigate in vivo effects of IL-6/IL-21 blockade on Th17 and arthritis development, antigen-induced arthritis (AIA) was induced in IL-6-/- x IL-21R-/- mice. The therapeutic potential of this combined blocking strategy was assessed by treating mice with collagen-induced arthritis (CIA) with anti-IL-6R antibodies and soluble (s)IL-21R.Fc. We demonstrated that combined IL-6/IL-21 blocking synergistically reduced in vitro Th17 differentiation. In mice with AIA, absence of IL-6 and IL-21 signaling more strongly reduced Th17 levels and resulted in stronger suppression of arthritis than the absence of either cytokine. Additionally, anti-IL-6/anti-IL-21 treatment of CIA mice during the arthritis induction phase reduced disease development more potent than IL-6 or IL-21 inhibition alone, as effective as anti-TNF treatment. Collectively, these results suggest dual IL-6/IL-21 inhibition may be a more efficacious therapeutic strategy compared to single cytokine blockade to suppress arthritis development.

In Vivo Imaging of Antileukemic Drug Asparaginase Reveals a Rapid Macrophage-Mediated Clearance from the Bone Marrow.

The antileukemic drug asparaginase, a key component in the treatment of acute lymphoblastic leukemia, acts by depleting asparagine from the blood. However, little is known about its pharmacokinetics, and mechanisms of therapy resistance are poorly understood. Here, we explored the in vivo biodistribution of radiolabeled asparaginase, using a combination of imaging and biochemical techniques, and provide evidence for tissue-specific clearance mechanisms, which could reduce the effectiveness of the drug at these specific sites. METHODS: In vivo localization of 111In-labeled Escherichia coli asparaginase was performed in C57BL/6 mice by both small-animal SPECT/CT and ex vivo biodistribution studies. Mice were treated with liposomal clodronate to investigate the effect of macrophage depletion on tracer localization and drug clearance in vivo. Moreover, macrophage cell line models RAW264.7 and THP-1, as well as knockout mice, were used to identify the cellular and molecular components controlling asparaginase pharmacokinetics. RESULTS: In vivo imaging and biodistribution studies showed a rapid accumulation of asparaginase in macrophage-rich tissues such as the liver, spleen, and in particular bone marrow. Clodronate-mediated depletion of phagocytic cells markedly prolonged the serum half-life of asparaginase in vivo and decreased drug uptake in these macrophage-rich organs. Immunohistochemistry and in vitro binding assays confirmed the involvement of macrophagelike cells in the uptake of asparaginase. We identified the activity of the lysosomal protease cathepsin B in macrophages as a rate-limiting factor in degrading asparaginase both in vitro and in vivo. CONCLUSION: We showed that asparaginase is rapidly cleared from the serum by liver-, spleen-, and bone marrow-resident phagocytic cells. As a consequence of this efficient uptake and protease-mediated degradation, particularly bone marrow-resident macrophages may provide a protective niche to leukemic cells.

The role of the Th17 cytokines IL-17 and IL-22 in Rheumatoid Arthritis pathogenesis and developments in cytokine immunotherapy.

Over the past few years, the importance of Interleukin (IL)-17 and T helper (Th)17 cells in the pathology of Rheumatoid Arthritis (RA) has become apparent. RA is a systemic autoimmune disease that affects up to 1% of the population worldwide. It is characterized by an inflamed, hyperplastic synovium with pannus formation, leading to bone and cartilage destruction in the joints. By the production of effector cytokines like IL-17 and IL-22, the T helper 17 subset protects the host against bacterial and fungal infections, but it can also promote the development of various autoimmune diseases like RA. Hence, the Th17 pathway recently became a very interesting target in RA treatment. Up to now, several therapies targeting the Th17 cells or its effector cytokines have been tested, or are currently under investigation. This review clarifies the role of Th17 cells and its cytokines in the pathogenesis of RA, and provides an overview of the clinical trials using immunotherapy to target this particular T helper subset or the two main effector cytokines by which the Th17 cells exert their function, IL-17 and IL-22.

Interleukin-21 receptor deficiency increases the initial toll-like receptor 2 response but protects against joint pathology by reducing Th1 and Th17 cells during streptococcal cell wall arthritis.

OBJECTIVE: The cytokine interleukin-21 (IL-21) can have both proinflammatory and immunosuppressive effects. The purpose of this study was to investigate the potential dual role of IL-21 in experimental arthritis in relation to Th17 cells. METHODS: Antigen-induced arthritis (AIA) and chronic streptococcal cell wall (SCW) arthritis were induced in IL-21 receptor-deficient (IL-21R(-/-) ) and wild-type mice. Knee joints, synovial tissue, and serum were analyzed for arthritis pathology and inflammatory markers. RESULTS: During AIA and chronic SCW arthritis, IL-21R deficiency protected against severe inflammation and joint destruction. This was accompanied by suppressed serum IgG1 levels and antigen-specific T cell responses. Levels of IL-17 were reduced during AIA, and synovial lymphocytes isolated during SCW arthritis for flow cytometry demonstrated that mainly IL-17+ interferon-γ (IFNγ)-positive T cells were reduced in IL-21R(-/-) mice. However, during the acute phases of SCW arthritis, significantly higher joint swelling scores were observed, consistent with enhanced tumor necrosis factor and IL-6 expression. Interestingly, IL-21R(-/-) mice were significantly less capable of up-regulating suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 messenger RNA. IL-21 stimulation also affected the Toll-like receptor 2 (TLR-2)/caspase recruitment domain 15 response to SCW fragments in vitro, indicating that impaired SOCS regulation in the absence of IL-21 signaling might contribute to the increased local activation during SCW arthritis. CONCLUSION: In contrast to the proinflammatory role of IL-21 in adaptive immunity, which drives IL-17+IFN+ cells and joint pathology during chronic experimental arthritis, IL-21 also has an important immunosuppressive role, presumably by inhibiting TLR signaling via SOCS-1 and SOCS-3. If this dual role of IL-21 in various immune processes is present in human disease, it could make IL-21 a difficult therapeutic target in rheumatoid arthritis.

GM-CSF as a therapeutic target in inflammatory diseases.

GM-CSF is a well-known haemopoietic growth factor that is used in the clinic to correct neutropaenia, usually as a result of chemotherapy. GM-CSF also has many pro-inflammatory functions and recent data implicates GM-CSF as a key factor in Th17 driven autoimmune inflammatory conditions. In this review we summarize the findings that have led to the development of GM-CSF antagonists for the treatment of autoimmune diseases like rheumatoid arthritis (RA) and discuss some results of recent clinical trials of these agents.

The Th17 pathway as a therapeutic target in rheumatoid arthritis and other autoimmune and inflammatory disorders.

Production of the pro-inflammatory cytokine interleukin (IL)-17 by Th17 cells and other cells of the immune system protects the host against bacterial and fungal infections, but also promotes the development of rheumatoid arthritis (RA) and other autoimmune and inflammatory disorders. Several biologicals targeting IL-17, the IL-17 receptor, or IL-17-related pathways are being tested in clinical trials, and might ultimately lead to better treatment for patients suffering from various IL-17-mediated disorders. In this review, we provide a clear overview of current knowledge on Th17 cell regulation and the main Th17 effector cytokines in relation to IL-17-mediated conditions, as well as on recent IL-17-related drug developments. We demonstrate that targeting the Th17 pathway is a promising treatment for rheumatoid arthritis and various other autoimmune and inflammatory diseases. However, improvements in technical developments assisting in the identification of patients suffering from IL-17-driven disease are needed to enable the application of tailor-made, personalized medicine.

IQCB1 mutations in patients with leber congenital amaurosis.

PURPOSE: Leber congenital amaurosis (LCA) is genetically heterogeneous, with 15 genes identified thus far, accounting for ∼70% of LCA patients. The aim of the present study was to identify new genetic causes of LCA. METHODS: Homozygosity mapping in >150 LCA patients of worldwide origin was performed with high-density SNP microarrays to identify new disease-causing genes. RESULTS: In three isolated LCA patients, the authors identified large homozygous regions on chromosome 3 encompassing the IQCB1 gene, which has been associated with Senior-Loken syndrome (SLSN), characterized by nephronophthisis and retinal degeneration. Mutation analysis of IQCB1 in these three patients and a subsequent cohort of 222 additional LCA patients identified frameshift and nonsense mutations in 11 patients diagnosed with LCA. On re-inspection of the patient's disease status, seven were found to have developed SLSN, but four maintained the diagnosis of LCA as the kidney function remained normal. CONCLUSIONS: Results show that the onset of renal failure in patients with IQCB1 mutations is highly variable, and that mutations are also found in LCA patients without nephronophthisis, rendering IQCB1 a new gene for LCA. However, these patients are at high risk for developing renal failure, which in early stages is often not recognized and can cause sudden death from fluid and electrolyte imbalance. It is therefore recommended that all LCA patients be screened for IQCB1 mutations, to follow them more closely for kidney disease.