Mattias N. D. Svensson

Fms-like tyrosine kinase 3 ligand controls formation of regulatory T cells in autoimmune arthritis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is known as the primary differentiation and survival factor for dendritic cells (DCs). Furthermore, Flt3L is involved in the homeostatic feedback loop between DCs and regulatory T cell (Treg). We have previously shown that Flt3L accumulates in the synovial fluid in rheumatoid arthritis (RA) and that local exposure to Flt3L aggravates arthritis in mice, suggesting a possible involvement in RA pathogenesis. In the present study we investigated the role of Flt3L on DC populations, Tregs as well as inflammatory responses in experimental antigen-induced arthritis. Arthritis was induced in mBSA-immunized mice by local knee injection of mBSA and Flt3L was provided by daily intraperitoneal injections. Flow cytometry analysis of spleen and lymph nodes revealed an increased formation of DCs and subsequently Tregs in mice treated with Flt3L. Flt3L-treatment was also associated with a reduced production of mBSA specific antibodies and reduced levels of the pro-inflammatory cytokines IL-6 and TNF-α. Morphological evaluation of mBSA injected joints revealed reduced joint destruction in Flt3L treated mice. The role of DCs in mBSA arthritis was further challenged in an adoptive transfer experiment. Transfer of DCs in combination with T-cells from mBSA immunized mice, predisposed naïve recipients for arthritis and production of mBSA specific antibodies. We provide experimental evidence that Flt3L has potent immunoregulatory properties. Flt3L facilitates formation of Treg cells and by this mechanism reduces severity of antigen-induced arthritis in mice. We suggest that high systemic levels of Flt3L have potential to modulate autoreactivity and autoimmunity.

IL-1 Receptor Antagonist Treatment Aggravates Staphylococcal Septic Arthritis and Sepsis in Mice

Background Interleukin-1 receptor antagonist (IL-1Ra) is the primary therapy against autoinflammatory syndromes with robust efficacy in reducing systemic inflammation and associated organ injury. However, patients receiving IL-1Ra might be at increased risk of acquiring serious infections. Aims To study whether IL-1Ra treatment deteriorates Staphylococcus aureus (S. aureus) septic arthritis and sepsis in mice. Method NMRI mice were treated with anakinra (IL-1Ra) daily for 7 days before intravenous inoculation with S. aureus strain Newman in both arthritogenic and lethal doses. The clinical course of septic arthritis, histopathological and radiological changes of the joints, as well as the mortality were compared between IL-1Ra treated and control groups. Results IL-1Ra treated mice developed more frequent and severe clinical septic arthritis. Also, the frequency of polyarthritis was significantly higher in the mice receiving IL-1Ra therapy. In line with the data from clinical arthritis, both histological and radiological signs of septic arthritis were more pronounced in IL-1Ra treated group compared to controls. Importantly, the mortality of IL-1Ra treated mice was significantly higher than PBS treated controls. Conclusion IL-1Ra treatment significantly aggravated S. aureus induced septic arthritis and increased the mortality in these mice.

CTLA4 Immunoglobulin but Not Anti–Tumor Necrosis Factor Therapy Promotes Staphylococcal Septic Arthritis in Mice

BACKGROUNDnThe development of biologics has greatly increased the quality of life and the life expectancy of many patients with rheumatoid arthritis. However, a large number of these patients have an increased risk of developing serious infections. The aim of this study was to examine differential effects of anti-tumor necrosis factor (TNF) treatment and CTLA4 immunoglobulin (Ig) treatment on both immunological response and host defense in a murine model of septic arthritis.nnnMETHODSnAbatacept (CTLA4-Ig), etanercept (anti-TNF), or phosphate-buffered saline were given to NMRI mice intravenously inoculated with Staphylococcus aureus. The clinical course of septic arthritis and histopathological and radiological changes of joints were compared among the groups.nnnRESULTSnMice receiving CTLA4-Ig treatment had more-severe septic arthritis, compared with controls and mice receiving anti-TNF treatment. Anti-TNF treatment led to more-severe weight loss and kidney abscesses, as well as a higher bacterial burden in the kidneys. Mice receiving CTLA4-Ig therapy had lower serum levels of interleukin 4, whereas mice receiving anti-TNF therapy had higher levels of TNF-α. Both iNOS and arginase-1 expression were reduced in peritoneal macrophages from mice receiving CTLA4-Ig, compared with expression in the anti-TNF group.nnnCONCLUSIONSnCTLA4-Ig therapy significantly increased the susceptibility to S. aureus septic arthritis in mice, whereas anti-TNF therapy deteriorated host bacterial clearance, resulting in more-severe weight loss and kidney abscesses.

Down-regulation of survivin alleviates experimental arthritis

Survivin is a proto‐oncogene that regulates cell division and apoptosis. It is a molecular marker of cancer. Recently, survivin has emerged as a feature of RA, associated with severe joint damage and poor treatment response. The present study examined if inhibition of survivin affects experimental arthritis, which was induced in mBSA‐immunized mice by an injection of mBSA in the knee joint or developed spontaneously in collagen type II‐immunized mice. The inhibition of survivin transcription by a lentivirus shRNA construct alleviated joint inflammation and reduced bone damage. The inhibition of survivin reduced the levels of metalloproteinases, β‐catenin, and vimentin, limiting the invasive capacity of synovia, while no inhibition of osteoclastogenesis could be found. The inhibition of survivin led to a p53‐independent reduction of T cell proliferation and favored the transcription and activity of Blimp‐1, which limited IL‐2 production and facilitated formation of regulatory Foxp3+CD4+ and effector CD8+ T cells. The study shows that the inhibition of survivin is sufficient to reduce joint inflammation and bone damage in preclinical models of arthritis. Antiarthritic effects of survivin inhibition are related to p53‐independent control of lymphocyte proliferation.

Trabecular bone loss in collagen antibody-induced arthritis

IntroductionPostmenopausal women with rheumatoid arthritis (RA) have increased risk of developing osteoporosis due to chronic inflammation and estrogen deprivation. Collagen antibody-induced arthritis (CAIA), an experimental polyarthritis model representing the effector phase of arthritis, is mainly mediated by the innate immune system. Compared to the widely used collagen-induced arthritis model, CAIA is conveniently short and can be used in C57BL/6 mice, enabling studies with knock-out mice. However, the impact on bone of the CAIA model in C57BL/6 mice has not previously been studied. Therefore, the aim of this study was to determine if CAIA can be used to study postmenopausal arthritis-induced osteoporosis.MethodsCAIA was induced by administration of collagen-type II antibodies and lipopolysaccharide to ovariectomized female C57BL/6J mice. Control mice received lipopolysaccharide, but no antibodies. Nine days later, femurs were collected for high-resolution micro-CT and histomorphometry. Serum was used to assess cartilage breakdown and levels of complement. Frequencies of immune cell subsets from bone marrow and lymph nodes were analyzed by flow cytometery.ResultsTrabecular bone mass was decreased and associated with increased number of osteoclasts per bone surface in the CAIA model. Also, the frequency of interleukin-17+ cells in lymph nodes was increased in CAIA.ConclusionThe present study show that CAIA, a short reproducible arthritis model that is compatible with C57BL/6 mice, is associated with increased number of osteoclasts and trabecular bone loss.

S100A4 regulates the Src-tyrosine kinase dependent differentiation of Th17 cells in rheumatoid arthritis

OBJECTIVESnTo evaluate the role of S100A4, a calcium-binding regulator of nonmuscle myosin assembly, for T-cell responses in rheumatoid arthritis.nnnMETHODSnArthritis was induced in the methylated bovine serum albumin (mBSA)-immunized mice lacking the entire S100A4 protein (S100A4KO) and in wild-type counterparts treated with short hairpin ribonucleic acid (shRNA)-lentiviral constructs targeting S100A4 (S100A4-shRNA). The severity of arthritis was evaluated morphologically. T-cell subsets were characterized by the expression of master transcription factors, and functionally by proliferation activity and cytokine production. The activity of the Scr-kinases Fyn and Lck was assessed by the autophosphorylation of C-terminal thyrosine and by the phosphorylation of the CD5 cytodomain. The interaction between S100A4 and the CD5 cytodomain was analysed by nuclear magnetic resonance spectrophotometry.nnnRESULTSnS100A4-deficient mice (S100A4KO and S100A4-shRNA) had significantly alleviated morphological signs of arthritis and joint damage. Leukocyte infiltrates in the arthritic joints of S100A4-deficient mice accumulated Foxp3(+) Treg cells, while the number of RORγt(+) and (pTyr705)STAT3(+) cells was reduced. S100A4-deficient mice had a limited formation of Th17-cells with low retinoic acid orphan receptor gamma t (RORγt) mRNA and IL17 production in T-cell cultures. S100A4-deficient mice had a low expression and activity of T-cell receptor (TCR) inhibitor CD5 and low (pTyr705)STAT3 (signal transducer and activator of transcription 3), which led to increased (pTyr352)ZAP-70 (theta-chain associated protein kinase of 70kDa), lymphocyte proliferation and production of IL2. In vitro experiments showed that S100A4 directly binds Lck and Fyn and reciprocally regulates their kinase activity towards the CD5 cytodomain. Spectrometry demonstrates an interaction between the CD5 cytodomain and EF2-binding sites of S100A4.nnnCONCLUSIONnThe present study demonstrates that S100A4 plays an important part in the pathogenesis of arthritis. It controls CD5-dependent differentiation of Th17 cells by regulating the activity of the Src-family kinases Lck and Fyn.

IGF-1R signalling contributes to IL-6 production and T cell dependent inflammation in rheumatoid arthritis

BACKGROUNDnSignalling through insulin-like growth factor 1 receptor (IGF-1R) is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated and supports expansion of the inflamed synovia.nnnAIMnIn the present study, we assess if disruption of IGF-1R signalling resolves arthritis.nnnMATERIAL AND METHODSnClinical associations of IGF-1R expression in leukocytes of the peripheral blood were studied in 84 RA patients. Consequences of the IGF-1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of insulin receptor substrates.nnnRESULTSnIn RA patients, high expression of IGF-1R in leukocytes was associated with systemic inflammation as verified by higher expression of NF-kB, serum levels of IL6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF-1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF-1R and STAT3 in synovia, and alleviated arthritis and joint damage in mice. It also reduced expression of IGF-1R and despaired ERK and Akt signalling in spleen T cells. This limited IL-6 production, changed RoRgt/FoxP3 balance and IL17 levels.nnnCONCLUSIONnIGF-1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF-1R on the level of insulin receptor substrates alleviates arthritis by restricting IL6-dependent formation of Th17 cells and may open for new treatment strategies in RA.

Impaired signaling through the Fms-like tyrosine kinase 3 receptor increases osteoclast formation and bone damage in arthritis

Osteoclasts are bone‐resorbing cells that accumulate in the joints of patients with rheumatoid arthritis causing severe bone damage. Fms‐like tyrosine kinase 3 ligand is enriched in the synovial fluid of patients with rheumatoid arthritis, and local exposure to Fms‐like tyrosine kinase 3 ligand aggravates arthritis in mice. Because Fms‐like tyrosine kinase 3 ligand has been suggested to facilitate osteoclast differentiation, we asked whether Fms‐like tyrosine kinase 3 ligand affects bone remodeling in arthritis. The effect of Fms‐like tyrosine kinase 3 signaling on osteoclast development was studied by immunohistochemistry in methylated bovine serum albumin–induced arthritis using mice that lack the gene for Flt3l (Flt3L−/−) and by an in vitro assay. Bone and joint changes were studied morphologically and by microcomputer tomography. We found that Flt3L−/− mice had increased accumulations of osteoclasts in the periarticular area of the arthritic joint. This triggered bone destruction and trabecular bone loss. The increased number of osteoclasts in Flt3L−/− mice may be a consequence of insufficient expression of interferon regulatory factor 8. Treatment of Flt3L−/− mice with Fms‐like tyrosine kinase 3 ligand increased expression of interferon regulatory factor 8, reduced the number of osteoclasts in arthritic mice, and promoted trabecular bone formation. Finally, the reduced number of regulatory T cells in the bone marrow of Flt3L−/− mice could further contribute to the increased osteoclastogenesis by reducing the ratio of regulatory T cells to T helper 17 cells. This study shows that Fms‐like tyrosine kinase 3 ligand may serve as a negative regulator of osteoclast development by promoting transcription of interferon regulatory factor 8 and sustaining a balance between protective regulatory T cells and pathogenic T helper 17 cells in the pathogenesis of arthritis.

Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination.

Significance The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development. Recent reports have shown that Flt3 is reexpressed on peripheral B cells during activation, however the function for Flt3 during B-cell activation is poorly understood. The present study describes a role for Flt3 during B-cell activation and antibody production. We show that Flt3 is reexpressed on activated germinal center B cells and that lack of functional Flt3 signaling in peripheral B cells results in a specific deficiency in production of the antibody subclass IgG1. Mechanistic studies indicate that Flt3 synergizes with the IL-4 receptor to promote proper activation of Stat6 and class switch to IgG1. This study provides evidence for a previously unknown function for Flt3 during peripheral B-cell activation. Switched antibody classes are important for efficient immune responses. Aberrant antibody production to otherwise harmless antigens may result in autoimmunity. The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has an important role during early B-cell development, but the role of Flt3 in peripheral B cells has not been assessed before. Herein we describe a previously unappreciated role for Flt3 in IgG1 class-switch recombination (CSR) and production. We show that Flt3 is reexpressed on B-cell lymphoma 6+ germinal center B cells in vivo and following LPS activation of peripheral B cells in vitro. Absence of Flt3 signaling in Flt3 ligand-deficient mice results in impaired IgG1 CSR and accumulation of IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed and functions in synergy with the common-gamma chain receptor family. B cells from Flt3 ligand-deficient mice have impaired IL-4R signaling, with reduced phosphorylation of signal transducer and activator of transcription (Stat) 6, and demonstrate a failure to initiate CSR to IgG1 with low expression of γ1 germ-line transcripts, resulting in impaired IgG1 production. Thus, functional synergy between Flt3 and IL-4R signaling is critical for Stat-mediated regulation of sterile γ1 germ-line transcripts and CSR to IgG1.

01.13 Igf1R signalling contributes to T cell dependent inflammation in rheumatoid arthritis

Background Signalling through insulin-like growth factor (IGF) 1 receptor is essential for cell survival, but may turn pathogenic in uncontrolled tissue growth in tumours. In rheumatoid arthritis (RA), the IGF-1R signalling is activated supporting expansion of the inflamed synovia. The aim of the present study was to understand the place of IGF-1R expression and signalling for development of adaptive immune responses in the setting of prospective RA cohort and in experimental arthritis. Material and methods Clinical associations of IGF1R expression in leukocytes of the peripheral blood were studied in 84 female RA patients with mean disease duration 8 years, all treated with methotrexate at the time of enrolment. Consequences of the IGF1R signalling inhibition for arthritis were studied in mBSA immunised Balb/c mice treated with NT157 compound promoting degradation of IRS-1/2. Results In RA patients, high expression of IGF1R in leukocytes was associated with and systemic inflammation as verified by higher transcription factor NF-kB, serum levels of IL-6 and erythrocyte sedimentation rate, and higher pain perception. Additionally, phosphorylated IGF1R and STAT3 enriched T cells infiltrate in RA synovia. Treatment with NT157 inhibited the phosphorylation of IGF1R and STAT3 in synovia, and alleviated arthritis and erosions in mice. It also reduced expression of IGF1R in spleen T cells and despaired ERK and Akt signalling. This limited the ability of mBSA-specific T cells to produce IL-6, IFNg and IL-17. The reduction of IL-6 and IL-17 levels was associated with changed RoRgt/FoxP3 balance. Conclusion IGF1R signalling contributes to T cell dependent inflammation in arthritis. Inhibition of IGF1R signalling alleviates arthritis by restricting IL-6 dependent formation of Th17 cells and may open for new treatment strategies in RA.