Tove Eneljung

Reactive Oxygen Species Produced by the NADPH Oxidase 2 Complex in Monocytes Protect Mice from Bacterial Infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47phox) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN+ mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b+Ly6G+ cells defined as neutrophils. MN+ mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN− mice. Most strikingly, MN+ mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN− mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.

Interleukin-17A during Local and Systemic Staphylococcus aureus-Induced Arthritis in Mice

ABSTRACT Staphylococcus aureus is one of the dominant pathogens that induce septic arthritis in immunocompromised hosts, e.g., patients suffering from rheumatoid arthritis treated with immunosuppressive drugs. S. aureus-induced arthritis leads to severe joint destruction and high mortality despite antibiotic treatment. Recently, interleukin-17A (IL-17A) has been discovered to be an important mediator of aseptic arthritis both in mice and humans, but its function in S. aureus-induced arthritis is largely unknown. Here, we investigated the role of IL-17A in host defense against arthritis following systemic and local S. aureus infection in vivo. IL-17A knockout mice and wild-type mice were inoculated systemically (intravenously) or locally (intra-articularly) with S. aureus. During systemic infection, IL-17A knockout mice lost significantly more weight than the wild-type mice did, but no differences were found in the mortality rate. The absence of IL-17A had no impact on clinical arthritis development but led to increased histopathological erosivity late during systemic S. aureus infection. Bacterial clearance in kidneys was increased in IL-17A knockout mice compared to the level in wild-type mice only 1 day after bacterial inoculation. During systemic S. aureus infection, serum IL-17F protein levels and mRNA levels in the lymph nodes were elevated in the IL-17A knockout mice compared to the level in wild-type mice. In contrast to systemic infection, the IL-17A knockout mice had increased synovitis and erosions and locally decreased clearance of bacteria 3 days after local bacterial inoculation. On the basis of these findings, we suggest that IL-17A is more important in local host defense than in systemic host defense against S. aureus-induced arthritis.

Disease-dependent local IL-10 production ameliorates collagen induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterised by periods of flare and remission. Today’s treatment is based on continuous immunosuppression irrespective of the patient’s inflammatory status. When the disease is in remission the therapy is withdrawn but withdrawal attempts often results in inflammatory flares, and re-start of the therapy is commenced when the inflammation again is prominent which leads both to suffering and increased risk of tissue destruction. An attractive alternative treatment would provide a disease-regulated therapy that offers increased anti-inflammatory effect during flares and is inactive during periods of remission. To explore this concept we expressed the immunoregulatory cytokine interleukin (IL)-10 gene under the control of an inflammation dependent promoter in a mouse model of RA - collagen type II (CII) induced arthritis (CIA). Haematopoetic stem cells (HSCs) were transduced with lentiviral particles encoding the IL-10 gene (LNT-IL-10), or a green fluorescence protein (GFP) as control gene (LNT-GFP), driven by the inflammation-dependent IL-1/IL-6 promoter. Twelve weeks after transplantation of transduced HSCs into DBA/1 mice, CIA was induced. We found that LNT-IL-10 mice developed a reduced severity of arthritis compared to controls. The LNT-IL-10 mice exhibited both increased mRNA expression levels of IL-10 as well as increased amount of IL-10 produced by B cells and non-B APCs locally in the lymph nodes compared to controls. These findings were accompanied by increased mRNA expression of the IL-10 induced suppressor of cytokine signalling 1 (SOCS1) in lymph nodes and a decrease in the serum protein levels of IL-6. We also found a decrease in both frequency and number of B cells and serum levels of anti-CII antibodies. Thus, inflammation-dependent IL-10 therapy suppresses experimental autoimmune arthritis and is a promising candidate in the development of novel treatments for RA.

Antigen-Specific Gene Therapy after Immunisation Reduces the Severity of Collagen-Induced Arthritis

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

Interleukin 15 Mediates Joint Destruction in Staphylococcus Aureus Arthritis

BACKGROUND Staphylococcus aureus arthritis causes severe and rapid joint damage despite antibiotics. Thus, there is a need to identify new treatment targets in addition to antibiotics. Lately, interleukin 15 (IL-15) has been implicated both in osteoclastogenesis and in bacterial clearance-2 important issues in S. aureus-induced joint destruction. This has prompted us to investigate the importance of IL-15 in S. aureus-induced arthritis. METHODS Toxic shock syndrome toxin-1 producing S. aureus was intravenously inoculated in IL-15 knockout and wildtype mice and in wildtype mice treated with anti-IL-15 antibodies (aIL-15ab) or isotype control antibody. RESULTS Absence of IL-15, either in knockout mice or after treatment with aIL-15ab, significantly reduced weight loss compared with controls during the infection. The severity of synovitis and joint destruction was significantly decreased in IL-15 knockout and aIL-15ab treated mice compared with controls. In IL-15 knockout mice there was a reduced number of osteoclasts in the joints. The hosts ability to clear bacteria was not influenced in the IL-15 knockout mice, but significantly increased after treatment with aIL-15ab. CONCLUSIONS IL-15 is a mediator of joint destruction in S. aureus-induced arthritis and contributes to general morbidity, which makes this cytokine an interesting treatment target in addition to conventional antibiotics.

Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis

BackgroundThe mechanisms underlying tolerance induction and maintenance in autoimmune arthritis remain elusive. In a mouse model of rheumatoid arthritis, collagen type II (CII)-induced arthritis, we explore the contribution of B cells to antigen-specific tolerance.MethodsTo generate expression of the CII-peptide specifically on B-cell major histocompatibility complex type II, lentiviral-based gene therapy including a B-cell-specific Igk promoter was used.ResultsPresentation of the CII-peptide on B cells significantly reduced the frequency and severity of arthritis as well as the serum levels of CII -specific IgG antibodies. Further, both frequency and suppressive function of regulatory T cells were increased in tolerized mice. Adoptive transfer of regulatory T cells from tolerized mice to naïve mice ameliorated the development of CII-induced arthritis.ConclusionOur data suggest that endogenous presentation of the CII-peptide on B cells is one of the key contributors to arthritis tolerance induction and maintenance.

Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis

Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

Influence of Blue Mussel (Mytilus edulis) Intake on Disease Activity in Female Patients with Rheumatoid Arthritis: The MIRA Randomized Cross-Over Dietary Intervention

Rheumatoid Arthritis (RA) is a chronic inflammatory disease. This study evaluates the effect of blue mussel intake on disease activity and quality of life in women with RA. Thirty-nine women with established RA and a disease activity score 28 (DAS28) >3.0 were recruited to a randomized 2 × 11-week cross-over dietary intervention. The participants continued with their medication and habitual diet and exchanged one cooked meal a day, five days a week, with a meal including 75 g blue mussels or 75 g meat. Diets were switched after an eight week washout period. Data regarding quality of life (SF-36), blood lipids, erythrocyte sediment rate (ESR), C-reactive protein (CRP) and tender and swollen joints were examined at the start and end of each dietary period. Thirty women completed one period, and twenty-three completed both. Intake of the blue mussel diet led to a significant reduction of DAS28-CRP (p = 0.048), but not DAS28. The number of EULAR (European League Against Rheumatism) criteria moderate and good responders were higher when consuming blue mussel diet (p = 0.036). Blood lipids did not change. To conclude, blue mussel intake reduced disease symptoms in women with RA and improved perceived health. The reported effects need to be confirmed by non-patient reported outcomes, such as inflammation markers.

OP0185 Endogenous B Cell-Targeted Antigen Expression Induces Tolerance to Collagen Type II Arthritis in Mice

Background Rheumatoid arthritis (RA) is a destructive autoimmune disease. Today’s treatment is associated with side effects and only suppresses the disease. A safe and effective therapy would be to re-establish tolerance, i.e. the ability to recognize self. Here, we have investigated the use of gene therapy to establish tolerance in CIA, a mouse model of RA. We have previously shown that presentation of the collagen type II peptide on all types of antigen-presenting cells (APCs) can prevent development of arthritis and ameliorate ongoing disease (1). Here, we assess how targeting B cells affect tolerance induction in the CIA model. Objectives To induce tolerance in collagen induced arthritis (CIA) by endognous B cells expression of the tolerogen. Methods As gene therapy, self-inactivating lentiviral particles were used. The T cell epitope of rat collagen type II (CII) were cloned into the CLIP position of Invariant chain to allow presentation on MHC class II. As control the naturally occurring CLIP peptide was used. B cell-specific targeting was achieved by using an Ig kappa promoter. We also used B cells from mice where all types of antigen presenting cells (APCs) had been target using the spleen focus forming virus (SFFV) promoter. Haematopoetic stem cells were transduced with the lentiviral particles containing either of the constructs and transplanted into lethally irradiated DBA/1 recipient mice. After repopulation for at least 12 weeks, CIA was induced. In addition, B cells were isolated from non-immunised mice transplanted with the SFFV driven construct and transferred to naive recipients before immunisation. Results Expression of the CII epitope on all APCs abolishes the development of arthritis and decreases number of splenic plasma cells as well as the serum levels of anti-CII specific antibodies. Adoptive transfer of CD19+ B cells expressing the CII epitope derived from transplanted non-immunised mice to naïve recipients two days before CII immunisation delayed the onset and decreased the severity of arthritis compared to controls. Further, when B cells were specifically targeted using the Igk-promoter there was also a substantially decrease in arthritis development and progression. These findings were accompanied by an increased number of T regulatory cells early during the course of arthritis, and reduced serum levels of CII-specific IgG antibodies. Thus, B cells are important mediators of tolerance in CIA. Further studies are warranted to delined the exact mechanisms behind the findings, however B cell targeted therapies in RA provide interesting treatment options in a future. Conclusions Thus, B cells are important mediators of tolerance in CIA. Further studies are warranted to delined the exact mechanisms behind the findings, however B cell targeted therapies in RA provide interesting treatment options in a future. References Gjertsson I, et al. Molecular Therapy 2009 Disclosure of Interest None Declared