Tania Mitera

Proinflammatory role of the Th17 cytokine interleukin-22 in collagen-induced arthritis in C57BL/6 mice.

OBJECTIVE To investigate the role of interleukin-22 (IL-22) in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis. METHODS C57BL/6 mice were immunized with type II collagen (CII) in Freunds incomplete adjuvant with added Mycobacterium tuberculosis, and levels of IL-22 and its specific receptor, IL-22 receptor type I (IL-22RI), were measured in sera and tissue by enzyme-linked immunosorbent assay and real-time quantitative polymerase chain reaction analysis. Clinical and histologic signs of arthritis were recorded and compared with those in C57BL/6 mice deficient in the IL-22 gene (IL-22(-/-)). Humoral and cellular immune responses against CII were analyzed. In vitro osteoclastogenesis assays were performed on splenocytes. RESULTS Upon immunization with CII in Freunds incomplete adjuvant plus heat-killed Mycobacterium tuberculosis, sera from C57BL/6 mice were found to contain high levels of IL-22, and the specific IL-22RI was expressed in lymphoid tissue, including splenocytes. IL-22(-/-) mice were less susceptible to CIA than were wild-type mice, as evidenced by their decreased incidence of arthritis and decreased pannus formation. Remarkably, the less severe form of arthritis in IL-22(-/-) mice was associated with increased production of CII-specific and total IgG antibodies, whereas cellular CII responses were unchanged. In vitro, IL-22 was found to promote osteoclastogenesis, a process that might contribute to its proinflammatory activity in CIA. CONCLUSION Endogenous IL-22 plays a proinflammatory role in CIA in C57BL/6 mice. Our data also indicate that IL-22 promotes osteoclastogenesis and regulates antibody production.

Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freunds adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4+CD25+ Treg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of Treg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4+CD25+ T cells in the central and peripheral lymphoid tissues. In addition, CD4+CD25+ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4+CD25+ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for Treg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both Treg cells and accessory cells. Our results demonstrate that the decrease in Treg cell activity in CIA is counter-regulated by endogenous IFN-γ.

Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis

IntroductionThe goal of this study is to analyze the potential immunosuppressive properties of mesenchymal stem cells (MSC) on T cell proliferation and in collagen-induced arthritis (CIA). An additional aim is to investigate the role of interferon-γ (IFN-γ) in these processes.MethodsMSC were isolated from bone marrow of DBA/1 wild type and IFN-γ receptor knock-out (IFN-γR KO) mice and expanded in vitro. Proliferation of anti-CD3-stimulated CD4+ T cells in the presence or absence of MSC was evaluated by thymidine incorporation. CIA was induced in DBA/1 mice and animals were treated with MSC by intravenous or intraperitoneal injections of wild type or IFN-γR KO MSC.ResultsPurity of enriched MSC cultures was evaluated by flow cytometry and their ability to differentiate into osteoblasts and adipocytes. In vitro, wild type MSC dose-dependently suppressed anti-CD3-induced T cell proliferation whereas IFN-γR KO MSC had a significantly lower inhibitory potential. A role for inducible nitric oxide (iNOS), programmed death ligand-1 (PD-L1) and prostaglandin E2 (PGE2), but not indoleamine 2,3-dioxigenase (IDO), in the T cell inhibition was demonstrated. In vivo, neither wild type nor IFN-γR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II.ConclusionsWhereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-γ, MSC do not influence in vivo T cell proliferation nor the disease course of CIA. Thus there is a clear discrepancy between the in vitro and in vivo effects of MSC on T cell proliferation and CIA.

Activated CD4+CD25+ regulatory T cells inhibit osteoclastogenesis and collagen-induced arthritis

Objectives: Patients with rheumatoid arthritis (RA) have defective CD4+CD25+ regulatory T (Treg) cells and increased osteoclastogenesis. A similar situation has been described in collagen-induced arthritis (CIA). In this study, it was investigated whether a single transfer of polyclonally activated Treg cells inhibits CIA and osteoclastogenesis. Methods: Purified Treg cells were expanded in vitro with anti-CD3 and anti-CD28 antibody-coated beads and injected into DBA/1 mice. Mice were immunised with collagen type II (CII) in complete Freund adjuvant (CFA) and scores of arthritis were recorded. In vitro osteoclastogenesis assays were performed on splenocytes by stimulation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)κB ligand (RANKL). Levels of anti-CII antibody and cytokines were determined in the supernatant using ELISA and Bio-Plex protein array system. Results: It was found that 106 activated Treg cells significantly counteracted the development of CIA, which was accompanied by decreased serum levels of TNFα and IL6, but not by inhibition of autoimmune antibody responses. The differentiation of osteoclasts in splenocyte cultures was significantly reduced in the presence of prestimulated Treg cells. Expression of cytokines that are described to inhibit osteoclastogenesis, including granulocyte macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ, interleukin (IL)5 and IL10, were dramatically increased upon addition of Treg cells. Furthermore, splenocytes from mice that had been treated with Treg cells displayed an impaired capacity to develop into mature osteoclasts, suggesting that Treg cells abrogated osteoclastogenesis in vivo. Conclusions: Activated CD4+CD25+ Treg cells improve clinical symptoms of CIA, regulate cytokine production and inhibit osteoclastogenesis in vitro and in vivo.

Anti‐IL‐12 antibody prevents the development and progression of collagen‐induced arthritis in IFN‐γ receptor‐deficient mice

In several models of inflammation, including collagen‐induced arthritis (CIA), the disease‐promoting effect of IL‐12 has been attributed to its well‐known ability to produce IFN‐γ. However, IFN‐γ receptor knockout (IFN‐γ R KO) mice of the DBA/1 strain have been reported to be more susceptible to CIA than corresponding wild‐type mice, indicating the existence of an IFN‐γ‐mediated protective pathway in this model. In the present study the development of CIA was found to be completely prevented by pretreatment with a neutralizing anti‐IL‐12 antibody, not only in wild‐type, but significantly also in IFN‐γ R KO mice. In both strains of mice, the protective effect of anti‐IL‐12 was associated with lower production of anti‐collagen type II antibodies. In vivo stimulation with anti‐CD3 antibody in arthritic IFN‐γ R KO mice resulted in production of higher levels of circulating IFN‐γ, TNF and IL‐2 than in corresponding control mice that had not received the arthritis‐inducing immunization. This was not the case in arthritis‐developing wild‐type mice. Furthermore, the protective effect of anti‐IL‐12 antibody in mutant, but not in wild‐type mice, was associated with lower circulating IFN‐γ, TNF and IL‐2 and higher IL‐4 and IL‐5 cytokine levels following an anti‐CD3 challenge. The data indicate that IL‐12 promotes the development of arthritis independently of its ability to induce or favor production of IFN‐γ. In fact, any IFN‐γ produced in the course of the disease process rather exerts a protective effect. Furthermore, our study suggests that, in the absence of a functional IFN‐γ system, endogenous IL‐12 exerts its disease‐promoting effect by favoring production of other Th1‐associated cytokines (IL‐2 and TNF), by inhibiting development of IL‐4‐ and IL‐5‐producing T cells and by stimulating production of anti‐collagen autoantibodies.

Effector mechanisms of interleukin-17 in collagen-induced arthritis in the absence of interferon-gamma and counteraction by interferon-gamma

IntroductionInterleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). Since interferon (IFN)-γ inhibits Th17 cell development, IFN-γ receptor knockout (IFN-γR KO) mice develop CIA more readily. We took advantage of this model to analyse the mechanisms of action of IL-17 in arthritis. The role of IFN-γ on the effector mechanisms of IL-17 in an in vitro system was also investigated.MethodsIFN-γR KO mice induced for CIA were treated with anti-IL-17 or control antibody. The collagen type II (CII)-specific humoral and cellular autoimmune responses, myelopoiesis, osteoclastogenesis, and systemic cytokine production were determined. Mouse embryo fibroblasts (MEF) were stimulated with IL-17, tumor necrosis factor (TNF)-α and the expression of cytokines and chemokines were determined.ResultsA preventive anti-IL-17 antibody treatment inhibited CIA in IFNγR KO mice. In the joints of anti-IL-17-treated mice, neutrophil influx and bone destruction were absent. Treatment reduced the cellular autoimmune response as well as the splenic expansion of CD11b+ cells, and production of myelopoietic cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-6. IL-17 and TNF-α synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NFκB ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-γ in a STAT-1 (signal transducer and activator of transcription-1)-dependent way.ConclusionsIn the absence of IFN-γ, IL-17 mediates its pro-inflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-γ profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-γ, this may be an additional mechanism through which IFN-γ attenuates autoimmune diseases.

Protective role of IFN‐γ in collagen‐induced arthritis conferred by inhibition of mycobacteria‐induced granulocyte chemotactic protein‐2 production

Mice with a disrupted IFN‐γ system are remarkably susceptible to experimental autoimmune diseases, such as collagen‐induced arthritis (CIA), which rely on the use of CFA. The inflammatory lesions of these IFN‐γ knockout (KO) mice are characterized by an excessive proportion of neutrophils. Here, we show that the increased severity of CIA in IFN‐γR KO as compared with wild‐type mice is accompanied by increased levels of the CXC chemokine granulocyte chemotactic protein‐2 (GCP‐2), a major neutrophil‐attracting chemokine in mice. We demonstrated that the heat‐killed mycobacteria present in CFA elicited production of GCP‐2 in mouse embryo fibroblast cultures and that this production was inhibited by IFN‐γ. Inhibition of GCP‐2 production by IFN‐γ was STAT‐1‐dependent. IFN‐γ receptor KO mice treated with neutralizing anti‐GCP‐2 antibodies were protected from CIA, indicating the in vivo importance of GCP‐2 in the pathogenesis of CIA. Our data support the notion that one of the mechanisms whereby endogenous IFN‐γ mitigates the manifestations of CIA consists of inhibiting production of GCP‐2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation. These results may also be applicable to other experimental models of autoimmunity that rely on the use of CFA.

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.

Emergence of cowpox: study of the virulence of clinical strains and evaluation of antivirals.

The last years, cowpox infections are being increasingly reported through Eurasia. Cowpox viruses (CPXVs) have been reported to have different genotypes and may be subdivided in at least five genetically distinct monophyletic clusters. However, little is known about their in vitro and in vivo features. In this report, five genetically diverse CPXVs, including one reference strain (CPXV strain Brighton) and four clinical isolates from human and animal cases, were compared with regard to growth in cells, pathogenicity in mice and inhibition by antivirals. While all CPXVs replicated similarly in vitro and showed comparable antiviral susceptibility, marked discrepancies were seen in vivo, including differences in virulence with recorded mortality rates of 0%, 20% and 100%. The four CPXV clinical isolates appeared less pathogenic than two reference strains, CPXV Brighton and vaccinia virus Western-Reserve. Disease severity seemed to correlate with high viral DNA loads in several organs, virus titers in lung tissues and levels of IL-6 cytokine in the sera. Our study highlighted that the species CPXV consists of viruses that not only differ considerably in their genotypes but also in their in vivo phenotypes, indicating that CPXVs should not be longer classified as a single species. Lung virus titers and IL-6 cytokine level in mice may be used as biomarkers for predicting disease severity. We further demonstrated the potential benefit of cidofovir, CMX001 and ST-246 use as antiviral therapy.