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

INTRODUCTION: The 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-gamma (IFN-gamma) in these processes. METHODS: MSC were isolated from bone marrow of DBA/1 wild type and IFN-gamma receptor knock-out (IFN-gammaR 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-gammaR KO MSC. RESULTS: Purity 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-gammaR 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-gammaR KO MSC were able to reduce the severity of CIA or the humoral or cellular immune response toward collagen type II. CONCLUSIONS: Whereas MSC inhibit anti-CD3-induced proliferation of T cells in vitro, an effect partially mediated by IFN-gamma, 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.status: publishe

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

Introduction Interleukin (IL)-17 is a pro-inflammatory cytokine in rheumatoid arthritis (RA) and collagen-induced arthritis ( CIA). Since interferon (IFN)-gamma inhibits Th17 cell development, IFN-gamma receptor knockout (IFN-gamma R 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-gamma on the effector mechanisms of IL-17 in an in vitro system was also investigated. Methods IFN-gamma R 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)-alpha and the expression of cytokines and chemokines were determined. Results A preventive anti-IL-17 antibody treatment inhibited CIA in IFN gamma R 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-alpha synergistically induced granulocyte chemotactic protein-2 (GCP-2), IL-6 and receptor activator of NF kappa B ligand (RANKL) in MEF. This induction was profoundly inhibited by IFN-gamma in a STAT-1 (signal transducer and activator of transcription-1)dependent way. Conclusions In the absence of IFN-gamma, IL-17 mediates its proinflammatory effects mainly through stimulatory effects on granulopoiesis, neutrophil infiltration and bone destruction. In vitro IFN-gamma profoundly inhibits the effector function of IL-17. Thus, aside from the well-known inhibition of the development of Th17 cells by IFN-gamma, this may be an additional mechanism through which IFN-gamma attenuates autoimmune diseases

Collagen-induced arthritis as an animal model for rheumatoid arthritis: role of IFN-gamma in immunomodulation by mesenchymal stem cells and in extra-articular manifestations.

Over an extended period of time, work in our laboratory focused on unravelling the role of interferon-γ (IFN-γ) in collagen-induced arthritis (CIA), a well characterised animal model for human rheumatoid arthritis (RA). IFN-γ was found to display anti-inflammatory properties in CIA since IFN-γ receptor knock-out (IFN-γR KO) mice developed CIA more rapidly and with a higher severity as compared to their wild-type counterparts. The protective effect of IFN-γ could be explained by its suppressive effects on neutrophil mobilisation, activation of macrophages/osteoclasts and its role in the activation of regulatory T cells. The dogma that IFN-γ acts as a proinflammatory cytokine was questioned by these findings. Moreover, IFN-γ could be considered a novel therapy for RA, a perception confirmed by numerous clinical trials concerning IFN-γ in RA. In the quest for novel therapies for RA, mesenchymal stem cells (MSCs) have recently been proposed. They are multipotent cells that combine interesting characteristics such as tissue regeneration and immunosuppression and might therefore prove to be ideal therapeutics for RA. Since IFN-γ has been identified as an activator of the immuosuppressive capacities of MSCs, in this thesis we investigated the potential of MSCs to suppress the joint pathology in CIA and the effect of IFN-γ on this suppression. Although the joint pathology in CIA resembles that of RA remarkably well, RA is associated with extra-articular manifestations in for instance lung tissue. In CIA no reports of pulmonary involvement were reported. In this thesis we therefore investigated whether mice with CIA would develop pulmonary manifestations and analysed the role of IFN-γ on this systemic pathology.MSCs were isolated from the bone marrow of wild-type and IFN-γR KO mice and after several passages homogenous populations of MSCs were obtained. By culturing anti-CD3-stimulated T cells in vitro in the presence of MSCs we could demonstrate that T cell responses were suppressed by MSCs in a dose-dependent manner. IFN-γR KO MSCs were less suppressive than wild-type MSCs, indicating an IFN-γ-dependent mechanism was involved in the T cell suppression. By stimulating MSCs with IFN-γ we could demonstrate that the production of programmed death ligand-1, inducible nitric oxide synthase and cyclo-oxygenase-2, but not indoleamine 2,3-dioxygenase was involved in the immunosuppression mediated by MSCs. Despite their potent in vitro suppressive effects, MSCs were unable to counteract in vivo anti-CD3-induced T cell proliferation in mice. MSCs also failed to suppress CIA, even when they were administered at multiple time points or via different routes. This was reflected in unchanged humoral and cellular responses towards collagen type II. Together these data demonstrate that the immunosuppressive potential of MSCs in vitro can not be extrapolated to an in vivo situation.When MSCs are considered as a therapeutic in RA, thorough investigation of the effect of MSCs on all cell types involved in the pathogenesis of RA is necessary. Therefore, we investigated the effect of MSCs on osteoclasts, the cell type responsible for bone degradation in the joints of RA patients. Using mouse embryo fibroblasts (MEFs) as a source of fibroblasts, we developed an in vitro co-culture system between MEFs and splenocytes from mice with CIA. We could demonstrate that MEFs could stimulate the development of osteoclasts from splenocytes even without addition of osteoclast growth factors. When MEFs were substituted by MSCs, no osteoclasts could be detected. Only when receptor activator of nuclear factor-kB ligand (RANKL), a potent osteoclast inducer, was added osteoclasts developed. In the presence of tumor necrosis factor-α (TNF-α), osteoclasts were also not formed. Thus, MSCs can only support osteoclast differentiation when RANKL is present. As a consequence, whether MSCs will induce osteoclast differentiation in the joint of RA patients will probably depend on the cytokine balance present in the joint and administration of these cels in inflammatory conditions (containing RANKL and TNF-α) possibly has detrimental effects.As a second main goal in this thesis, we investigated whether pulmonary manifestations, which regularly complicate RA, were also present in CIA in mice. On macroscopic examination of the lungs 21 days after the induction of CIA, lesions could be detected on the surface of the lungs in IFN-γR KO mice but not in wild-type mice. Elevated numbers of macrophages and neutrophils were detected in bronchoalveolar lavage fluid of IFN-γR KO mice. Upon histological examination of the lungs, perivascular and peribronchial lymphocytic infiltrates as well as subpleural nodular accumulations of neutrophils and histiocytes could be visualised in lungs of arthritic IFN-γR KO mice but not wild-type mice. In IFN-γR KO mice, this pulmonary infiltration was accompanied by elevated mRNA levels of proinflammatory cytokines and chemokines. Upon functional assessment of the lungs, impaired lung function was ascertained which presented as a stiffening of the lungs. Treating the mice with anti-TNF-α therapy resulted in a complete prevention of joint pathology and a partial but significant reduction of the pulmonary complications. These data indicate that extra-articular manifestations in CIA can be provoked in CIA by interfering with one cytokine signaling pathway i.e. IFN-γ.Previous doctoral research in our laboratory demonstrated that upon immunisation with complete Freund s adjuvant (CFA) without the cartilage antigen collagen type II, IFN-γR KO mice develop arthritic symptoms which are identical to the joint pathology observed in CIA. Upon examination of the lungs, we could demonstrate that IFN-γR KO mice with CFA-induced arthritis displayed similar pulmonary pathology as IFN-γR KO mice with CIA. An important component of CFA is heat-killed Mycobacterium butyricum. Since this M. butyricum is the common factor in both arthritis models, we focused our attention to the M. butyricum to find a possible cause of the lung involvement. After fluorescent labelling of the M. butyricum, we could determine that the M. butyricum was present in the lungs of immunised animals. Thus, from these data we can conclude that M. butyricum used for the induction of CIA and CFA-induced arthritis translocates to the lungs after immunisation of IFN-γR KO mice and might locally cause production of proinflammatory cytokines and chemokines, followed by pulmonary inflammation.Summarising our investigations, we can state that although MSCs display potent immunosuppressive potential in vitro, they were unable to counteract in vivo T cell proliferation or CIA. Furthermore, MSCs could, depending on the local cytokine balance, possibly stimulate the development of osteoclasts. These findings are important to keep in mind when considering MSCs as potential therapy for RA. Although CIA is generally considered not to be associated with extra-articular manifestations, we could demonstrate that IFN-γR KO mice with CIA did present with pulmonary pathology. This finding adds relevance to the animal model and offers clear future perspectives both on fundamental and clinical level. We feel that on fundamental level, the model is suited to investigate the association between environmental factors, such as smoking, citrullination in the lungs and the development of arthritis. The fact that these extra-articular manifestations mainly occur in mice that have a mutation in the IFN-γ receptor, might offer new perspectives to the clinic such as treatment of RA patients with IFN-γ, possibly combined with current therapies.status: publishe

Induction of Tolerance to Therapeutic Proteins With Antigen-Processing Independent T Cell Epitopes:Controlling Immune Responses to Biologics

The immune response to exogenous proteins can overcome the therapeutic benefits of immunotherapies and hamper the treatment of protein replacement therapies. One clear example of this is haemophilia A resulting from deleterious mutations in the FVIII gene. Replacement with serum derived or recombinant FVIII protein can cause anti-drug antibodies in 20-50% of individuals treated. The resulting inhibitor antibodies override the benefit of treatment and, at best, make life unpredictable for those treated. The only way to overcome the inhibitor issue is to reinstate immunological tolerance to the administered protein. Here we compare the various approaches that have been tested and focus on the use of antigen-processing independent T cell epitopes (apitopes) for tolerance induction. Apitopes are readily designed from any protein whether this is derived from a clotting factor, enzyme replacement therapy, gene therapy or therapeutic antibody

Ameliorated course of glucose-6-phosphate isomerase (G6PI)-induced arthritis in IFN-γ receptor knockout mice exposes an arthritis-promoting role of IFN-γ

The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen.status: publishe

Thrombin Generation in Zebrafish Blood

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis

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 Freund's 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 Freund's 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

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 Freund's 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 Freund's 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.status: publishe

Near Patient Thrombin Generation in Patients Undergoing Elective Cardiac Surgery

Background: Measuring thrombin generation (TG) in plasma increasingly gained attention as a diag-nostic tool in the field of thrombosis and hemostasis. To include the contribution of all blood cells, recently, the whole blood TG method was developed. Methods: We changed the calculation method of the standard calibrated automated thrombography (CAT) to a method only taking into account the data until the peak of TG, thereby considerably reducing the time from blood draw to result. By redesigning the method, the blood volume per test was reduced to 15 μL. Results: For all TG parameters, the interassay variation proved to be below 15%. The interindividual variation of all parameters was comparable to the CAT method. Thirty-three patients undergoing cardiothoracic surgery were included to investigate whether our assay correlates with postoperative blood loss. On dividing patients into severe and mild bleeders, significant differences between both groups were found for the peak endogenous thrombin potential (peakETP) and peak values deter-mined by our near patient device. Importantly, patients with a peakETP below the median experienced significantly more blood loss compared to those with a peakETP above the median. A similar division based on the peak as well as the body mass index of the patient yielded similar significant differences. A combination of the peakETP, the body mass index, and the lag time even resulted in a better predictor of blood loss compared to each parameter separately. Conclusions: Our adapted whole blood TG assay can be used near patients and is indicative for the amount of blood loss post cardiothoracic surgery. IMPACT STATEMENT The near patient thrombin generation device allows for a fast