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

Study of Camelpox Virus Pathogenesis in Athymic Nude Mice

Camelpox virus (CMLV) is the closest known orthopoxvirus genetically related to variola virus. So far, CMLV was restricted to camelids but, recently, three human cases of camelpox have been described in India, highlighting the need to pursue research on its pathogenesis, which has been hampered by the lack of small animal models. Here, we confirm that NMRI immunocompetent mice are resistant to intranasal (i.n.) CMLV infection. However, we demonstrate that CMLV induced a severe disease following i.n. challenge of athymic nude mice, which was accompanied with a failure in gaining weight, leading to euthanasia of the animals. On the other hand, intracutaneous (i.c.) infection resulted in disease development without impacting the body weight evolution. CMLV replication in tissues and body fluids was confirmed in the two models. We further analyzed innate immune and B cell responses induced in the spleen and draining lymph nodes after exposure to CMLV. In both models, strong increases in CD11b+F4/80+ macrophages were seen in the spleen, while neutrophils, NK and B cell responses varied between the routes of infection. In the lymph nodes, the magnitude of CD11c+CD8α+ lymphoid and CD11c+CD11b+ myeloid dendritic cell responses increased in i.n. challenged animals. Analysis of cytokine profiles revealed significant increases of interleukin (IL)-6 and IL-18 in the sera of infected animals, while those of other cytokines were similar to uninfected controls. The efficacy of two antivirals (cidofovir or HPMPC, and its 2, 6-diaminopurine analog) was evaluated in both models. HPMPC was the most effective molecule affording 100% protection from morbidity. It appeared that both treatments did not affect immune cell responses or cytokine expression. In conclusion, we demonstrated that immunodeficient mice are permissive for CMLV propagation. These results provide a basis for studying the pathogenesis of CMLV, as well as for evaluating potential antiviral therapies in an immunodeficiency context

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 Freund's 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(+ )T(reg )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 T(reg )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 T(reg )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 T(reg )cells and accessory cells. Our results demonstrate that the decrease in T(reg )cell activity in CIA is counter-regulated by endogenous IFN-γ

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

Acute primary infection with mouse cytomegalovirus represents a natural animal model for virus-associated secondary hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a complex, life-threatening immune disorder, characterized by systemic inflammation, an out-of-control cytokine storm and widespread organ damage. In contrast to primary HLH, secondary HLH occurs without any known mutation in granule-mediated cytotoxicity, in a context of infections, malignancies or autoimmune/autoinflammatory disorders. Both subtypes are generally precipitated by an infectious agent, predominantly a member of the Herpesviridae. Exploiting this knowledge, we created an animal model for virus-associated secondary HLH by administering mouse cytomegalovirus (MCMV) to immunocompetent wild-type mice. Infected mice displayed the clinicopathologic features of HLH as set forth in the Histiocyte Society diagnostic guidelines: fever, cytopenia, hemophagocytosis, hyperferritinemia and elevated serum levels of soluble CD25. Additionally, mice developed lymphadenopathy, coagulopathy, liver dysfunction and hypercytokinemia. Strikingly, MCMV-infected interferon (IFN)-gamma-deficient mice were more prone to develop HLH-like symptoms, challenging the major pathogenic role attributed to IFN-gamma in most mouse models of primary HLH. As secondary HLH is known to occur in patients with primary infection or reactivation of human cytomegalovirus, our research demonstrates MCMV infection of wild-type mice as a promising natural model with utility for elucidating the poorly understood pathogenesis of secondary HLH and exploring novel treatment options.status: publishe

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

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

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