UVM Theses and Dissertations
Format:
Print
Author:
Goodwin, Meagan
Dept./Program:
Cell and Molecular Biology Program
Year:
2011
Degree:
Ph. D.
Abstract:
Mesenchymal stromal cells (MSCs) are adult, multi-potent progenitor cells, capable of tri-lineage differentiation into bone, fat and cartilage. Recently, it has been shown that MSCs can also suppress immune responses by inhibiting proliferation, maturation and effector functions of T and B lymphocytes and dendritic cells. The use of allogeneic MSCs in animal models and clinical trials has been proven to be well-tolerated and successful in suppressing a variety of inflammatory diseases, including that of the lung.
Since allergic asthma is an immune-mediated inflammatory disease of the lung in which Th2 CD4 T lymphocytes are central to the pathogenesis, we hypothesized that systemic administration of MSCs would suppress allergic airways disease, the mouse model of asthma. To mduce allergic airways disease, mice were immunized with the antigen ovalbumin and the Th2-promoting adjuvant alum on days 0 and 7 followed by systemic administration of syngeneic or allogeneic MSCs, saline or control cell type. Mice were challenged with aerosolized ova on days 14, 15, and 16 and euthanized two days later at which point lung function and Th2-mediated inflammation was assessed. We found that administration of MSCs decreased airways hyperresponsiveness, numbers of BAL eosinophils and levels of Th2-associated cytokines. MSC administration also decreased airways inflammation as assessed by the number of peribronchial cell infiltrates.
Contrary to published in vitro data, MSCs do not inhibit CD4 lymphocyte proliferation or IL-4 production in vivo, however promote a Th1-mediated immune response as seen by the increase in Th1-associated antigen-specific immunoglobulins and IFNy production in ovastimulated CD4 lymphocytes. To assess whether the generation of a Th1-mediated response was necessary in order for the MSCs to exert their suppressive effects, allergic airways disease was induced in IFNyR -/-mice and MSCs or saline control were systemically administered immediately following immunization with ova/alum. We found that MSC administration was no longer effective in decreasing lung inflammation as assessed by BAL eosinophils levels of Th2-associated cytokines and numbers of peribronchial cell infiltrates. In conclusion, promotion of Th1differenation in antigen-specific CD4T lymphocytes is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNy-dependent process.
We next investigate the mechanisms by which MSCs promote a Th1-mediated response. We found that MSCs express mRNA for IL-18 (IFNy inducible factor) both in vitro and ex vivo however no measurable levels of protein were detected. Furthermore, CD4+ lymphocytes undergoing either polyclonal or antigen-specific stimulation in the presence of MSCs did not develop a Th1 phenotype. Systemic administration of MSCs did not result in detectable levels of the Th1-promoting cytokines IFNy or IL-12 in lung, liver, spleen or blood and MSC administration was still effective in abrogating allergic airways disease in mice when TLR9 deficient mice were used as recipients. Both of these results suggest that MSC administration does not result in the activation of an NK, NKT or dendritic cell. MSCs were able to internalize OVA and activate primary T lymphocytes in vitro, however no detectable levels of IFNy were detected s ggesting that while MSCs can activate T cells, they do not induce a Th1 phenotype, in vitro.
Since allergic asthma is an immune-mediated inflammatory disease of the lung in which Th2 CD4 T lymphocytes are central to the pathogenesis, we hypothesized that systemic administration of MSCs would suppress allergic airways disease, the mouse model of asthma. To mduce allergic airways disease, mice were immunized with the antigen ovalbumin and the Th2-promoting adjuvant alum on days 0 and 7 followed by systemic administration of syngeneic or allogeneic MSCs, saline or control cell type. Mice were challenged with aerosolized ova on days 14, 15, and 16 and euthanized two days later at which point lung function and Th2-mediated inflammation was assessed. We found that administration of MSCs decreased airways hyperresponsiveness, numbers of BAL eosinophils and levels of Th2-associated cytokines. MSC administration also decreased airways inflammation as assessed by the number of peribronchial cell infiltrates.
Contrary to published in vitro data, MSCs do not inhibit CD4 lymphocyte proliferation or IL-4 production in vivo, however promote a Th1-mediated immune response as seen by the increase in Th1-associated antigen-specific immunoglobulins and IFNy production in ovastimulated CD4 lymphocytes. To assess whether the generation of a Th1-mediated response was necessary in order for the MSCs to exert their suppressive effects, allergic airways disease was induced in IFNyR -/-mice and MSCs or saline control were systemically administered immediately following immunization with ova/alum. We found that MSC administration was no longer effective in decreasing lung inflammation as assessed by BAL eosinophils levels of Th2-associated cytokines and numbers of peribronchial cell infiltrates. In conclusion, promotion of Th1differenation in antigen-specific CD4T lymphocytes is sufficient to inhibit Th2-mediated allergic airways inflammation through an IFNy-dependent process.
We next investigate the mechanisms by which MSCs promote a Th1-mediated response. We found that MSCs express mRNA for IL-18 (IFNy inducible factor) both in vitro and ex vivo however no measurable levels of protein were detected. Furthermore, CD4+ lymphocytes undergoing either polyclonal or antigen-specific stimulation in the presence of MSCs did not develop a Th1 phenotype. Systemic administration of MSCs did not result in detectable levels of the Th1-promoting cytokines IFNy or IL-12 in lung, liver, spleen or blood and MSC administration was still effective in abrogating allergic airways disease in mice when TLR9 deficient mice were used as recipients. Both of these results suggest that MSC administration does not result in the activation of an NK, NKT or dendritic cell. MSCs were able to internalize OVA and activate primary T lymphocytes in vitro, however no detectable levels of IFNy were detected s ggesting that while MSCs can activate T cells, they do not induce a Th1 phenotype, in vitro.