Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/ BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1b-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1β stimulation for 24 h. AS-1 treatment significantly attenuated IL-1β-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.

Osteoarthritis (OA) is the most prevalent arthritis in the world with increasing numbers of people expected to acquire the disease as the population ages. Therapies commonly used to manage the disease have limited efficacy and some carry significant risks. Current data suggest that the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) can alter the inflammatory response and cartilage erosion present in OA. Intra-articular gene expression of IL-1Ra has shown promising results in animal models to provide symptomatic improvement and minimize osteoarthritic changes. Orthogen AG (Dusseldorf, Germany) has developed a method to produce an autologous conditioned serum (ACS) rich in IL-1Ra marketed as Orthokine®. Study participants treated with ACS have improved pain and function; however, these results are preliminary and need confirmation. If ongoing trials prove that ACS can retard cartilage degeneration and reduce inflammation, the management of OA would be dramatically altered, perhaps providing a mechanism to prevent the disease or at least its progression.

Homeostasis of human T cells is regulated by many factors that control proliferation, differentiation of effector cells and generation of memory. Our current knowledge of the mechanisms controlling human T cell homeostasis in vivo is based on experiments in small animal models. However many differences exist between immune systems of mice and humans, including cell composition, function, and gene expression. Humanized mouse models have shown great value in the study of human immunobiology. I have used novel humanized mouse models to examine the role of human MHC (HLA) and human IL2 in CD8 T cell and CD4 regulatory T cell (Treg) homeostasis. To study human CD8 T cells I engrafted CD8 T cells from healthy donor PBMC into NOD-scid IL2rgnull (NSG) mice that lacked expression of murine MHC and that expressed HLA-A2. My data demonstrate that CD8 T cell survival and effector function required the presence of HLA-A2, helper function from human CD4 T cells and exogenous human IL2. To study human Treg homeostasis I used NSG mice engrafted with human fetal thymus and hematopoietic stem cells (BLT model). NSG-BLT mice support the growth of human thymic tissue and enable the efficient development of HLA-restricted Treg and conventional T cells. Using an AAV vector to express human IL2, I demonstrated that functional human Treg but not conventional T cells increased in number in NSG-BLT mice and that this coincided with increases in activated human NK cells. Overall my research has revealed that HLA and human IL2 have an essential role in human T cell survival and function in vivo.

Breast cancer is the second most common malignancy in the world, accounting for over 1.7 million new diagnoses and an estimated 500,000 deaths per year (1). Overexpression of the receptor tyrosine kinase ErbB2, also known as Her2 or Neu, occurs in over 30% of breast cancers and correlates with metastasis, poor prognosis, and decreased survival (1, 2). Although therapeutics targeting ErbB2 show clinical efficacy, many patients display no initial response or develop drug resistance over time (2). A deeper understanding of the molecular basis of ErbB2-driven tumorigenesis is thus required for the development of improved therapeutic strategies. In vitro experiments suggest that activation of the c-Jun NH2-terminal kinase (JNK) pathway, a mitogen-activated protein kinase pathway, promotes proliferation, cellular invasion, and stem cell expansion in ErbB2-driven breast cancer (3, 4). Furthermore, unpublished data from our lab using mammary epithelial cells expressing activated ErbB2 show that JNK is required for acinus formation in in vitro 3D cultures. In contrast to these studies showing a tumorigenic role for the JNK pathway, other data from our lab show that JNK loss results in accelerated breast tumor growth, suggesting a tumor suppressive role (5, 6). However, these studies were performed in p53 knockout mice with or without a Kras mutation, where the latter required extensive aging and genomic instability to occur before differences in tumor growth were observable. To date, limited in vivo studies exist to confirm the role of JNK in more biologically relevant breast tumor models, such as in ErbB2-mediated cancer, which accounts for over 30% of all human breast cancers. In addition, the molecular mechanisms by which JNK signaling promotes ErbB2-driven tumorigenesis remains poorly understood. To address the discrepancy in JNK function between the in vitro ErbB2-driven breast cancer data and the in vivo p53 knockout tumor data, I began the development of an in vivo murine model to confirm the role of JNK in ErbB2-driven breast cancer. This mouse model will also allow us to test a potential mechanism by which JNK regulates tumorigenesis. Studies show that ErbB2-mediated secretion of the inflammatory cytokine IL6 promotes transformation and tumor growth by activation of the STAT3 transcription factor, triggering an IL6/STAT3 autocrine signaling loop (7,8). A major regulator of Il6 gene expression includes activator protein 1 (AP-1), a transcription factor composed of downstream JNK targets in the Jun protein family (9). In vitro experiments using ErbB2-overexpressing mammary epithelial cell lines show that chemical inhibition of JNK suppresses secreted IL6 protein levels, supporting a role for the JNK pathway in IL6 regulation (7). Thus, I hypothesize that JNK drives ErbB2-driven breast cancer by promoting IL6-mediated tumor progression. Addressing this will increase our understanding of the role of JNK in ErbB2-driven breast cancer and reveal a potentially new mechanism by which JNK functions in tumor progression. Additionally, I began the development of a mouse model that will allow us to investigate the role of JNK in macrophage polarization as an alternative mechanism by which JNK regulates ErbB2-driven breast cancer. In addition to promoting STAT3-dependent tumor growth, IL6 can indirectly drive tumorigenesis by promoting expression of the IL4 receptor in macrophages, triggering STAT6-mediated macrophage polarization towards the pro-tumorigenic M2 phenotype (10, 11). Unlike classically activated M1 macrophages, which promote inflammation and anti-tumor immunity, alternatively activated M2 macrophages function in immunosuppression and metastasis and correlate with advanced stages of breast cancer (12, 13). Further evidence supporting a role for the JNK pathway in macrophage polarization includes a recent study suggesting that JunB, a downstream JNK target and component of the AP-1 complex, plays a crucial role in the induction of M2 macrophage polarization in human alveolar macrophages (13). I hypothesize that activation of the JNK signaling pathway induces IL6-dependent macrophage polarization towards the pro-tumorigenic M2 phenotype. Addressing this hypothesis will determine for the first time whether JNK functions in regulating macrophage polarization within the tumor microenvironment, offering a potentially new mechanism by which JNK can promote ErbB2-driven breast cancer. Determining the role of JNK in ErbB2-mediated breast cancer will have direct therapeutic relevance, as targeting JNK has the potential to inhibit ErbB2-driven breast cancer and other IL6-mediated diseases. Investigating the underlying mechanisms by which JNK functions in ErbB2-positive breast cancer can also offer new molecular targets and further contribute to effective drug design.

Interleukin-7 is an essential cytokine that plays a major role in the development and homeostatic maintenance of T-cells. The presence of soluble forms of various cytokine receptors have been proposed to be involved in the endogenous regulation of cytokine activity. Due to the natural ability of soluble CD127 (sCD127) to bind to IL-7, there is an interest in its potential application as an immunotherapeutic agent in diseases, where IL-7 has been found to be relevant, including HIV infection. In this study, I hypothesize that by administering sCD127 to healthy mice, IL-7 activity should be enhanced, thus enhancing T cell proliferation in vivo. The work presented here focuses on three main objectives: 1) evaluating the effect of IL-7 with or without sCD127 on T cell proliferation in healthy mice; 2) validating a mouse model of T cell depletion using anti-CD4 and CD8 antibodies; and 3) determining the effect of sCD127 treatment with or without IL-7 on T cell reconstitution and proliferation in the T cell depletion model. To assess the effect of administering exogenous sCD127, IL-7 or the combination on T cell proliferation, peripheral blood mononuclear cells and spleen were isolated, and stained to characterize T cell number, proliferation, and surface CD127 expression by flow cytometry. For the T cell depletion model, wild type C57BL/6 mice were injected intra-peritoneally with 150 μg single dose of anti-CD4 and anti-CD8 depleting antibodies. Consequently, mice were bled weekly to demonstrate the kinetics of T cell reconstitution following depletion (from d7 to d63). Our results demonstrated that in healthy mice daily treatment with murine IL-7 significantly stimulated T cell proliferation and consequently increased cell number. This observation was further boosted by pre-complexing IL-7 with sCD127. For T cell depletion experiments, the kinetics of T-cell reconstitution was different between the CD4+ and CD8+ T cells. CD4+ T cell reconstitution was almost complete 6 weeks following T cell depletion, while CD8+ T cells were only partially reconstituted at this time point. Treatment with IL-7 or combined therapy had a transient and significant effect on T cell proliferation and reconstitution, and this influence was abrogated after treatment discontinuation. Interestingly, CD8+ T cells exert greater responses to our treatments in that a more pronounced proliferation and significant increase in cell number was observed relative to the effect seen on CD4+ T cells in both healthy and depleted mice. In conclusion, antibody-mediated T cell depletion is a potentially valuable tool to investigate lymphopenia-induced proliferation and potential therapies thereof. This study suggests that combining sCD127 and IL-7 therapies enhances IL-7-mediated T cell proliferation, and provides important information for the potential therapeutic use of sCD127 and its impact on IL-7 function.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18425号 / 生博第305号 / 新制||生||40(附属図書館) / 31283 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 稲葉 ｶﾖ, 教授 米原 伸, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19930号 / 医博第4150号 / 新制||医||1017(附属図書館) / 33016 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 小川 誠司, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20084号 / 医博第4177号 / 新制||医||1018(附属図書館) / 33200 / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 山田 亮, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20533号 / 生博第375号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 杉田 昌彦, 教授 米原 伸, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20971号 / 医博第4317号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM