京都大学 / 新制・課程博士 / 博士(医学) / 甲第24503号 / 医博第4945号 / 新制||医||1064(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 羽賀 博典, 教授 椛島 健治, 教授 後藤 慎平 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The interrogation of T cell signaling over the past fifty years has led to the discovery of amazingly intricate cascade networks and elaborate descriptions of individual proteins' domains and functions. A complex landscape has been rendered in which proteins relay messages from the extracellular ligation of the TCR by a cognate peptide loaded MHC via changes in sub-cellular location, phosphorylation, and binding affinities and partners to enact nuclear localization of three key transcription factors required for cellular effector function and proliferation: AP-1, NF-AT, and NF-κB. Dogma has favored activation of each of these transcription regulating elements to be a linear and parallel activity, thus very little interaction between pathways has been highlighted by previous findings in the molecular immunology community. The focus of this dissertation explores the role of NF-κB in T cell signaling with emphasis on subunits p50, cRel, IκBα, and IKKβ, and with respect to NF-κB’s ability to modulate calcium and NF-AT signaling, proximal TCR phosphorylation, and CRAC and purinergic calcium channel proteins. The role of NF-κB in T cells can be a difficult thing to establish, as this thirteen member family innervates almost every cellular process from homeostasis to activation, and even functions in the opposing processes of survival and apoptosis. To convolute the investigation further, many family members also fulfill redundant tasks, as a result of their high evolutionarily conserved sequence homology. To this end, we discovered the best way to evaluate the function of NF-κB in the activation of T cells was to knockdown two family members: p50 and cRel. In doing this, we rendered mice that were viable (unlike knockdown of RelA) and fertile, but possessed T cells that were highly unresponsive to strong stimulation (anti CD3/CD28) or foreign antigen (OVA) presented to mice bearing the correct transgenic TCRs (OT-1) by professional antigen presenting cells (APC). Through in vitro assays, we discovered that in addition to the specific defects in NF-κB activation, NF-AT signaling was also greatly disrupted in these cells, sequela to retarded calcium influx and signaling. This was of great interest, as while several studies have shown that calcium signaling has the ability to amplify and fine tune NF-κB activation, there is a dearth of studies and publications highlighting the effect of an activated NF-κB pathway on calcium influx and signaling leading to the activation of NF- AT. Another fascinating discovery, that explicated the calcium reduction and NF-AT inhibition, was that ablation of p50 and cRel led to decreases in mRNA and protein levels of two additional NF-κB family members: IKKβ and IKKγ. The results presented here suggest that it is the reduction in IKKβ and IKKγ that leads to impaired phosphorylation of the key TCR proximal proteins: Zap70 and PLC&gamma1, and it is the decrease in activated PLCγ1 that renders less IP3 and ultimately abrogates calcium signaling. Overall, this thesis highlights the ability of IKKβ to enhance general proximal TCR protein phosphorylation (and specifically Zap70) leading to a greater influx of calcium (perhaps aided by IKKβ also augmenting the function of the CRAC protein, STIM1) which leads to superior activation of NF- AT, and amplifies downstream cellular effector functions such as IL-2 production and proliferation. Moreover, this work demonstrates that NF-κB subunits likely form supermolecular clusters, and ablation of certain subunits (i.e. p50 and cRel) can lead to instability and decreased levels of other family members (i.e. IKKβ and IKKγ.)

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22595号 / 生博第428号 / 新制||生||57(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 松田 道行, 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

Growing evidence supports the role of transcription factor activation in the pathophysiology of inflammatory disorders, sepsis, ARDS, SIRS, and shock. Kinase mediated phosphorylation of IκBα is a crucial step in the NFκB activation pathway. We investigated IκBα phosphorylation in murine liver and lung extracts after cecal ligation and puncture (CLP) in the presence and absence of a glucan ligand. ICR mice were subjected to CLP. Unoperated and sham-operated mice served as the controls. Glucan phosphate (50 mg/kg) was administered 1 h before or 15 min after CLP. CLP increased hepatic and pulmonary levels of phospho-IκBα by 48-192%. Pre-or post-treatment with glucan phosphate decreased (P < 0.05) tissue phospho-IκBα levels in CLP mice. Phospho-IκBα in the glucan-CLP group were not significantly different from the unoperated controls. To investigate mechanisms we examined IKKβ kinase activity, IκBα phosphorylation and degradation, and NFκB activity in a murine macrophage cell line, J774a.1, treated with LPS (1 μg/mL) and/or glucan phosphate (1 μg/mL) for up to 120 min. The glucan ligand blunted LPS-induced IKKβ kinase activity, phosphorylation and degradation of IκBα, and NFκB nuclear binding activity. The data indicate that one mechanism by which (1→3)-β-D-glucan may alter the response to endotoxin or polymicrobial sepsis involves modulation of IKKβ kinase activity with subsequent decreases in IκBα phosphorylation and NFκB activation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19223号 / 医博第4022号 / 新制||医||1010(附属図書館) / 32222 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 開 祐司, 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

No description available.

Azithromycin improves clinical outcomes in patients with cystic fibrosis (CF), specifically in patients infected with Pseudomonas aeruginosa. Azithromycin shifts macrophage programming away from a pro-inflammatory classical (M1) phenotype, and towards an anti-inflammatory alternative (M2) phenotype; however, little is known about this mechanism, nor of its impact upon immune response to pulmonary infection. We set out to determine the mechanism by which azithromycin is able to alter macrophage phenotype, and assess the effect of azithromycin induced macrophage polarization on inflammation during pulmonary infections. Utilizing macrophage cell culture, we found that azithromycin increased IKKβ, a signaling molecule in the NFκB pathway, which likely is altering macrophage programming. Using a Pseudomonas infection model in mice that lack physiologic alternative macrophage activation, we showed that azithromycin’s ability to alter macrophage function and decrease lung damage was independent of interleukin control of macrophage programming. Azithromycin increased fibrotic protein production both in vivo and in vitro, but blunted immune-driven fibrotic damage. We extended our study to patients with CF, describing gene expression in macrophages isolated from sputum samples. We found markers consistent with a shift toward M2 polarization in these patients. These data suggest potential mechanisms by which azithromycin benefits patients with CF.

No description available.