京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23609号 / 医科博第132号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 濵﨑 洋子, 教授 森信 暁雄, 教授 上野 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Acute inflammatory response is caused by the rapid recruitment of leukocytes, mainly neutrophils and monocytes, from blood to the tissue site. Diverse agents, including invading pathogens, injured or dead cells, and other irritants, may stimulate this response. In the ensuing inflammatory response, the recruited leukocytes and their secreted molecules help in eliminating or containing the injurious agents and promoting tissue regeneration. But often this response is imprecise and can lead to bystander tissue damage. Unchecked neutrophil activation is implicated in the pathology of many inflammatory conditions. An in-depth understanding of the pathways regulating this response, therefore, becomes critical in identifying therapeutic targets for these diseases. In this study, we investigate the role of intestinal commensal bacteria in regulating the acute inflammatory response. Furthermore, we examine the mechanism by which Interleukin-1 (IL-1) controls the inflammatory response to sterile agents. Inflammatory responses have been studied in the context of host defense against pathogens. However, we report that the innate immune system needs to be primed by intestinal flora to enable neutrophil recruitment to diverse microbial or sterile inflammatory signals. This priming requires myeloid differentiation primary response gene (88) (MyD88) signaling. In antibiotic-treated mice, which have depleted intestinal flora, we show that neutrophils get released into the blood from the bone marrow, but have a specific defect in migration into the inflammed tissue. This deficiency can be restored by pre-stimulating the mice with a purified MyD88 ligand. Despite having reduced number of infiltrating neutrophils, antibiotic-treated mice make higher levels of pro-inflammatory cytokines in the tissue, after inflammatory challenge. This suggests that antibiotic-treated mice produce some anti-inflammatory molecule(s) that counteract the effect of the pro-inflammatory cytokines. However, this effect is not due to the overproduction of the anti-inflammatory cytokine, Interleukin-10 (IL-10). In summary, our findings highlight the role of commensals in the development of acute inflammatory responses to microbial and sterile particles. The inflammatory response to sterile dead cells has been shown to be critically dependent upon IL-1. However, several key aspects of the IL-1 signaling cascade including the source of IL-1 and the cellular target of IL-1 were unresolved. We find that in most cases, the injured cells are not a major contributor of IL-1 that is required to propagate the inflammatory signal. On the contrary, we demonstrate that both the isoforms of IL-1, IL-1α/IL-1β are generated by bone marrow-derived, tissue-resident responding cells, upon sensing the injury. We also sought to determine the identity of the cellular target of IL-1 signaling. Previous studies have shown that for cell death-induced neutrophil recruitment, interleukin-1 receptor (IL-1R) expression is required on parenchymal cells. To identify this parenchymal cell, we are currently in the process of making the conditional knockout mouse of IL-1R. The latter would facilitate the parenchymal tissue-specific deletion of IL-1R. In summary, this study reports our progress in unraveling key aspects of IL-1 signaling during sterile inflammation. Taken together, we have identified key modulators of the acute inflammatory response and their mechanisms of regulation. These findings would facilitate the development of new therapies for inflammatory diseases triggered by both microbe and sterile agents.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammation is a major driver of tumor progression in over fifty percent of breast cancers. Tumors activate inflammatory processes by secreting factors that recruit and trigger inflammatory cells to release cytokines such as Interleukin 6 (IL-6). IL-6 stimulates the activity of signal transducers and activators of transcription 3 (STAT3), a transcription factor that has been extensively studied for its role in promoting breast cancer. Recently, downregulated HIPPO signaling was shown to drive the pro-growth effects of IL 6. Reduced HIPPO signaling allows for the nuclear translocation of transcriptional co-activator yes associated protein (YAP), implicating IL-6 in the co-activation of several transcription factors such as the TEADs that trigger pro growth programs. While IL-6/STAT3 stimulation has been shown to increase YAP activity, the mechanism driving this remains undocumented. The Angiomotins (Amots) are adapters of the HIPPO pathway that directly bind and regulate YAP activity. Molecular characterization of Amot transcriptional regulation unexpectedly revealed a single promoter controlling the expression of its two major isoforms: Amot 130 and Amot 80. Through immunofluorescent analysis, this study found that total Amot levels were elevated across multiple breast tumor subtypes and highest in samples with increased presence of stromal inflammatory cells. Further, the induction of total Amot expression by IL 6 was found to be essential for YAP dependent growth of breast cancer cells. The activation of Amot transcription by IL-6 was found to be through Specificity Protein 1 (Sp1), a transcription factor that is activated by STAT3. This work connects the activation of YAP1 by IL-6/STAT3 through the elevation of Amot expression by Sp1. Taken together, this explains a new avenue whereby breast cancer cells acquire enhanced oncogenic properties in response to inflammatory signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20808号 / 医博第4308号 / 新制||医||1025(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 椛島 健治, 教授 玉木 敬二, 教授 生田 宏一 / 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(医科学) / 甲第21027号 / 医科博第88号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 杉田 昌彦, 教授 濵﨑 洋子, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In summary, this study will be focused on Il-21 and its implications in the antibody response in influenza. The isotype classes primarily involved in this process will also be examined. This will be accomplished by looking at the serum of mice and analyzing the present influenza specific antibodies using ELISA. Another goal was to optimize the ELISA in order to make it sensitive enough to catch small differences in the results. This topic is important due to its implications for improving influenza vaccinations and preventions as current vaccines are not 100% effective. Influenza contributes to significant disease and death around the world every year and each piece of this puzzle is significant in order for the scientific community to be able to eventually make strides to improve the burden of this disease.

Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.

The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases. For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R. This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist. For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability. The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab. / Die Zytokine Interleukin-4 (IL-4) und IL-13 sind zentrale Mediatoren in der humoralen Immunantwort und sind wesentlich an der Entstehung chronisch inflammatorischer Erkrankungen, wie Asthma, Allergien und atopischer Dermatitis beteiligt. Daher werden IL- 4 und IL-13 als wichtige therapeutische Angriffspunkte für die Behandlung atopischer Erkrankungen betrachtet. Zur Signaltransduktion aktiviert IL-4 zwei heterodimere Rezeptorkomplexe, den Typ I Rezeptor bestehend aus den Untereinheiten IL-4R und γc und den Typ II Rezeptor, der sich aus IL-4R und IL-13R1 zusammensetzt. Der Typ II Rezeptor wird auch von IL-13 zur Signalweiterleitung genutzt, wodurch sich die teilweise überschneidenden Funktionen der beiden Zytokine erklären lassen. Die überlappende Rezeptornutzung erlaubt es durch eine gezielte Blockade des IL-4R-Rezeptors sowohl IL-4, als auch IL-13 gleichzeitig zu inhibieren. Ziel dieser Arbeit war die Herstellung neuartiger IL-4 basierter Antagonisten. Dazu wurden ausgewählte Positionen innerhalb der IL-4 Bindestelle für γc and IL-13R1, jedoch außerhalb des Bindeepitops für IL-4R gezielt chemisch modifiziert. Im Gegensatz zu bereits existierenden Studien, die synthetische Gruppen wie Polyethylenglykol (PEG) zur chemischen Modifizierung von IL-4 nutzten, wurden in dieser Arbeit Glykane als natürliche Alternative zu PEG an IL-4 gekoppelt. Da sich Glykosylierung auf wichtige pharmakologische Eigenschaften proteinbasierter Therapeutika, wie Immunogenität oder Serum Halbwertszeit, positiv auswirken kann, würde der Zucker in dieser Strategie nicht nur den auf sterischer Hinderung basierenden inhibitorischen Effekt vermitteln, sondern könnte gleichzeitig zu einer Optimierung der pharmakologischen Eigenschaften des IL-4 Inhibitors beitragen. Zur chemischen Zucker-Kopplung wurden zusätzliche Cystein-Reste in IL-4 eingebracht, deren freie Thiol-gruppen eine hochspezifische Modifizierung der IL-4 Mutanten erlauben. Die IL-4 Cystein-Mutanten wurden rekombinant in prokaryotischen und eukaryotischen Expressionssystemen hergestellt. Anschließend wurden verschiedene Strategien entwickelt, die eine ortsspezifische Kopplung Amin- und Thiol-haltiger Zucker an die eingebrachten Cystein-Reste in IL-4 ermöglichen. Eine Linker-basierte Reaktion, sowie ein Kopplungsansatz, der eine Aktivierung der Thiol-Gruppe mittels Bromselenobenzol erforderte, wiesen einige Nachteile auf, die eine erhebliche Einschränkung ihrer technischen Durchführbarkeit zur Folge hatte. Eine dritte Strategie hingegen, die eine Rückfaltung derIL-4 Cystein-Mutanten in Anwesenheit von Thiol-Glykanen involvierte, erlaubte eine effiziente Herstellung von IL-4 Glykokonjugaten in Form gemischter Disulfide im Milligrammbereich. Dieser Ansatz könnte somit zur Produktion homogen glykosylierter IL-4 Antagonisten im Großmaßstab eingesetzt werden. Die Herstellung homogener Glykokonjugate mit klar definierten Glykosylierungsmuster würde es erlauben über die gekoppelten Glykanstrukturen die pharmakologischen Eigenschaften von IL-4, wie Absorption und metabolische Stabilität, gezielt zu modulieren. Die hergestellten IL-4 Glykokonjugate erwiesen sich als hochwirksame Antagonisten, die die Aktivität von IL-4 und IL-13 in zellbasierten Experimenten inhibieren konnten. Glykosylierte IL-4 Antagonisten stellen somit eine vergleichbare Alternative zu gängigen IL-4 Inhibitoren dar, die zur Behandlung von atopischen Erkrankungen eingesetzt werden, wie beispielweise der neutralisierende anti-IL-4R Antikörper Dupilumab.

Indiana University-Purdue University Indianapolis (IUPUI) / CD4 T cells are critical regulators of inflammatory diseases and play an important role in allergic airway diseases (AAD) by producing type 2 cytokines including IL-4, IL- 13, IL-5 and IL-9. In chronic AAD models, IL-9 producing CD4 T-helper (TH9) cells lead to accumulation of eosinophils and mast cells in the airway, increase levels of type-2 cytokines, stimulate ILC2 cell proliferation, and induce mucus production from airway epithelium. However, the transcriptional network that governs the development of TH9 cells and their function during allergic responses is not clearly understood. Naïve CD4 T cells differentiate into TH9 cells in the presence of IL-2, IL-4 and TGFβ, activating a complex network of transcription factors that restricts their development to TH9 lineage. In this study a variety of approaches were utilized, including characterizing Il9 reporter mice, to identify an additional Ets-transcription factor termed ERG (Ets-related gene) that is expressed preferentially in the TH9 subset. Knock-down of Erg during TH9 polarization led to a decrease in IL-9 production in TH9 cells in vitro. Deletion of Erg at the later stage of TH9 induced pathogenesis resulted in reduced IL-9 production in the airways in chronic AAD. Chromatin immunoprecipitation assays revealed that ERG interaction at the Il9 promoter region is restricted to the TH9 lineage and is sustained during TH9 polarization. In the absence of PU.1 and ETV5, ERG regulated IL-9 production independent of other Ets-transcription factors and the deletion of Erg further lead to a decrease in IL-9 production by lung-derived CD4-T cells in chronic AAD model. Lastly, I also identified IL-9 secreting CD4 tissue resident memory cell population that play an instrumental role in allergic recall responses. In summary, in this study novel transcription factors were identified that can regulate TH9 function and the role of IL-9 in allergic airway recall responses. / 2022-12-28

Interleukin-33 (IL-33) is a multifunctional cytokine that mediates local inflammation upon tissue damage. IL-33 is known to act on multiple cell types including group 2 innate lymphoid cells (ILC2s), Th2 cells, and mast cells to drive production of Th2 cytokines including IL-5 and IL-13. IL-33 signaling activity through transmembrane ST2L can be inhibited by soluble ST2 (sST2), which acts as a decoy receptor. Previous findings suggested that modulation of IL-13 levels in mice lacking decoy IL-13Rα2, or mice lacking IL-13, impacted responsiveness to IL-33. In this study, we used Il13-/- mice to investigate whether IL-13 regulates IL-33 activity by modulating the transmembrane and soluble forms of ST2. In Il13-/- mice, the effects of IL-33 administration were exacerbated relative to wild type (WT). Il13-/- mice administered IL-33 i.p. had heightened splenomegaly, more immune cells in the peritoneum including an expanded ST2L+ ILC2 population, increased eosinophilia in the spleen and peritoneum, and reduced sST2 in the circulation and peritoneum. In the spleen, lung, and liver of mice given IL-33, gene expression of both isoforms of ST2 was increased in Il13-/- mice relative to WT. Because IL-13 and IL-4 signal through a shared receptor complex IL-13Rα1/IL-4Rα, we also studied the combined deficiency of IL-4 and IL-13 using Il4rα-/- mice which are defective in both IL-4 and IL-13 signaling. Responses of Il4rα-/- mice were indistinguishable from those of Il13-/- mice in our model system of IL-33-induced inflammation, suggesting that IL-4 does not play a distinct role separate from IL-13 in regulation of IL-33 activity. Through in vitro experiments, we confirmed fibroblasts to be an IL-13-responsive cell type that can regulate IL-33 activity through production of sST2. This study elucidates the important regulatory activity that IL-13 exerts on IL-33 through induction of IL-33 decoy receptor sST2 and through modulation of ST2L+ ILC2s.