Research Doctorate - Doctor of Philosophy (PhD) / Asthma involves chronic inflammation of the airways that is heterogeneous in nature. Eosinophilic airway responses are well described in asthma, however non-eosinophilic subtypes of asthma have been recently reported, and can involve the influx of neutrophils into the airways (neutrophilic asthma). Neutrophils are important effector cells of the innate immune system. These cells are the first to migrate to inflammatory sites, where they contain and eliminate pathogenic microorganisms. Neutrophils also release cytokines and chemokines that initiate and amplify inflammatory responses. The mechanisms of neutrophilic asthma remain largely unknown; however activation of the innate immune response is implicated, particularly increased levels of proinflammatory cytokines Interleukin (IL)-8 and IL-1beta and gene expression of Toll Like Receptor (TLR)-4 and TLR2 have been demonstrated in induced sputum samples. This thesis examines innate immune responses of airway and circulating neutrophils, with a focus on neutrophilic asthma. Innate immune neutrophil activation occurs in response to exposure to Lipopolysaccharide (LPS), which activates TLR4. The activation response consists of the release of preformed granule associated mediators such as Matrix Metalloproteinase (MMP)-9 and Oncostatin M (OSM), new gene transcription and release of inflammatory cytokines such as IL-8, IL-1beta and Tumor Necrosis Factor (TNF)-alpha, and new gene transcription of TLR2 & TLR4 which serve to amplify neutrophil responses. In addition, this thesis examines whole genome gene expression profiles of circulating neutrophils in neutrophilic and eosinophilic asthma. The aims of this thesis are based on the hypothesis that dysregulation of innate immune neutrophil responses occurs with ageing and airway disease, particularly neutrophilic asthma and chronic obstructive pulmonary disease (COPD). With advancing age, there were alterations in the innate immune responses of neutrophils, which were characterised by enhanced spontaneous activation of both airway and circulating neutrophils, and a decreased response of circulating neutrophils to LPS. There was a decreased activation of airway neutrophils in airway disease that was most pronounced in neutrophilic asthma and COPD, with decreased production and release of proinflammatory cytokines most likely due to a downregulation of TLR4. TLR2 was downregulated in resting and LPS stimulated circulating neutrophils in asthma, particularly neutrophilic asthma. Circulating neutrophils had a decreased spontaneous release of total MMP-9, and downregulation of OSM, TLR2 and TLR4 at rest in COPD. However when stimulated with LPS, subjects with COPD had an enhanced proinflammatory cytokine release, with increases in IL-8 and TNF-alpha compared to subjects with asthma or healthy controls. Analysis of whole genome gene expression of circulating neutrophils in asthma revealed distinct gene profiles relating to asthma subtype. There was upregulation of genes relating to cell motility, inhibition of apoptosis and the NF-kB in neutrophilic asthma, which would contribute to their accumulation in the airways. The innate immune response is critical in controlling infections by bacteria and viruses. The reduced innate immune response of airway neutrophils in airway disease could contribute to impaired local defense, which may lead to an increased susceptibility to infection by invading pathogens. Systemically, the molecular mechanisms of neutrophilic asthma are distinct from eosinophilic asthma and may involve the enhancement of neutrophil chemotaxis and survival, contributing to their accumulation in the airways.

Induced and activated as part of the innate immune response, the first line of defence against bacterial or viral infections, Inhibitor of Kappa-B Kinase ε (IKKε) triggers NF-κB and IFNβ signalling. Whilst not expressed at basal levels in healthy cells and tissue, the kinase is overexpressed in roughly 30% of human breast cancer cases, driving oncogenesis through aberrant activation of NF-κB. The impracticality of therapeutic targeting of NF-κB for cancer treatment has led to a requirement for greater understanding of IKKε's oncogenic potential to treat tumours driven by the kinase. Considering that IKKε alters cellular metabolism in dendritic cells, promoting aerobic glycolysis akin to the metabolic phenotype observed in cancer, it was hypothesised that the kinase would play a similar role in breast cancer. Using a Flp-In 293 model of IKKε induction and suppressing IKKε expression in a panel of breast cancer cell lines using siRNA, IKKε-dependent changes in cellular metabolism were characterised using labelled metabolite analysis. IKKε was found to induce serine biosynthesis, an important pathway in breast cancer development that supports glutamine-fuelling of the TCA cycle and contributes to one carbon metabolism to maintain redox balance. Promotion of serine biosynthesis occurred via a dual mechanism. Firstly, PSAT1, the second enzyme of the pathway, was found to be phosphorylated in an IKKε-dependent manner, promoting protein stabilisation. Secondly, an IKKε-dependent transcriptional upregulation of all three serine biosynthesis enzymes, PHGDH, PSAT1 and PSPH, was observed, induced by the inhibition of mitochondrial activity and the subsequent induction of ATF4-mediated mitochondria-to-nucleus retrograde signalling. These data demonstrate a previously uncharacterised mechanism of metabolic regulation by IKKε and highlight new potential therapeutic targets for the treatment of IKKε-driven breast cancer in the form of the enzymes of the serine biosynthesis pathway.

Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (2 mg/Kg BW; dissolved in 0.9% saline) or vehicle (0.9% saline) to stimulate the immune system. Two hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. The data show 628 significantly upregulated transcripts in the hypothalamus, as well as 439 and 121 in the spleen and RBCs, respectively, relative to controls. Also, 134 transcripts in the hypothalamus, 517 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated, suggesting a potential trade-off in expression of genes that regulate immunity and metabolism. Unlike mammals, birds have nucleated RBCs, and these results suggest a novel transcriptomic response of RBCs to immune challenge. Lastly, molecular biomarkers could be developed to rapidly screen bird populations by simple blood sampling in the field.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13454号 / 論医博第2247号 / 新制||医||1055(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 竹内 理, 教授 杉田 昌彦, 教授 生田 宏一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUCTION: Seasonal human coronaviruses (HCoV) are endemic to the human population, regularly infecting and reinfecting humans while typically causing asymptomatic to mild respiratory infections. The human coronavirus OC43 (HCoV-OC43) is one of the most common causes of the common cold but can lead to fatal pneumonia in children and the elderly. However, no vaccines or antiviral treatments are available against this virus. Animal models available to study HCoV-OC43 and test antiviral counter measures do not accurately recapitulate the respiratory symptoms and physiopathology observed in humans. These limitations impede our understanding of HCoV-OC43 pathogenesis and the development of efficient antiviral therapies or vaccines. Objective: Animal models are crucial for enhancing our understanding of HCoV-OC43 pathophysiology and pathogenesis, and to enable the development of vaccines or therapeutics. In this study, we tested the susceptibility of various mice models to HCoV-OC43 infection and identified type-I interferon signaling as an immune barrier that restricts HCoV-OC43 infection in mice. Utilizing mice defective for type-I interferon signaling (IFNAR -/ -mice), we established virological and histopathological readouts that could assist in identifying avenues for this model to be used for vaccines and therapeutic evaluations. Methods: C57BL/6, IFNAR -/-, and IFNAR -/- mice treated with anti-IFN-λ (antibodies blocking type-III Interferon cytokines) were infected with different doses of HCoV-OC43. Nasal passages and lung tissues were analyzed at different time points during the course of the infection. Focus forming assay and RT-qPCR were utilized to determine viral titers and loads in the lung, respectively. Tissues were stained with Hematoxylin and eosin for histopathological evaluation and immunohistochemistry was performed for quantification of HCoV-OC43 spike protein via image analysis. Whole slide images were generated using a Vectra PolarisTM whole slide scanner and digital analysis with area quantification (AQ) was completed using HALOTM v3.5.3.2577 The region of interests included the olfactory and respiratory epithelium of the nasal cavity. Algorithms to quantify the spike protein were designed specifically for each slide. Signal intensity was selected by pixel pigmentation and using the real-time tuning function in HALOTM v3.5.3.2577 allowing capture of accurate biological signal. Statistical analysis was conducted using GraphPad PrismTM 9.5.1. Results: IFNAR -/- mice intranasally inoculated with HCoV-OC43 displayed greater viral antigen in the olfactory epithelium compared to C57BL/6 mice at three-and-five days post infection. IFNAR -/- mice also displayed mild histopathological manifestations in the respiratory epithelium compared to infected C57BL/6 wild-type mice. Minor histological characteristics seen in the IFNAR -/- mice were characterized by mild rhinitis with neutrophilic and mononuclear influx including edema at the level of the respiratory epithelium, scarce numbers of denuded olfactory epithelium, and mild squamous metaplasia at the level of the respiratory epithelium. No differences in lung viral loads were observed between the two models throughout the infection course, suggesting that additional immune barriers or absence of specific human factors prevent viral dissemination to the lower respiratory tract in mice. Interestingly, treatment of IFNAR -/- mice with antibodies targeting type III interferon cytokines increased viral replication in the olfactory epithelium and extended viral dissemination to the respiratory epithelium of the nasal cavity compared to control IFNAR -/- mice. Altogether, our findings indicate that IFNAR -/- mice represent a potential mouse model of HCoV-OC43 infection, albeit viral replication is restricted to the nasal cavity. More research is needed to identify additional immune barriers, including type III interferon signaling, restricting viral replication in the mouse respiratory epithelium. Conclusion: Combining virological, molecular biology, and histopathological techniques, our study identify type I and III interferon signaling as restriction mechanisms of HCoV-OC43 replication in the mouse nasal cavity. Our work highlights IFNAR -/- mice as a potential model to study early HCoV-OC43 pathogenesis, and open avenues for developing advanced mouse models enabling the evaluation of vaccine candidates. / 2026-02-28T00:00:00Z

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L’infection chronique est à l’origine de 15-20% des cancers dans le monde. Dans la plupart des cas, les infections sont éliminées par le système immunitaire, sans incidence importante sur les hôtes infectés. Toutefois, les oncovirus peuvent échapper au système immunitaire et induire une transformation cellulaire, ce qui constitue deux éléments clés de la cancérogenèse associée aux virus. L’EBV est un herpesvirus ubiquitaire à ADN double brin qui infecte plus de 90% de la population, avec un tropisme spécifique pour les cellules B. Après primo-infection, le virus persiste dans l’hôte pour toujours. L’EBV est responsable de la mononucléose infectieuse bénigne et est associé à plusieurs tumeurs malignes telles que le lymphome de Burkitt, le lymphome de Hodgkin et certaines formes de cancers gastriques. Les récepteurs Toll-like (TLRs) mammaires jouent un rôle important dans la défense de l’hôte lors de l’infection pathogène en régulant et reliant les réponses immunitaires innées et adaptatives. Dans cette étude, nous avons montré que l’EBV pouvait altérer la régulation et l’expression de TLR9, une des molécules effectrices majeures de la réponse immunitaire innée. L’infection par l’EBV des lymphocytes B primaires humains a entraîné l’inhibition de la fonctionnalité de TLR9. Nous avons observé que l’EBV exerçait sa fonction inhibitrice en diminuant les niveaux d’ARNm et de la protéine du récepteur TLR9. De plus, nous avons établi que LMP1, oncoprotéine majeure de l’EBV, inhibait fortement la transcription de TLR9. La sur-expression de LMP1 par transfection transitoire ou transduction des cellules B réduit l’activité du promoteur de TLR9, l’ARNm et les niveaux protéiques. Bloquer la voie de signalisation de NF-κB induite par la signalisation de LMP1 permet de récupérer l’activité du promoteur de TLR9 et l’expression de la protéine. L’ensemble de nos résultats mettent en évidence un nouveau mécanisme utilisé par l’EBV pour supprimer la réponse immunitaire de l’hôte en dérégulant la transcription de TLR9 via l’activation de NF-κB par LMP1 / Chronic infection causes about 15-20% of cancer worldwide. In most cases, infections are cleared by the immune system with no dramatic consequence for the infected hosts. However, oncoviruses can escape the immune system and induce cellular transformation, two key events in cancer mediated by viruses. EBV is a ubiquitous double-stranded DNA herpesvirus, which infects more than 90% of the population with a specific tropism to B-cells. Upon primo-infection the virus persists in the host for lifetime. EBV is responsible of the benign infectious mononucleosis and is associated to several malignancies such as the Burkitt lymphoma, Hodgkin’s lymphoma and some forms of gastric cancers. Mammalian Toll-like receptors (TLRs) play a key role in host defense during pathogen infection by regulating and linking the innate and adaptive immune responses. TLRs belong to a family of receptors that recognize pathogen-associated molecular patterns and are expressed on immune and non-immune cells, endowing them with the capacity to sense pathogen-derived products and to alert the immune system . In this study we show that EBV can alter the regulation and expression of TLR9, one of the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6, TNFα and IgG, which was inhibited in cells infected with EBV. We observed that EBV exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed twelve hours post EBV infection of primary cells as well as in an immortalized B cell line, demonstrating the specific role of the virus to turn down TLR9 levels. In addition, we determined that the EBV oncoprotein LMP1 is a strong inhibitor of TLR9 transcription. Over expression of LMP1 by transient transfection or transduction of B cells, reduced TLR9 promoter activity, mRNA and protein levels. Blocking the NF-κB pathway induced by LMP1 signaling, recovered TLR9 promoter activity and protein expression. Moreover LMP1 mutants deficient in activating the NF-κB pathway inversely restored TLR9 transcription. Taken together, our study reveals a novel mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation

A resposta inflamatória aguda é uma importante defesa contra o Streptococcus pneumoniae, mas a persistência na inflamação pode causar danos aos tecidos. Duas linhagens de camundongos geneticamente selecionadas para resposta inflamatória aguda mínima (AIRmin) e máxima (AIRmax) foram avaliadas frente a um desafio respiratório invasivo com pneumococo. O desafio induziu a morte de 100% dos camundongos AIRmin, e apenas 36,4% dos camundongos AIRmax. A caracterização da resposta imune inata mostrou que ambas as linhagens de camundongos responderam ao desafio com secreção de citocinas pro-inflamatórias. Entretanto, apenas os camundongos AIRmax controlaram a inflamação. Diferenças significativas quanto à expressão de metaloproteases de matriz sugerem o papel destas proteínas no controle da infecção. Além disso, os camundongos AIRmin apresentaram um aumento no número de macrófagos expressando o receptor de manose CD206, após o desafio. Uma menor resistência de macrófagos e neutrófilos dos camundongos AIRmin à morte celular programada, após o desafio, também foi observada. / Acute inflammatory response is an important defense against Streptococcus pneumoniae, but persistence of inflammation may result in tissue damage. The susceptibility against an invasive respiratory pneumococcal challenge was evaluated in two outbred mice strains, genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory responses. The challenge induced the death of 100% of the AIRmin mice and only 36.4% of the AIRmax mice. Characterization of the innate immune responses showed that both mice strains responded to the challenge with the secretion of inflammatory cytokines. However, only the AIRmax mice controlled the inflammation. Significant differences on the expression of matrix metalloproteinases suggest a role of these proteins in the control of the infection. Moreover, the AIRmin mice presented an increase in the number of macrophages expressing the CD206 mannose receptor after the challenge. A reduced resistance of macrophages and neutrophils from AIRmin mice to programmed cell death, after the challenge, was also observed.

A resposta inflamatória aguda é uma importante defesa contra o Streptococcus pneumoniae, mas a persistência na inflamação pode causar danos aos tecidos. Duas linhagens de camundongos geneticamente selecionadas para resposta inflamatória aguda mínima (AIRmin) e máxima (AIRmax) foram avaliadas frente a um desafio respiratório invasivo com pneumococo. O desafio induziu a morte de 100% dos camundongos AIRmin, e apenas 36,4% dos camundongos AIRmax. A caracterização da resposta imune inata mostrou que ambas as linhagens de camundongos responderam ao desafio com secreção de citocinas pro-inflamatórias. Entretanto, apenas os camundongos AIRmax controlaram a inflamação. Diferenças significativas quanto à expressão de metaloproteases de matriz sugerem o papel destas proteínas no controle da infecção. Além disso, os camundongos AIRmin apresentaram um aumento no número de macrófagos expressando o receptor de manose CD206, após o desafio. Uma menor resistência de macrófagos e neutrófilos dos camundongos AIRmin à morte celular programada, após o desafio, também foi observada. / Acute inflammatory response is an important defense against Streptococcus pneumoniae, but persistence of inflammation may result in tissue damage. The susceptibility against an invasive respiratory pneumococcal challenge was evaluated in two outbred mice strains, genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory responses. The challenge induced the death of 100% of the AIRmin mice and only 36.4% of the AIRmax mice. Characterization of the innate immune responses showed that both mice strains responded to the challenge with the secretion of inflammatory cytokines. However, only the AIRmax mice controlled the inflammation. Significant differences on the expression of matrix metalloproteinases suggest a role of these proteins in the control of the infection. Moreover, the AIRmin mice presented an increase in the number of macrophages expressing the CD206 mannose receptor after the challenge. A reduced resistance of macrophages and neutrophils from AIRmin mice to programmed cell death, after the challenge, was also observed.

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.