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Cardiac dysfunction is a major consequence that contributes to the high mortality of trauma-hemorrhage (TH) patients. Recent evidence suggests that innate immune and inflammatory responses mediated by Toll-like receptors (TLRs) play a critical role in the pathophysiologic mechanisms of acute organ dysfunction during TH. This study investigated the role of TLR4 in cardiac dysfunction following TH. Toll-like receptor 4-deficient (TLR4-/-, n = 7/group) and age-matched wild-type (WT, n = 8/group) mice were subjected to TH that was induced by soft tissue injury and blood withdrawal from the jugular vein to a mean arterial pressure of 35 ± 5 mmHg. Cardiac function and mean arterial pressure were measured with a Millar system before, during, and after blood withdrawal. Sham surgical-operated mice served as control (WT, n = 9/group; TLR4-/-, n = 10/group). Cardiac function in WT mice was significantly reduced following TH. However, cardiac function was well preserved in TLR4-/- mice. Administration of a TLR4 antagonist (3 mg/kg) to WT mice also significantly attenuated TH-induced cardiac dysfunction. Western blot showed that either TLR4-/- or TLR4 antagonist markedly attenuated TH-induced decreases in the levels of phosphorylated-Akt in myocardium. In addition, inhibition of TLR4 attenuated TH-induced myocardial nuclear factor κB-binding activity as well as lung myeloperoxidase activity and tumor necrosis factor α production. The data indicate that TLR4 plays a central role in TH-induced cardiac dysfunction. Toll-like receptor 4 deficiency or TLR4 inhibition attenuated cardiac dysfunction following TH, which may involve activation of the phosphoinositide 3-kinase/Akt signaling and decrease in nuclear factor κB-binding activity. Toll-like receptor 4 antagonism may be a new and novel approach for the treatment and management of cardiac dysfunction in TH patients.

Tim-3 and PD-1 are powerful immunoinhibitory molecules involved in immune tolerance, autoimmune responses, and antitumor or antiviral immune evasion. A current model for Tim-3 regulation during immune responses suggests a divergent function, such that Tim-3 acts synergistically with TLR signaling pathways in innate immune cells to promote inflammation, yet the same molecule terminates Th1 immunity in adaptive immune cells. To better understand how Tim-3 might be functioning in innate immune responses, we examined the kinetics of Tim-3 expression in human CD14 + M/M 4 in relation to expression of IL-12, a key cytokine in the transition of innate to adaptive immunity. Here, we show that Tim-3 is constitutively expressed on unstimulated peripheral blood CD14 + monocytes but decreases rapidly upon TLR stimulation. Conversely, IL-12 expression is low in these cells but increases rapidly in CD14 + M/M.J, in correlation with the decrease in Tim-3. Blocking Tim-3 signaling or silencing Tim-3 expression led to a significant increase in TLR-mediated IL-12 production, as well as a decrease in activation-induced up-regula-tion of the immunoinhibitor, PD-1; TNF-a production was not altered significantly, but IL-10 production was increased. These results suggest that Tim-3 has a role as a regulator of pro- and anti-inflammatory innate immune responses.

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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Gut microbiota has been shown to regulate the growth and development of the central and enteric nervous systems (CNS and ENS) after birth. There is ample evidence to suggest that intestinal bacteria can influence behavior of the host through both immune and immune-independent mechanisms. Gut-microbiota-regulated CNS structural changes are focused in the limbic system, at centres associated with memory, social and emotional behaviour. Several studies using germ-free (GF) and specific pathogen free (SPF) mice demonstrated microbial influence on behaviour development accompanied by neurochemical changes in the brain. Higher exploratory and lower anxiety-like behavior was found in GF mice compared to SPF mice with lower central expression of neurotrophins, such as nerve growth factor and BDNF. The mechanisms by which the microbiota influences behavior are unknown but could be immune-mediated, neural, or humoral in origin. In this study I investigated the role of immune system maturation on mouse behaviour after bacterial colonization. I showed that mono-colonization of GF mice with E. coli normalizes behaviour similar to colonization with complex microbiota (SPF and ASF) and the continuous presence of bacteria is not required to maintain this normal behaviour. I also showed that innate immunity through the MyD88/Ticam pathway is crucial for the development of normal behaviour and that multiple innate immunity and neuronal genes are involved in this process. Together these results suggest that bacterial colonization primes and matures the innate immunity and development of the central nervous system ultimately leading to normal behaviour. I believe that bacterial colonization at birth is not only important for the epithelial barrier function, gut homeostasis, and immune functions, but also for the development of normal behaviour. Altered immune priming during the postnatal period due to abnormal microbial colonization may have wider implications when considering psychiatric disorders in humans. / Thesis / Doctor of Philosophy (PhD)

Innate immune memory has become an increasingly popular area of research in the last decade. However, much of the work done on innate immune memory using inflammatory agents such as BCG, C. albicans, and β-glucan has been pursued through systemic administration, which has been shown to induce training in circulating monocytes. In addition, little is known about whether microbial ligands can induce training. Here, we show that local mucosal exposure to an acute dose of LPS induces long-lasting phenotypic changes in airway macrophage populations. LPS-exposed macrophages display increased glycolytic metabolism and differential cytokine expression upon restimulation, whereas circulating monocytes are not affected. Finally, we show that LPS exposure provides long-lasting protection against Streptococcus pneumoniae in the lung, likely due to the higher acquisition of CD11b, which is indicative of macrophage activation and phagocytosis. As much of the work on innate immune memory has been done through systemic administration of training agents, this project aims to fill existing knowledge gaps in the induction of innate immune memory upon local mucosal exposure to inflammatory agents. / Thesis / Master of Science in Medical Sciences (MSMS) / The innate immune system is one of the first defenders in our bodies that fight against a variety of pathogens. In the last decade, the innate immune system was found to be capable of having memory, meaning it reacts faster or at a heightened magnitude in response to a wide range of subsequent pathogens after it is trained by an agent. This project explores the effect a bacteria wall component, LPS, has on the lung environment and examines if it will induce memory in the lung. Our findings show that intranasal exposure to LPS changes the cellular landscape in the lung. LPS-exposed airway innate immune cells become more activated and provide subsequent protection against bacterial infections. This work has implications for using LPS as a vaccine adjuvant in order to provide protection against a variety of pathogens in addition to specific protection brought by the vaccine.

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.