京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22810号 / 生博第444号 / 新制||生||59(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 野田 岳志, 教授 朝長 啓造, 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

Extracellular histones have been implicated as a cause of tissue inflammatory injury in a variety of disorders including sepsis, lung and liver diseases. However, little is known about their interactions with neutrophils and how this might contribute to injury. We used human neutrophils isolated from healthy donors in this study, and we conducted functional and mechanistic studies of the effects of histone H4 on neutrophils in vitro. We found that histone H4 acts as a strong neutrophil activator, inducing cytokine release, hydrogen peroxide production, adhesion and degranulation. H4 had a distinctive profile as compared to other neutrophil agonists in that it caused a marked and sustained elevation of intracellular calcium, which resulted from permeabilization of the neutrophil plasma membrane. Prior studies showed that neutrophils predominate in the early response to influenza A virus (IAV) infection in the lung, that IAV induces formation of neutrophil extracellular traps (NETs), and IAV infection in vivo is worsened by release of free histones in the lung. Neutrophils predominate in the early response in the lung to IAV infection. We found that IAV-induced NETs contain histones. Of interest, we found that histone H4 binds to and aggregates IAV particles, inhibits IAV infectivity in vitro, and increases the uptake of IAV by neutrophils. On the other hand, histone H4 potentiated IAV-induced neutrophil responses, including hydrogen peroxide production and calcium flux. These findings may in part explain the injurious effect of histones during IAV infection in vivo. We found that C-reactive protein (CRP) and surfactant protein-D (SP-D) or its carbohydrate binding domain bound to histone H4. CRP markedly reduced all of the effects of histone H4 on IAV and/or neutrophils. We also found that SP-D or its isolated binding domain reduced neutrophil activation induced by histone H4 which may in part account for its ability to reduce lung inflammation. We hope these findings provide a better understanding of the pro-inflammatory effects of histones (in IAV or other types of injury) and how the body protects itself against histone-induced injury. We also hope our results will aid development of novel strategies to ameliorate tissue damage caused by histones.

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Influenza A virus (IAV) is a zoonotic pathogen which consists of a large genetically and antigenically diverse viral population. Swine IAVs not only cause disease outbreaks among swine, but can also be transmitted to humans, causing sporadic infections and even pandemic outbreaks apart from human seasonal IAV. Antigenic variant identification is fundamental for an effective vaccination program. Red blood cell based immunological tests have been used to identify antigenic variants among circulating IAV strains. Because these assays require viral isolation, they are time consuming and labor intensive. Thus only limited numbers of virus isolates are subjected to antigenic characterization in influenza surveillance studies and much of this important information is lost. In this project, a novel polyclonal antibody based proximity ligation assays (polyPLA) was developed and validated to characterize IAV antigenic variants directly using clinical samples. The application of this method with clinical samples from influenza surveillance had aided in the understanding of the antigenic evolution of IAV in human and swine populations.

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Periodic introductions of influenza A viruses (IAVs) from wild birds contribute to emergence of novel strains that infect domestic poultry, lower mammals, and humans, but the mechanisms of emergence are unclear. The objectives of this dissertation research are to infer the genesis of two emerging IAVs, low pathogenic avian influenza (LPAI) H10N8 and highly pathogenic avian influenza (HPAI) H7N8 viruses, and to characterize the antigenic diversity and genetic evolution of contemporary H7 avian influenza viruses (AIVs) from North America. First, AIVs that are genetically close to the human H10N8 isolate were recovered at the live poultry market (LPM) visited by the first H10N8 patient. High seroprevalence of H10 virus was observed in ducks and chickens from five LPMs in the region. These findings suggested that LPM was the most probable source of human infection with the H10N8 virus, and this virus appeared to be present throughout the LPM system in the city. Second, the novel H7N8 virus most likely circulated among diving ducks in the Mississippi flyway during autumn 2015 and was subsequently introduced to Indiana turkey, in which it evolved from LPAI into HPAI. H4N8 IAVs from diving ducks possess a gene constellation comprising five H7N8–like gene segments. These findings suggest that viral gene constellations circulating among diving ducks could contribute towards the emergence of IAVs that can affect poultry. Diving ducks may serve as a unique reservoir, contributing to the maintenance, diversification, and transmission of IAVs in wild birds. Third, antigenic and genetic characterization of 93 H7 AIVs from North America showed limited antigenic diversity. Gradual accumulation of nucleotide and amino acid substitutions in the H7 gene of AIVs from wild and domestic birds caused a wide genetic diversity. These findings suggested that continuous genetic evolution has not led to significant antigenic diversity for contemporary H7 AIVs isolated from wild and domestic birds in North America. In summary, these findings not only improve our understanding of the ecology and evolution of IAVs but also provide information for formulation of effective disease prevention and control strategies.

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Influenza A virus (IAV) causes annual seasonal epidemics and occasional pandemics resulting in significant levels of mortality and socio-economic costs worldwide. PB1-F2 is a small non-structural protein encoded by an alternate +1 open reading frame in the PB1 gene. PB1-F2 is considered to play important roles in primary influenza virus infection and post-influenza secondary bacterial pneumonia in mice. It is a multifunctional and enigmatic protein with diverse functions attributed to it and the precise contribution of PB1-F2 to the IAV life cycle in avian and mammalian hosts remains largely unknown. In the triple-reassortant H3N2 (TR H3N2) swine influenza virus (SIV) background, we found that PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology in pigs. On the other hand, in turkeys, deletion of PB1-F2 resulted in early induction of clinical disease and effective transmission among the turkey poults. Interestingly, the virulence associated 66S mutation in PB1-F2 abolished the ability of the IAV to successfully infect turkeys and transmit to in-contacts. These results highlight the strain- and species-specific role of PB1-F2 protein. We also demonstrated that specific amino acid residues in the C-terminal of PB1-F2 determine the pathogenicity of 2009 swine-origin pandemic H1N1 virus in a mouse model. The C-terminal residues 73K, 75R, and 79R together with 66S increased virus replication, decreased type I interferon response, increased infiltration of neutrophils and myeloperoxidase production in lungs resulting in acute respiratory distress syndrome (ARDS) in mice with characteristic clinical and pathological features of acute lung injury (ALI). Further, we found that PB1-F2 induces mitochondrial superoxide production and mitochondrial damage in a sequence dependent manner in IAV-infected lung epithelial cells. PB1-F2-mediated mitochondrial damage promotes Parkin-mediated mitophagy but suppresses the autophagic degradation of damaged mitochondria in the infected lung epithelial cells. Accumulated dysfunctional mitochondria likely to aggravate host cell death and inflammatory responses. Taken together, the present findings enhance our understanding of PB1-F2 protein as a virulence determinant in IAV infection in a species- and strain-specific manner and provide new insights into the impact of genetic changes in PB1-F2 on the host pathogenesis of virulent IAV strains. / Ph. D.