Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) are recognized by different cellular pathogen recognition receptors (PRRs), which are expressed on cell membrane or in the cytoplasm of cells of the innate immune system. Nucleic acids derived from pathogens or from certain cellular conditions represent a large category of PAMPs/DAMPs that trigger production of type I interferons (IFN-I) in addition to pro-inflammatory cytokines, by specifically binding to intracellular Toll-like receptors or cytosolic receptors. These cytosolic receptors, which are not related to TLRs and we call them "Toll-free" receptors, include the RNA-sensing RIG-I like receptors (RLRs), the DNA-sensing HIN200 family, and cGAS, amongst others. Viruses have evolved myriad strategies to evoke both host cellular and viral factors to evade IFN-I-mediated innate immune responses, to facilitate their infection, replication, and establishment of latency. This review outlines these "Toll-free" innate immune pathways and recent updates on their regulation, with focus on cellular and viral factors with enzyme activities.

Inflammasome signaling during infections results in cell death and processing and secretion of cytokines from the IL-1 family, which facilitates control over an infection. Pseudomonas aeruginosa and Salmonella Typhimurium are opportunistic bacterial pathogens which may induce acute infections and activate various innate immune signaling pathways, including inflammasomes. However, under favourable conditions these pathogens may evade immune clearance resulting in the establishment of a chronic infection. In this study, I evaluated the modulation of host inflammasome signaling induced by P. aeruginosa and S. Typhimurium during chronic infections. I used a collection of P. aeruginosa clinical isolates obtained from the sputum of cystic fibrosis patients collected during stable and exacerbation periods of disease. I demonstrated that the majority of isolates displayed poor inflammasome signaling and only a small proportion of isolates retained their ability to induce inflammasome activation, which may be associated with pulmonary exacerbations in cystic fibrosis. Sequencing and bioinformatics revealed genetic variations within the type III and type VI secretion systems of P. aeruginosa. While an inactivation of the type III secretion system is expected to impair inflammasome signaling, my results indicate that the type VI secretion system inhibits inflammasome signaling in eukaryotic cells. Due to the lack of chronic animal models for P. aeruginosa, I utilized a murine model of chronic S. Typhimurium infection to assess the modulation of inflammasome signaling throughout the course of a chronic infection. I observed that S. Typhimurium isolated during the acute phase of infection displayed an increased potential to activate inflammasome signaling and this ability progressively declined during the chronic phase of infection. This reduction in inflammasome activation was associated with reduced expression of bacterial virulence factors, such as flagella and the type III secretion system, and was dependent on the NLRP3 inflammasome. Overall, these results reveal that the expression of virulence factors is modulated during chronic bacterial infections, which results in a reduction of inflammasome activation leading to co-survival of the pathogen and host.

Innate immunity operates independently of prior exposure to pathogens. There are several signal transduction pathways that play a key role in inflammatory and immune responses. Critical signaling cascade in the interest of my research is type-1 interferon (IFN) signaling pathway in response to infection with Salmonella Typhimurium (ST). The role of type-I interferons is well established in the context of a viral infection; however, their role in bacterial infections is not clear. In my thesis I aimed to understand the role of type-1 IFNs in bacterial pathogenesis, and scrutinize the mechanism adopted by various components of type-1 IFN signaling, especially ISGF3 complex in response to Salmonella Typhimurium. My results indicate that type-I IFN signaling is detrimental to host survival. I further investigated the mechanism through which type-1 IFN signaling results in host susceptibility against Salmonella. My results indicated that the three transcription factors downstream of IFNAR1 have different impacts in mounting an innate immune response against ST. IRF9 and STAT2 promote susceptibility against ST whereas STAT1 through IFNAR1-signaling, promotes enhanced expression of pro inflammatory cytokines and protection against ST. I also observed that the monocytes/macrophages lineage in Ifnar1⁻ᐟ⁻ mice is responsible for conferring the enhanced resistance against ST. Furthermore, my work determined that expression of type-I IFN signaling compromises the fitness of macrophages by reducing mitochondrial respiration, glycolysis and myelopoiesis.

Toll-like receptor (TLR) ligands and phytochemical feed additives (PFAs) were evaluated in this study to determine the effects of immune stimulation on gut immunity in the zebrafish, Danio rerio, and the channel catfish, Ictalurus punctatus. Rag1-/- (MT) zebrafish were used to study how the TLR ligands β-glucan and resiquimod (R848) affect the innate immune system in the gut of MT zebrafish. Enhanced expression of marker genes (NITR9, NCCRP-1 and MPEG-1) indicated stimulation of Natural Killer (NK) cells, non-specific cytotoxic cells (NCCs) and macrophages. After challenge with Edwardsiella ictaluri, MT zebrafish stimulated with β-glucan demonstrated higher survival and the presence of more macrophages/monocytes in the gut than control MT zebrafish. A PFA test diet containing a blend of prebiotic fiber, oregano, thyme, cinnamon essential oils, and Yucca schidigera (ONE Current™, OC) was fed to channel catfish for 3 months in ponds to determine the effect on channel catfish fingerling growth. Fish were fed in ponds and a tank bacterial challenge followed to test the efficacy of the product. Catfish fed OC demonstrated greater weight gain and feed conversion ratios, higher survival after challenge with E. ictaluri, greater phagocytosis or binding by macrophages and cytotoxic cells. Catfish fed OC also demonstrated greater gut surface area after 2 months feeding OC. To elucidate the effect(s) of each of the compounds in the OC diet on gut immune responses and to determine if PFAs can decrease bacterial colonization and replication within gut tissues, WT and MT zebrafish were fed diets containing different compounds included in OC. Quantification of live bacteria from gut and kidney tissue was determined after challenge with E. ictaluri. Expression levels of immune response genes were evaluated after ingestion of PFAs. Actifibe, Essential oil 25 ppm (EO 25) and Actifibe + EO demonstrated the lowest infection and colonization rate, upregulation of immune response genes, and significantly higher survival when challenged with E. ictaluri. This study demonstrates the potential for application of TLR ligand and feed administered PFAs to improve fish health. Our findings provide a more comprehensive understanding of host gut/pathogen interactions as well as suggestions for novel disease control measures.

Streptococcus pneumoniae (the pneumococcus) is one of the leading causes of death due to infectious disease in the world, with over one million deaths being attributed to this bacterium each year. While the majority of these deaths occur in children in developing nations, significant morbidity and mortality in the developed world, especially in the elderly, can be attributed to pneumococcal diseases such as bacterial pneumonia and meningitis. This is despite the near-universal use of anti-pneumococcal vaccines in these parts of the world. The work presented in this thesis describes the ways in which resident nasal macrophages respond to nasopharyngeal pneumococcal colonization, allowing for the protection of immunocompetent individuals from these diseases. This thesis describes the role of the macrophage scavenger receptor MARCO in recognizing the bacterium upon colonization, and the chain of events that are subsequently established. I have found that MARCO is vital in orchestrating the clearance of pneumococci from the nasopharynx in an expedient manner, as well as preventing the swift spread of bacteria to other tissues of the body early on in colonization. I also outline a role for regulatory micro-RNAs present in macrophages in the mounting of this anti-pneumococcal response via the induction of specific T cell populations. The collection of data found herein is an important resource for those attempting to understand the complex narrative that takes place between the pneumococcus and the innate immune system during a colonizing event and will lead to further discovery on how healthy individuals escape fatal pneumococcal disease. / Thesis / Doctor of Philosophy (PhD) / The bacterium Streptococcus pneumoniae is one of the most dangerous pathogens in the world, accounting for more one million deaths every year worldwide. This bacterium is also very common, with approximately one third of all people having some S. pneumoniae in their noses at any given time. The goal of this thesis is to provide a better understanding of how our immune cells interact with S. pneumoniae when it first enters our noses and how these initial interactions prevent healthy people from becoming sick. I have found that white blood cells called macrophages are crucial to these interactions. Macrophages are able to ‘eat’ the bacteria using a specialized protein called MARCO to grab onto them. This information will be vital in trying to develop new vaccines and treatments for S. pneumoniae-related diseases like bacterial pneumonia (lung infection) and meningitis (brain infection).

LysM-domain containing (LysMD) proteins are widespread in nature and associated with host-pathogen interactions, often-binding peptidoglycan and chitin. However, the functions of mammalian LysMD proteins have not been fully defined. Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, chitin recognition by mammals remains enigmatic at best. The principal receptor(s) on epithelial cells for chitin recognition remain to be determined. In this study, we demonstrate that LYSMD3 is expressed on the surface of human airway epithelial cells. Interestingly, LYSMD3 is able to bind chitin and β-glucan as well as fungal spores. Knockdown and knockout of LYSMD3 markedly impaired chitin and fungi-induced inflammatory cytokine production in lung epithelial cells. Antagonization of LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 all significantly blocked chitin signaling. Taken together our study identifies LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and is required for the epithelial inflammatory response to chitin and fungal spores. / Doctor of Philosophy / Chitin is the main ingredient in the crustacean shells (e.g. crab, shrimp, lobster). It can also be found in fungal cell walls and insect exoskeletons like house dust mites and cockroaches. Many people are allergic to seafood, fungal spores, house dust mites, and cockroach. These allergies are thought to be driven at least partially by a response to chitin. However, how mammals sense and response to chitin is largely unknown. In plants, LysM-domain (LysMD, chitin binding domain) containing receptors are the primary receptors for chitin. These receptors can bind directly to chitin and/or mediate the innate immune response against chitin-containing pathogens such as fungi. Mammals also have LysMD containing proteins, but the functions of these proteins are unclear. In this study, we demonstrate that human LYSMD3 is a novel receptor for chitin. LYSMD3 is essential for chitin recognition and chitin induced inflammatory responses by airway epithelial cells. Our characterization of LYSMD3 as the elusive human epithelial cell receptor for chitin, resolves a long-standing mystery and provides a new insight into the context of innate immunity in mammals against chitin-containing organisms and allergic inflammation.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24514号 / 医博第4956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 長尾 美紀, 教授 中川 一路, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

microRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression. This class of 18-25 nucleotide-long non-coding RNAs has been found to play critical roles in the modulation of a wide spectrum of cellular processes including immunity, development, and metabolism. They modulate their interactions by binding to the 3’ untranslated region of the target messenger RNA to mediate the repression of gene expression. Given their emerging critical roles in the regulation of biological processes, it is not surprising that miRNAs play a significant part in modulating host-virus interactions. Viruses are obligate parasites that hijack the host cellular machinery and processes to promote their life cycle and their propagation. Emerging evidence suggests that miRNAs add an extra regulatory layer to fine-tune viral pathogenesis. This offers novel opportunities not only to delineate the crosstalk between the host and the virus but also allows for the development of novel therapeutics and the identification of novel potential biomarkers of viral infection. Herein, we examine the roles of various miRNAs in the modulation of host-virus interactions. In this thesis, we identify a polycistronic miRNA cluster (miR-183, miR-96, and miR-182) to possess antiviral properties against RNA viruses by augmenting innate immune responses to viral infection. We as well identify miR-383 to possess novel antiviral potential against Dengue virus (DENV), through its targeting of PLA2G4A, a pro-viral host factor essential for the production of infectious particles. Finally, we examine miR-185’s role in the modulation of SARS-CoV-2 infection where we show that miR-185’s regulation of fatty acid and cholesterol metabolism suppresses the virus’s entry and propagation in lung and liver cells. Collectively, the findings in this thesis demonstrate the critical role that miRNAs play in the modulation of host-virus interaction through modifying the host’s cellular environment essential for the regulation of viral pathogenesis.

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