In the last decade, the potential of β-glucan, a fungal cell wall component, to induce epigenetic and functional modification of innate immune cells, signified as trained innate immunity (TII) has been demonstrated in several pre-clinical and clinical studies. Parenteral administration of β-glucan has resulted in centrally induced TII in the bone marrow/circulating monocytes. Such trained innate immune cells play a critical role in protection against secondary infections. However, there are now indications that inducing local long-lasting immunity at mucosal barrier tissues such as the lung is warranted for protective immunity against respiratory pathogens. Currently, it remains unclear whether respiratory mucosal administration of β-glucan will induce long-lasting resident-memory macrophages and TII and if so, what are the underlying mechanisms of development and maintenance of memory macrophages at respiratory mucosa. To address this, and kinetics of immune responses in the lung were studied. Profound changes in airway macrophage (AM) pools were observed starting from 3 days post-exposure, which was associated with monocyte recruitment, and this was followed by a series of phenotypic shifts in AMs. The altered AM phenotype profile persisted for up to 8 weeks post-exposure. Importantly, β-glucan-trained AMs demonstrated heightened MHC II expression, enhanced responses to secondary stimulation and improved capacity to perform bacterial phagocytosis. Furthermore, mice with, β-glucan-trained AMs displayed higher rates of survival and improved bacterial control, in the lung and periphery, following a lethal S. pneumoniae infection. Our findings together indicate that a single intranasal delivery of β-glucan is able to train AMs. Further work into epigenetics, metabolism, and the contribution of AMs in protection is needed. / Thesis / Master of Health Sciences (MSc) / The immune system has been classically divided into two major compartments known as the innate and adaptive immune system. For decades, the predominant consensus amongst the field was that only the adaptive immune system can form memory against any pathogens encountered. It has been well established that plants and invertebrates only possess an innate immune system and still show boosted responses and enhanced protection against previously encountered as well as new pathogens. Recently, such capacity for innate immune memory has also been demonstrated in humans and pre-clinical animal models. Innate immune memory provides non-specific, broad- spectrum protection whereas adaptive memory is specific to a singular pathogen. Inducing broad-spectrum protection can be crucial for the future of human medicine. Activation of both adaptive and innate immune arms could prove to be extremely beneficial in vaccination strategies. Through the use of a pre-clinical model, we have found that administering β-glucan, a component of fungal cell wall, directly into the lung significantly alters the phenotype and functionality of lung immune cells, and also provides enhanced protection against a heterologous infection.

Introduction: Atopic dermatitis (AD) is characterized by chronic pruritic relapsing eczematous lesions of the skin. Eosinophilic inflammation in AD is driven by activation of type 2 inflammatory cells including CD4+ T cells and type 2 innate lymphoid cells (ILC2s). We have shown that type 2 cytokines, namely interleukin (IL)-5 and IL-13, stimulate migration and terminal differentiation of eosinophil progenitor cells (EoPs). We propose that these cytokines are important drivers of tissue eosinophilia in AD lesional skin. This study aimed to quantify, by flow cytometry, cells that produce type 2 cytokines in lesional skin compared to peripheral blood from moderate-severe AD patients. Methods: In a cross-sectional study of patients with moderate-to-severe AD (n=16), type 2 inflammatory cells were enumerated in blood and cells extracted from excised skin biopsies. By flow cytometry, live, singlet CD45+cells were identified as ILC2 (lin-CD127+CD294+), EoP (CD34+125+), and CD4+ T cells (Lin+CD3+CD4+). Intracellular expression of type 2 cytokines (IL-5 and IL-13) were evaluated in each cell population. In addition, we developed a protocol to enumerate ILC2s by fluorescence immune-histochemistry in lesional versus non-lesional skin samples and skin biopsies taken 24h post-intradermal challenge with allergen versus diluent. Data are expressed as median (interquartile range [IQR]) unless otherwise stated. Cross compartmental comparisons were made using the Wilcoxon rank-sum test and where applicable, correlational analyses were performed using a Spearman’s rank-correlational test. Results: There was a significantly higher number of total ILC2s in lesional skin compared to blood from AD subjects (556 [99 – 5501] vs 235 [67 – 569] cells/mL, p=0.03). Similarly, IL-5+, IL-13+ ILC2s, were significantly greater in skin compared to blood (6 [1 – 666] vs 1 [1 – 19] cells/mL, p=0.03; 28 [1 – 1357] vs 1 [1 – 7] cells/mL, p=0.01, respectively). We found higher numbers of total and type 2 cytokine positive EoP in lesional skin biopsies from AD patients compared to blood (Total EoP: 815 [285 – 2794] vs 112 [46 – 247] cells/mL, p<0.01; IL-5+EoP: 36 [1 – 129] vs 1 [1 – 23] cells/mL, p=0.07; IL-13+EoP: 92 [10 – 182] vs 1 [1 – 8] cells/mL, p<0.01 and IL-5+IL-13+ILC2: 70 [1 – 158] vs 1 [1 – 12] cells/mL, p=0.02, respectively). In contrast, significantly higher numbers of total and type 2 cytokine positive CD4+ cells were found in blood compared to lesional skin biopsies from AD patients (Total CD4+: 1092 [650 – 1742] vs 58.3 [35.3– 152.4] x 103 cells/mL, p<0.01 and IL-5+IL-13+CD4+ cells: 13.5 x 103 [2.1 x 103 – 42.9 x 103] vs 3.8 x 103 [1.6 x 103 – 4.9 x 103] cells/mL, p=0.02, respectively). For IF staining, there was a significant higher number of ILC2s in lesional compared to non-lesional skin biopsies and biopsies taken 24h post allergen- compared to diluent challenge (1 [0 – 2] vs 0 [0 - 0] cells/mm2, p=0.008, and 2 [1 – 2] vs 0 [0 – 0] cells/mm2, p=0.0002, respectively). Interestingly, in sex analyses we found significantly greater levels of blood ILC2 in females compared to males, but this not was found in the skin. Importantly, we found a significant correlation between lesional skin levels of ILC2 measured by flow cytometry and clinical measures of disease severity/symptoms as reported/calculated from the Patient-Oriented Eczema Measure questionnaire (POEM) score (total ILC2: r=0.55, p=0.04; IL-13+ ILC2s, r=0.61, p=0.02 and IL-5+ IL-13+ ILC2s: r=0.75, p=0.002). Conclusions: Preferential increases in skin-resident ILC2 that produce a type 2 rich environment were found in AD subjects. These levels correlated with patient-oriented measure of disease severity. We propose that this increase may encourage recruitment of mature eosinophils and EoP and possibly drive localized differentiation of EoP into mature eosinophils that may drive the pathology of AD lesions. Furthermore, immunofluorescence staining may be a suitable alternative to flow cytometry for identification of ILC2 in the event of a low cell count. These techniques can be used in future studies that target ILC2 biology to fully understand the role of these cells in driving AD. / Thesis / Master of Science (MSc)

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) is the leading cause of infectious disease-related death worldwide. The critical role of adaptive immunity in anti-TB host defence has been firmly established; thus, current efforts in developing novel vaccination strategies against TB are primarily focused on generating protective adaptive immunity at the infection site, the lungs. Innate immunity has not been a target for vaccination strategies against TB due to the belief that innate immune cells cannot exhibit memory-like characteristics which are known to be central to the long-lasting immunity created by vaccines. Also, the importance of innate immunity in anti-TB immunity has been overlooked. However, over 25% of individuals that are heavily exposed to M.tb clear infection without any detectable conventional T cell immune responses, suggesting a crucial role for innate immune cells in bacterial clearance. Interestingly, the early protection in these individuals is associated with their Bacillus Calmette-Guerin (BCG) vaccination status. Epidemiological studies have shown that BCG is capable of providing protection against numerous infections unrelated to TB in an innate-immune dependent manner. Such observations suggest that the innate immune system exhibits memory-like characteristics, capable of remembering the exposure to the vaccine and thereby responding in an augmented manner to future systemic infections. Nonetheless, it still remains unknown whether parenteral BCG immunization modulates the innate immune cells in the lung and airways, and if so, what role the trained innate immune cells play in early protection against pulmonary TB. Using a subcutaneous BCG immunization and pulmonary TB challenge murine model, we show that early protection against M.tb is independent of adaptive responses in the BCG immunized host. Our data suggest that enhanced early protection is mediated by the BCG-trained memory alveolar macrophages that we have shown to be functionally, phenotypically, metabolically, and transcriptionally altered following immunization. These novel findings suggest a significant anti-TB immune role for the innate immune memory established in the lung following parenteral BCG immunization and have important implications for the development of novel vaccination strategies against TB. / Thesis / Master of Science (MSc) / Pulmonary tuberculosis (TB) is a disease of the lung and is now one of the leading causes of human mortality worldwide. For more than eight decades, parenterally administered Bacillus Calmette–Guérin (BCG) vaccine has been globally used as the only approved vaccine against TB. Recently, it has also been observed that BCG vaccination provides protection against other diseases unrelated to TB and reduces childhood mortality in many developing countries where it is routinely administered to children shortly after birth. The mechanisms underlying the off-target protective effects of BCG vaccine remains largely under-investigated. In this project, we investigated how BCG vaccination enhances the immune system responses against TB and other unrelated infectious diseases. A better understanding of how the BCG vaccination modulates our immune system will provide us with the knowledge that will be useful in the development of more effective vaccination strategies against infectious diseases.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / Enterococci are gram-positive bacteria typically found as commensals in the gastro-intestinal tracts of most mammals. Enterococci, most notably Enterococcus faecalis and Enterococcus faecium, have become problematic causative agents of several nosocomially acquired infections including urinary tract infections, bacteremia, surgical sight infections, and endocarditis. These bacteria must first overcome the innate immune response in order to establish infection. Many bacteria produce capsular polysaccharides that contribute to pathogenesis by helping the microbe evade the host innate immune response. The capsular polysaccharide produced by E. faecalis has been shown to play a role in pathogenesis; however the mechanisms of innate immune avoidance were unknown. Moreover, the number of capsule serotypes produced by E. faecalis and the genetic differences that contribute to capsule serospecificity were in doubt. In the current study it is made clear that only two capsule serotypes are produced by E. faecalis and that both capsule serotypes contribute to evasion of the host innate immune system. This work shows two mechanisms by which the capsule of E. faecalis contributes to immune evasion. First, the presence of capsule inhibited complement mediated phagocytosis through limiting the detection of opsonic complement protein C3 on the surface of the bacteria. Secondly, the presence of capsule altered cytokine signaling of macrophages by shielding bacterial components from detection. Many pathogenic strains of E. faecalis also produce an extracellular protease known as gelatinase (GelE). This work also shows a novel mechanism involving GelE in innate immune evasion through the degradation of the anaphylatoxin C5a. Degradation of C5a by GelE resulted in decreased neutrophil recruitment in vitro. A rabbit model of endocarditis was employed to assess the effect of GelE production on disease development and progression. Rabbits infected with GelE producing strains had increased bacterial burdens in the heart compared to rabbits infected with strains that were GelE negative. Reduced phagocyte infiltration at primary and secondary infection sites was also observed in rabbits infected with GelE producing strains compared to GelE negative strains. The work presented here demonstrates that both the capsular polysaccharide and GelE play roles in E. faecalis evasion of innate immune responses. Moreover, these pathogenic determinants would be suitable targets for developing alternative therapeutics used to treat E. faecalis infections.

The mammalian immune system is capable of detecting and responding to different infectious conditions with specificity at the adaptive level, however whether this ability extends to individual cells of the innate immune system is unclear. The hypothesis of this thesis is that macrophages, as individual cells, can distinguish between productive and non-productive virus infections and respond differently at the gene expression and secreted protein level. To test the hypothesis, mouse bone marrow derived macrophages (BMDMs) were infected in parallel with either a productive (live) and non-productive (attenuated) strain of murine cytomegalovirus (MCMV) and profiled temporally using a range of techniques. Both productive and non-productive MCMV infection resulted in strong type I IFN induction in BMDMs, however induction was significantly more rapid in response to productive infection. In addition, chemoattractant and pro-inflammatory cytokines TNFα, IL-6, RANTES, MIG and MIP-2 were secreted to significantly higher levels in response to productive MCMV infection, and curtailed in response to non-productive MCMV infection. Furthermore, genome-wide microarray profiling revealed a number of co-expressed gene networks regulated differentially in response to the two conditions. This consisted of macrophage gene networks targeted for modulation by de novo MCMV proteins, and late macrophage response genes regulated specifically in response to productive MCMV infection. To further explore the mechanisms of transcriptional regulation during macrophage antiviral response, BMDMs from mice lacking either the type I IFN receptor (Ifnar1) or the IFNβ (Ifnb1) gene were profiled using a similar approach. The resulting genome-wide transcriptional data provided a unique insight into the relationship between type I IFN regulation and the macrophage transcriptome in response to MCMV infection. Overall, the study utilizes a combination of genetic mutants from both host and pathogen to investigate mechanisms of virus detection and host transcriptional regulation during the innate immune response to MCMV infection in macrophages.

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.

Arboviruses are transmitted between vertebrate hosts by arthropod vectors, such as mosquitoes or ticks. In vertebrates arboviruses cause cytopathic effects and disease, however, arbovirus infection of arthropods usually results in persistence. Control of arboviral infection is mediated by the arthropod’s immune system. Pathways such as RNAi, JAK/STAT, Toll and IMD have previously been implicated in controlling arbovirus infections. In contrast, the antiviral role of other pathways in mosquitoes, such as melanisation, is unknown. Using high through output 454 sequencing the transcriptome of U4.4 cells infected with the model arbovirus Semliki Forest virus (SFV)(Togaviridae, Alphavirus) was generated. This experiment revealed intriguing patterns of differential transcript abundance that suggest a broad impact of SFV infection in U4.4 cells, such as in metabolism, cell structure and nucleic acid processing. SFV infection induces differential expression of genes in pathways such as apoptosis, stress response and cell cycle. Most interestingly, this study indicated that melanisation might have an antiviral role in mosquitoes. In arthropods, melanisation is a process involved in wound healing and antimicrobial defences. Phenoloxidase (PO), a key enzyme involved in melanisation, is cytotoxic and therefore kept in its inactive form, prophenoloxidase (PPO), until activation is triggered. The PPO activation process is tightly regulated by serine protease inhibitors (serpins) which inhibit the proteolytic activation reaction. In this thesis I demonstrate that the supernatant of cultured Aedes albopictus-derived U4.4 cells contains a functional proPO-activating system, which is activated by infection with bacteria and virions of SFV. Activation of this pathway reduces the spread and infectivity of SFV in vitro and in vivo. In order to further characterise the PO cascade and its antiviral role the serpins in Ae. albopictus were also investigated. Using the transcriptome sequencing and bioinformatics we identified and classified 11 serpins. We silenced each of the serpins and monitored PPO levels and antiviral activity showing that homologues to drosophila’s serpin- 27a plays a role in melanisation against SFV in vitro. Collectively, these results characterise the mosquito PO cascade as a novel immune defence against arbovirus infection in mosquitoes.

Over the past four years, a previously unrecognised family of innate effector cells has been identified. Their comprehensive functional capabilities range from lymphoid organogenesis, tissue remodelling, wound healing, immune protection and homeostasis to contribution to inflammation and allergic responses. Here we investigate the presence and function of type 2 innate lymphoid cells (ILC2) in the skin. We show that human ILC2 are resident in human skin and express RORA and GATA3, and skin homing receptors. ILC2 further infiltrate the skin after allergen challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2 express the IL-33 receptor ST2, which is up-regulated during activation. Signalling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Atopic dermatitis (AD) is a chronic inflammatory skin disorder. Current evidence suggests that both skin barrier dysfunction and immune system abnormalities, particularly those of a type 2 phenotype, contribute to disease pathogenesis. We demonstrated that ILC2 are enriched in lesional skin biopsies from atopic patients and show higher expression of cytokine receptors, reflecting an activated phenotype. Down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of AD. Interestingly, E-cadherin binding to KLRG1 on human ILC2 dramatically inhibits IL-5 and IL-13 production. ILC2 may contribute to increases in type 2 cytokine production in the absence of the inhibitory E-cadherin ligation through this novel mechanism of barrier sensing. CRTH2, a receptor for prostaglandin D₂ (PGD₂), is expressed by human ILC2. However, the function of CRTH2 in these cells is unclear. We sought to determine the role of PGD₂ and CRTH2 in human ILC2 and compare it with that of the established ILC2 activators IL-25 and IL-33. PGD₂ binding to CRTH2 induced ILC2 migration and production of type 2 cytokines IL-4, IL-5, IL-13 and release of other pro-inflammatory cytokines IL-3, IL-8, IL-9, IL-21, GM-CSF, and CSF-1 in a dose-dependent manner. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA) suggesting a synergistic role. The effects of PGD₂ on ILC2 could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist. Therefore, PGD₂ can be considered as an important and potent activator of ILC2 through CRTH2 mediating strong inflammatory responses. Cell surface interaction mechanisms that activate ILC2 function are unknown. We observed the expression of NKp30 on ILC2 ex vivo and after culture. Using quantitative PCR we confirmed that ILCs express NKp30c splice variant, an immune-modulatory isoform. Incubation of ILC2 with the NKp30 ligand B7H6 and tumour cell lines expressing this protein induced production of type 2 cytokines. This interaction can be inhibited by NKp30 blocking antibodies. We further established that activation of NKp30 induces the canonical pathway of NFƙB signalling. Overall the work in thesis shows for the first time that ILC2 are resident in human skin and infiltrate rapidly after allergen challenge and in AD lesional skin. We have defined cytokine and lipid mediators that contribute to migration and activation of ILC2 and shown that KLRG1 and NKp30 act as inhibitory and activatory receptors respectively. The work defines novel pathways for barrier sensing and cutaneous inflammation, and identifies potential new targets for therapeutic intervention.

The use of antibiotic growth promoters has been limited all around the world because of the concerns about antibiotic resistant bacteria and the presence of antibiotic residues in poultry products. Yeast-derived products are rich sources of ß1,3-1,6-glucan, mannan polysaccharides, and nucleotides and are considered as possible antibiotic alternatives due to their potential intestinal health benefits, growth promotion, and immune system stimulation. The objectives of the current research were: (1) to the evaluate effect of yeast products derived from yeast Saccharomyces cerevisiae on growth performance, gut histomorphology, and innate immune response of broiler chickens; (2) to investigate the effect of yeast products, including distillers dried grains with solubles (DDGS), on innate and antibody-mediated immune response following immunization with different antigens; and (3) to examine the effect of yeast-derived products and DDGS on growth performance, incidence of necrotic enteritis (NE), and local innate immunity in broiler chickens challenged with Clostridium perfringens. Overall, supplementation of diets with yeast products did not affect growth performance of broilers. However, the diets containing yeast cell walls (YCW) and nucleotides increased the villus height in the jejunum and enhanced the number of goblet cells in the ileum. Inclusion of diets with yeast products did not activate the innate immune response of birds under non-pathogen challenge conditions. However, the diet containing YCW activated Th2 cell-mediated immune response in birds immunized with sheep red blood cells and bovine serum albumin. Furthermore, supplementation of diets with YCW and DDGS in birds challenged with Escherichia coli lipopolysaccharide, activated the systemic innate immune response. Regarding antibody-mediated immune response, when compared to the control, serum antibody titer and specific antibody response against different antigens were not affected by dietary treatments. In the C. perfringens challenge study, growth performance, NE lesions and C. perfringens counts in the intestine were not affected by yeast-derived products. However, diets containing YCW and nucleotides stimulated the local innate immune response of birds by upregulation of cytokines and receptors involved in innate immunity. Such findings suggest that the immune-adjuvant like properties of YCW and nucleotides activate the innate immunity of broiler chickens following immunization or challenge with different antigens. / October 2015