<p>Several labs have previously demonstrated that humoral immune responses at one mucosal tissue can disseminate to other mucosal sites, giving rise to the theory of the common mucosal immune system (CMIS). Evidence that demonstrates a similar link between systemic immune responses and mucosal protection is lacking despite indications that both mucosal and systemic memory B cells share common circulatory pathways. The focus of this study is to determine the contribution of blood-borne B cells to mucosal immunity in humans, and how B cells trafficking through blood can be induced to traffic to mucosal tissues.</p> <p>To address these aims, I have performed several analyses of active and inactive peripheral blood memory B cell (MBC) populations in normal, healthy humans. Analysis of recently activated blood B cells confirmed the revealed that the majority of pre-plasma cells (PPC) in blood of healthy, normal humans secrete IgA, and that the majority of IgA- positive PPC secrete primarily polymeric IgA. A large fraction of blood PPC also express CCR10, and this population contains the highest fraction of IgA-expressing and pIgA-secreting PPC in blood. In contrast, most CCR10- PPC secrete IgG, although a small fraction secretes and stains positive for IgA, and analysis of α4β7expression by blood MBC revealed that CCR10 is more inclusive of IgA-switched and pIgA-secreting PPC in blood. The data presented in this study demonstrate that IgA+/CCR10+ PPC represent the mucosal subset of PPC in the blood of healthy humans, and can be investigated as representative of recent or ongoing mucosal immune responses.</p> <p><em>In vitro </em>polyclonal stimulation of blood MBC through CD40 was able to induce CCR10 expression by blood MBC treated with IL-21, IL-2, IL-10 with additions of other germinal center (GC) cytokines such as IL-5 and TGF-β enhancing CCR10 induction. Surprisingly, CCR10 expression by IgG MBC stimulated under these conditions was similar to that of IgA MBC, demonstrating that CCR10 expression is not limited to IgA- switched B cell clones. The results of this study demonstrate that CCR10 expression is inducible by many GC factors, and importantly, is not limited to IgA-switched B cells.</p> <p>Systemic immunization of healthy volunteers with tetanus toxoid/diphtheria vaccine induced a robust systemic IgG response, but also resulted in a post-immunization mobilization of IgA PPC in blood. These PPC were not specific for tetanus or diphtheria, and in several volunteers showed specificity for mucosal pathogens such as poliovirus (PV) and herpes simplex virus (HSV) glycoproteins. In addition, stimulation of anti-HSV IgG memory was also observed. Thus we have demonstrated that a systemic immune response is capable of inducing antibody relevant to mucosal immunity, possibly exposing a mechanism through which systemic and mucosal humoral immune responses are linked.</p> <p>The studies presented here demonstrate the presence of mucosal B cell memory in blood and thus provide new insight into ways to assess and manipulate mucosal immunity.</p> / Doctor of Philosophy (Medical Science)

<p>The recent and dramatic rise in allergic disease, coupled with the manifestation of the disease within the first years of life, suggests that <em>in utero</em> events are likely critically important to the inception of allergy. Epidemiological and experimental evidence suggest that both genetic predisposition and prenatal environmental exposures (e.g., <em>in utero</em> microbial exposures) play a role in modulating neonatal immunity and subsequent development of allergy. Of relevance to the work in this thesis, reports suggest that bacterial agents can directly alter myelopoiesis and, in connection to allergy, we have previously shown that cord blood (CB) progenitors from high-atopic risk infants demonstrate altered hematopoietic responses. However, whether CB progenitor cell hematopoietic responses are directly altered by microbial stimulation, and what effect maternal atopy has on these responses are unclear. Therefore, this thesis examines the influences of bacterial lipopolysaccharide (LPS) stimulation (innate immunity), maternal atopy, and adaptive immune stimuli (representative of an atopic milieu) on CB progenitor cell eosinophilopoiesis. We show that CB progenitors from healthy, pregnant women respond to LPS through increased eosinophil-basophil (Eo/B) colony forming units (CFU) via the mitogen-activated protein kinase (MAPK) signalling pathway (Chapter 2), whereas the presence of maternal atopy (as defined by skin prick test positivity) is associated with reduced CB CD34⁺ cell LPS-induced Eo/B CFU formation (Chapter 3). To investigate the potential mechanism of reduced eosinophilopoiesis in high-atopic risk infants, CB progenitors stimulated with IL-4 (a surrogate <em>ex vivo</em> for maternal atopy), but not IL-13, demonstrate reduced LPS-induced MAPK activation and Eo/B CFU formation (Chapter 4). This novel work provides insight into mechanisms relating to the influence of maternal atopy and/or potential intrauterine exposures (e.g., prenatal cytokines) on the responsiveness of CB progenitor cells to LPS, which may be of key importance for the development of atopic illnesses. These observations may help in the generation of novel biomarkers and therapeutic targets for childhood atopy.</p> / Doctor of Philosophy (PhD)

<p>The incidence and severity of infectious diseases increases in elderly people (>60 years of age). It is believe that the age-associated changes in the immune system, termed immunosenescence, lead to diminished effectiveness of the immune system leaving the aged susceptible to infectious pathogens and associated diseases. The limited efficacy of the currently available vaccines in elderly populations contribute immensely to the frequency of infectious diseases in the globally growing aging population. As such, the demographic shift warrants the development of effective prophylactic vaccines for the elderly.</p> <p>West Nile virus (WNV) became endemic in North America in 1999, and although it infects people of all ages, the incidence of severe neuroinvasive disease is more prevalent in the elderly. I hypothesized that the susceptibility of the elderly towards severe WNV disease is a consequence of aberrant immune function, and specifically lack of functional virus-specific CD8+ T cells. Contrary to my hypothesis, I found that the magnitude, breadth and functionality of WNV-specific CD8+ T cells were not different between the age cohorts. These results argue that advanced age does not limit the development of functional CD8+ T cell responses following primary infections with an acute virus. Furthermore, the aged members of our cohort maintained functional CD8+ T cells to cytomegalovirus (CMV) and Epstein-Barr virus (EBV), common persistent viruses.</p> <p>Collectively, my results demonstrate that development of vaccines designed to elicit CD8+ T cell immunity may be warranted for elderly individuals and perhaps live vectors should be considered for this population.</p> / Doctor of Philosophy (Medical Science)

<p>BACKGROUND: The ontogeny of peanut allergy (PA) is poorly understood, and the treatment of its most severe manifestation, peanut-induced anaphylaxis (PIA), remains limited to rescue epinephrine. We argued that an untargeted metabolomic analysis would be a useful hypothesis-generating tool to identify novel biomarkers, mediators and possibly therapeutic targets in PA and PIA.</p> <p>METHODS: Models of PA and PIA used in this thesis involved either the oral administration of peanut along with cholera toxin or the topical application of peanut on tape-stripped skin. Liquid-chromatography mass-spectrometry (LC-MS) was performed to identify chemical changes in the serum of mice undergoing sensitization and anaphylaxis. Flow cytometry as well as <em>in vivo</em> gain-of-function and loss-of-function immunological studies were used to determine the biological significance of particular molecules in sensitization.</p> <p>RESULTS: LC-MS followed by multivariant analysis showed that the purine metabolism pathway was altered with elevated levels of uric acid (UA) in sensitized mice. UA depletion using allopurinol and uricase fully prevented the development of the allergic and anaphylactic phenotype. Conversely, administration of UA crystals, instead of cholera toxin or tape stripping along with peanut induced a typical allergic and anaphylactic phenotype. The effects of UA and UA crystals are likely a consequence of effects on the activation of resident dendritic cells. Post-challenge metabolic analysis also revealed a distinct metabolic signature in sensitized mice, highlighted by an increase in several metabolites such as histamine. Likewise, peanut allergic patients display a distinct metabolic profile after oral peanut challenge.</p> <p>CONCLUSION: We identified UA, released after damage to the mucosa and/or skin, as a critical alarmin that facilitates the development of Th2 immunity, specifically PA and PIA. Metabolomics analyses of either mice undergoing anaphylaxis or peanut allergic children subjected to a peanut oral challenge provided an extensive overview of metabolomic changes underlying these conditions. Further studies may lead to the identification of novel biomarkers and mediators.</p> / Master of Science in Medical Sciences (MSMS)

The gastrointestinal tract houses one of the most dense and diverse communities of bacteria on the planet. The mutualistic relationship between the host and commensal microbe permits the microbe an ideal environment to grow and provides the host with increased caloric intake, maturation of the adaptive immune system, and resistance against invading pathogens. To maintain a system in which both parties benefit, the epithelium has evolved numerous strategies to ensure epithelial cells respond to microbes appropriately and that potentially hazardous commensals remain distanced from the soma proper. Breakdown of these propitiating mechanisms elicits unchecked inflammation and can lead to pathology and reduction of host fitness. We show that oral and intestinal epithelial cells respond to the circulating hormone adiponectin in the presence of bacterial constituents, and that adiponectin has the potential to downregulate NF-κB signaling. We also show many commensal bacteria have no effect on TNF or IL-8 proinflammatory gene expression in intestinal cells. Commensals within the family Enterobacteriaceae can increase TNF and IL-8 expression, but also expression of the NF-κB regulator A20 and MAPK phosphatase MKP-1. Importantly, Enterobacteriaceae also increased expression of the IgA transporter pIgR. In the mouse model, we show pIgR expression along the intestinal epithelium is necessary for SIgA accumulation in the outer mucus layer where commensal bacteria reside. Loss of the mucus layer, but not pIgR is sufficient to allow direct bacterial-epithelial cell contact and induce spontaneous inflammation along the colon. Secretory IgA is supplied maternally through breast milk early in life to compensate for the neonate’s inability to produce sufficient endogenous amounts. By utilizing a breeding scheme in which mouse dams were unable to provide their offspring with SIgA, we show the necessity of maternally-supplied SIgA to control bacterial invasion to mesenteric lymph nodes before weaning. In addition, 8-10 week old adult offspring not receiving SIgA as neonates showed both a unique intestinal microbiota and different patterns of intestinal epithelial cell gene expression with and without chemically-induced acute colitis. In summary, we reveal new mechanisms the mammalian host utilizes in order to maintain peace between the commensal microbe and host immune system.

Periodontitis is a polymicrobially-induced, chronic inflammatory disease of the tissues that support and surround the tooth. While greater than 500 organisms are found in dental plaque, Treponema denticola is one of only a few species associated with disease. It has been hypothesized that oral bacteria disrupt host homeostasis through manipulation of the innate immune system. In this study, we examine the impact of binding and subsequent cleavage of factor H, a complement regulator, by T. denticola in the evasion and subversion of complement. The molecular interaction between the sole FH-binding protein, FhbB, and FH was detailed by x-ray crystallography, site-directed mutagenesis, and inhibition analyses. Negatively-charged amino acids of FhbB formed salt-bridges with positively-charged residues of FH within the complement control protein domains (CCP) 6 and 7. In support of its critical role in disease, FhbB was universal among T. denticola isolates, although, different strains produce highly divergent FhbB proteins. Despite extensive sequence variation, predominantly within the FH-binding determinant, all FhbB proteins have similar structure and interact with FH using nearly identical molecular mechanisms. In addition to differences in FhbB, many isolates of T. denticola displayed significant variation in the activity of the chymotrypsin-like protease, dentilisin. Neither FhbB type produced nor dentilisin activity influenced serum resistance, as all strains tested were highly serum resistant. While sequence diversity of FhbB did not influence the interaction with FH or serum resistance, FhbB proteins elicited type-specific antibodies that blocked FH binding. Collectively, these analyses indicate that FH binding is an essential complement evasion mechanism of T. denticola and characterizes the uniquely complex interaction between T. denticola and complement. The data presented here provide novel insight into the pathogenesis of disease and begins to explore a hypothetical molecular mechanism by which this key periopathogen disrupts the host innate immune system, leading to periodontitis.

Mast cells respond to a variety of signals, are associated with both increased inflammation and regulation of the immune response, and are able to interact with a variety of hematopoietic and non-hematopoietic cells. The majority of the work that highlights mast cell pleiotropic abilities has been completed in murine models. Though these models have significantly advanced our understanding of what mast cells can do, they cannot inform us as to what mast cells actually do in human beings. The goal of this dissertation is to assess fully mature, primary human mast cell function beyond the well-defined type 1 hypersensitivity function and place mature human mast cells in the context of interactions with other immune cells. The first project addresses the ability of IFNγ, a historically Th1 associated cytokine, to dramatically alter mast cell phenotype. In particular, IFNγ stimulation allows mast cells to act as antigen presenting cells to CD4+ T cells. The second project describes and addresses the T cell suppressive function of myeloid derived suppressor cells in Mastocytosis, a disease of clonal mast cells.

A Via Alternativa é a principal via de ativação do sistema complemento (SC), sendo o Fator H (FH) um de seus principais reguladores. No presente estudo, nós investigamos os mecanismos moleculares pelo qual o paciente com a mutação Arg127His no FH possui deficiência do SC. Para isto, utilizamos fibroblastos de paciente e individuo normal estimulados com IFN-<font face=\"Symbol\">g e verificamos que as células do paciente eram capazes de produzir FH, contudo a maior parte das proteínas estava retida no retículo endoplasmático (RE). Em paralelo, transfectamos células Cos-7 com plasmídeos contendo a mutação CG453T<font face=\"Symbol\">&#174; CA453T e observamos que a mutação foi responsável pelo retardo na secreção de FH. Apesar da mutação reduzir a secreção de FH, observamos que a capacidade de FH atuar como co-factor não foi afetada. Assim, avaliamos se o uso de chaperonas químicas poderia induzir a secreção da proteína e observamos que houve aumento na secreção de FH nos fibroblastos. Desta forma, propomos o uso desses fármacos como alternativa de tratamento para melhorar a sobrevida do paciente. / Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system (CS). In this study, we investigated the consequences of FH Arg127His mutation to the secretion ratio of this protein by skin fibroblasts in vitro. We stimulated the FH synthesis from patient and normal control with IFN<font face=\"Symbol\">g when we observed that the patient cells were able to synthetize FH, however this mutant protein was mainly retained at the endoplasmic reticulum. In parallel, we transfected Cos-7 cells with plasmids containing CG453T<font face=\"Symbol\">&#174; CA453T mutation and observed that the mutation was responsible for the delay in the FH secretion. Although the mutation reduced the FH secretion, we observed that the FH function was not affected. Thus, we evaluated whether the treatment with chemical chaperones could release FH to the culture supernant. We observed that patients fibroblasts treated increased the secretion of FH. In conclusion, we suggest the use of these chemical chaperones as a potential alternative therapeutic to improve the patients survival.

Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.

The proper regulation of inducible costimulator (ICOS) and its ligand (ICOSL) have been shown to be essential for maintaining immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant antibody production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that ADAM10 is the primary physiological sheddase of ICOSL in both mouse and human. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B-/-), elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wildtype (WT) mice, interaction of ICOSL/ICOS results in ADAM10 induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper (TFH) development and Th2 polarization, seen in a house dust mite exposure model. In addition, enhanced Th1 and Th1 immune responses are seen in experimental allergic encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and at least partially rescues both TFH numbers and the abnormal antibody production previously reported in these mice. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL as well as indirectly regulating ICOS, thus controlling ICOS:ICOSL-dependent responses. Additionally, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using mir146a-/- mice and a model of lymphoproliferative disease using the well characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared to control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA antibody production. In line with this, we found a significant reduction in follicular helper T cells (TFH) and germinal center (GC) B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only shows a role of B cell ADAM10 in controlling autoimmunity, but also increases our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity. Additionally, we found that ADAM17 is important for marginal zone (MZ) B cell development as well as responses to T-independent type 2 (TI2) immunizations. Mice which lack ADAM17 on B cells (A17B) have decreased MZ B cell numbers but have increased levels of antigen specific antibodies in response to TI2 Immunizations. ADAM17 also regulates the level of several surface molecules on plasma cells and MZ B cells necessary for their function and survival. We also show a role for ADAM17 in ILC2 responsiveness to IL-33. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2-/-) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. However, ADAM17ILC2-/- mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, ADAM17 inhibited ILC2s have an increased level of apoptosis and less IL-13 production in response to IL-33 compared to vehicle treated ILC2s. The defect in cytokine production following ADAM17 inhibition is not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the defective survival and IL-13 production following ADAM17 inhibition in these cells. Additionally, ADAM17 regulates the level of surface IL1R2 which may affect IL-33 signaling in ILC2s.