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Major histocompatibility complex (MHC) class I molecules present fragments of the cellular proteome, in the form of short peptides, to the cell surface for the inspection by cytotoxic T cells. This process is a crucial immunosurveillance mechanism used to induce appropriate immune responses against intracellular pathogens and cancer. In order to generate optimal T cell-mediated immune responses, prior to their export to the cell surface, MHC class I molecules undergo a process known as peptide selection. Optimal peptide selection is facilitated by two intracellular peptide editors, tapasin and TAPBPR. TAPBPR was shown to shape the peptide repertoire presented on MHC class I at the cell surface, either by directly catalysing peptide exchange on MHC class I molecules or by associating with the quality control enzyme UDP-glycoprotein glucosyltransferase 1 (UGT1), which selects optimally-loaded MHC class I molecules for export to the cell surface. Given that unlike tapasin, TAPBPR could catalyse peptide editing on MHC class I on its own in solution, I sought to test whether TAPBPR could also function as a peptide exchange catalyst on MHC class I molecules present on the surface of cells. By examining the artefactual expression of TAPBPR at the cell surface upon over-expression, I developed two novel cellular assays which allowed me to explore the function of TAPBPR as a peptide exchange catalyst on plasma membrane-expressed MHC class I molecules. I showed that, when given access the cell surface, TAPBPR can promote efficient peptide exchange on surface expressed MHC class I molecules. These assays allowed me to demonstrate that the 22-35 loop of TAPBPR was essential for its peptide exchange function. Moreover, I revealed that residue L30 within the loop was both necessary and sufficient for the efficient ability of TAPBPR to dissociate peptides from MHC class I molecules that typically accommodate hydrophobic anchor residues in their F pocket. This enabled me to propose a new mechanistic model for TAPBPR-mediated peptide editing. I further addressed the molecular basis governing the compatibility between TAPBPR and MHC class I molecules, by screening a wide panel of human leukocyte antigen (HLA) class I allotypes for their relative propensities to undergo peptide editing by TAPBPR. TAPBPR displayed a clear functional preference for HLA-A molecules, particularly for members of the A2 and A24 supertypes, over HLA-B and -C molecules. This preference appears to be driven by specific molecular features of the MHC class I F pocket, in particular residues H114 and Y116. Finally, I explored the potential translational applications of using TAPBPR as a peptide exchange catalyst on surface-expressed MHC class I molecules. I demonstrated that recombinant TAPBPR can be utilised to load immunogenic peptides of choice directly onto plasma-membrane expression MHC class I, thus overriding the internal antigen presentation pathway. Subsequently, I revealed that, TAPBPR can be used to induce T cell-mediated killing of tumour cells. These findings highlight a potential therapeutic application of TAPBPR in increasing the immune recognition of tumours.

Human trichuriasis is a disease caused by infection with the soil-transmitted gastrointestinal (GI) nematode Trichuris trichiura (T. trichiura), accountable for approximately half a billion infections worldwide. Natural infections in man are often chronic, with the parasite able to regulate the host immune response to facilitate its own survival. Exactly how Trichuris facilitates chronicity is unknown. However, work using the murine colonising species of Trichuris, Trichuris muris (T. muris), a species of Trichuris that is genetically and phylogenetically parallel to T. trichiura, permits laboratory investigations to decipher the immunological responses evoked upon parasite infection. Work within this thesis aimed to investigate the regulatory mediators of chronic T. muris infection in C57BL/6 mice, in an effort to understand regulatory mechanisms that facilitate chronicity. Firstly, it was shown that counter to expectations goblet cell expansion was evoked in C57BL/6 mice following chronic T. muris infection; a Th1 mediated response. Paradoxically, goblet cell hyperplasia was dependent on adaptive derived IL-13 and IL-22, showing that Th2 immune responses can operate within a Th1 setting. These mice also provided an excellent platform to assess the mucus associated proteome during homeostasis and chronic infection, as the mucus gel may provide a source of regulatory proteins that influences parasite and host survival. Initially, it was important to optimise a protocol to extract the mucus gel with minimal disruption to the tissue epithelium. Sequential washes with PBS and 2M urea were utilised to remove the mucus gel of the large intestine and samples were extracted at day 35 from naive and chronically infected mice. After tandem mass spectrometry analysis, several mucus associated proteins found to be exclusive to infected caecal and colonic mucus samples were identified, including Complement 3, Murinoglobulin-1 and Haptoglobin (Hp). The most notable finding was the identification of Hp, a protein that is predominantly expressed by liver hepatocytes and found in serum. Investigations to determine the functional role for Hp following T. muris infection showed that increased levels of Hp were not simply derived from the serum. mRNA and protein levels of Hp were increased during infection, locally within the intestine at the site of infection (i.e. the caecum), but not at non-infected sites (i.e. the colon). Using Hp knock-out (KO) mice and wild-type (WT) counterparts it was demonstrated that Hp deficiency increased Th2 associated responses during a chronic T. muris infection, including an increase in GATA3+CD4+ T cells, total IgE and parasite specific IgG1 production. It was also shown that neutrophils were the cellular source of increased Hp mRNA expression in the caecum following T. muris infection. These results imply that neutrophils can influence the immune response to T. muris and that neutrophil derived Hp may help promote chronic infection. Upon further investigation into the T. muris induced neutrophil response it was demonstrated that the cell surface expression of PD-L1 (a check point inhibitor ligand) was increased on populations of caecal neutrophils. Experiments also showed that caecal neutrophils maintained PD-1 expression within CD4+ T populations within the infected caecum and that neutrophils regulated caecal crypt hyperplasia. Transcriptomic analysis of caecal neutrophils during chronic T. muris supported these observations with significant increase in transcripts for Pd-l1 and Hp and genes associated with intestinal crypt hyperplasia (i.e. cxcl10 and Ido1). Taken together, data from this thesis extends our understanding of immune regulation during chronic T. muris infection and identifies novel roles for intestinal neutrophils in immunity to parasitic infection and the regulation of intestinal inflammation in the wider sense.

Osteosarcoma (OS) is the most common primary bone tumor with an annual incidence rate of 3-4 individuals per million particularly affecting children and young adults. The 5-year survival rate stands at 60-80% with the current standard of care for human OS patients who do not have metastatic disease at presentation, but this drops to 20% for patients with metastatic disease which frequently occurs in the lungs. OS is much more common in canines, with metastasis being the major contributor to mortality, the same as in humans. Metastatic OS warrants novel treatment strategies to improve prognosis and survival. High-frequency irreversible electroporation (H-FIRE) is a promising, non-thermal, minimally invasive technique that induces cell death by applying pulsed electric fields in targeted regions, potentially triggering an anti-tumor immune response that could also target and prevent metastases. Such a dual functionality of H-FIRE is uniquely suited to treat pulmonary metastatic OS. The goal of this thesis was to study the ablative and immunomodulatory effects of H-FIRE on OS in-vitro with the overall hypothesis that H-FIRE completely ablates OS cells, induces the release of damage-associated molecular patterns (DAMPs), and promotes pro-inflammatory immune activating signatures in macrophages and T cells. Using an in-vitro model, my master's thesis focused on 1) Determining the electric field strength that completely ablates OS cells 2) Evaluating the immunomodulatory effects of H-FIRE by co-culturing H-FIRE treated OS cells with macrophages and T cells separately. Our study has utilized murine, canine, and human OS and immune cells, thus demonstrating a unique cross-species approach, 3) Evaluating DAMPs (ATP, calreticulin, and HMGB1) post-H-FIRE ablation of human OS cells. Overall, our study showed that H-FIRE successfully ablated OS cells in-vitro, induced the release of DAMPs from treated cells, and promoted activation signatures in immune cells. This thesis provides foundational data for future investigations developing H-FIRE as an immunomodulatory strategy for treating metastatic OS.

Parasitic helminths infect millions of people and animals worldwide. A key feature of their lifecycle is the longevity of survival within a single host, which is often attributed to the ability of the parasite to divert or modulate the immune response against it. The excretory-secretory (ES) products released by helminths are of interest as the mediators of such immunomodulation. Heligmosomoides polygyrus is an excellent model of gastrointestinal (GI) helminth infection in rodents, and has been used here to investigate several aspects of the immune response, and the manipulation of these, in mice. Firstly, the roles of B cells and antibodies in infection with H. polygyrus and towards the adult ES (HES) were investigated. Using several B cell-deficient mouse strains, a minimal effect on immunity to primary infection with H. polygyrus was demonstrated. However, primary infection serum binds to a select set of highly immunodominant components of the complex protein mixture of HES, which were identified as venom allergen-like proteins (VALs). Utilising four strains of mice that vary in their resistance phenotype to H. polygyrus, several aspects of immunity towards the worm were investigated. Increased levels of markers of alternatively activated macrophages, which are a key component of the granulomatous inflammatory response around invading H. polygyrus larvae, were found in the most resistant strains, SJL and BALB/c. Depletion of macrophages, by administration of clodronate, severely disrupted the granuloma and parasite clearance. Numbers of innate lymphoid cells and the subsequent Th2 response, specificity range and titre of antibody, and activation of regulatory T cells all correlate with a resistant phenotype. A deficiency in the cytokine macrophage migration inhibitory factor (MIF) renders a resistant BALB/c mouse completely susceptible to infections with H. polygyrus, and Nippostronygylus brasiliensis, an acute model of GI helminth infection. This is accompanied by a failure to induce both ILCs and an early myeloid-derived cell population upon infection. The influx of alternatively activated macrophages around larvae in the mucosa of the small intestine is delayed in MIF-/- mice, although all immunological parameters are comparable to wild-type by day 14 post-infection. The susceptible phenotype of MIF-/- mice can be replicated using a chemical inhibitor of MIF in BALB/c mice. Finally, the previously documented transforming growth factor-β (TGF-β) activity of HES was dissected out further using two methods of fractionation. Distinct fractions with TGF-β activity were subjected to mass spectrometry to identify protein components that could be potential candidates for this activity.

Neuromodulation of the immune system has been described to be influenced by hemispheric lateralisation (HL), the stable tendency to relatively utilise one hemisphere or its functions over another. To date there has not been a systematic review of research in this phenomenon conducted, and only one study has examined the effects of HL on the progression of a disease – Human Immunodeficiency Virus (HIV). That research was conducted on a small sample with little control for confounders. The present work sought to compile a systematic review of literature concerning HL and immunity in humans, using effect size analysis. Further, the present work also describes an empirical advancement of this earlier HIV study with stricter control over confounds in a larger sample. The findings corroborated the theory of asymmetrical immune influence by HL via the systematic review showing clear, relatively consistent and strong relationships between left-HL and immunopotentiation. The empirical prospective study extended current knowledge of this relationship in HIV to identify a moderator – HAART treatment. Specifically, left-HL predicted better immunity in HIV-1 patients independent of confounders, with further findings of the same pattern in untreated patients, but not in HAART-treated patients. Further observations were made between HL and HIV-relevant behaviours, again adding to current knowledge. The finding of left-HL being associated with fewer sexual partners in Europeans presents new information of relevance to public health. The combined findings of the present work suggest that left-HL has predictive value in illness (HIV-1) and in general immunity. The present work adds to the existing knowledge new information concerning a moderating factor of the HLimmunity relationship in HIV, and behavioural implications of HL which impact upon HIV disease. Potential explanations for moderation, proposals for neurobiological mechanisms and direction towards future, more rigourous study in the field, both in HIV and immunity, are discussed.

Azithromycin (AZM) is a macrolide antibiotic that is commonly used in respiratory infections. In addition to this, AZM exhibits immunomodulatory properties and has shown utility in a spectrum of inflammatory conditions, including cystic fibrosis and chronic obstructive pulmonary disease. However, the emergence of antibiotic resistance with increased use of AZM and other macrolides for their secondary anti-inflammatory effects is of major concern. To address this, our studies aimed to pinpoint AZM’s mechanism of action while addressing antibiotic resistance issues through generation of nonantibiotic immunomodulatory AZM derivatives. We strategically designed these derivatives by modifying sugar moieties known to be responsible for antibiotic activity while also improving its anti-inflammatory efficacy through targeted synthesis. Minimum inhibitory concentration assays showed a significant loss of antibiotic potency against Staphylococcus aureus in 10 out of 11 analogs. Immunomodulatory potential was assessed through both assays testing NF-κB translocation in monocytes as well as interleukin- 12 secretion from macrophages. These assays both indicated that the majority of the nonantibiotic AZM derivatives exerted superior anti-inflammatory effects compared to their parent compound. Together, these data suggest that the anti-inflammatory mechanisms of AZM can be uncoupled from its antibiotic effects using a targeted approach and offer a path forward for rational design of further immunomodulatory agents. Chapter 4 delves into the nuanced anti-inflammatory mechanisms of AZM, seeking to address the unresolved structure-activity relationship (SAR) and the dearth of identified drug targets. Employing a meticulously designed series of novel AZM analogs and probes, our research aimed to elucidate the intricacies of AZM's immunomodulatory mechanism. Two noteworthy biotinylated AZM derivatives, namely compounds 11 and 12, demonstrated anti-inflammatory potential. Exhibiting the capacity to diminish NF-κB activity, suppress IL-12/23 (p40) secretion, and maintain cytotoxicity below 5%, these compounds emerge as promising candidates for sophisticated in vitro assays aimed at target identification. A groundbreaking enhancement to our toolkit includes the biorthogonal clickable AZM derivative, HS-4-58 (P), and the fluorescent AZM probe, HS-4-86. These probes are meticulously crafted to unravel the intricate network of AZM's binding partners and enable real-time tracking in a zebrafish model of inflammation. Chapter 5 embarks on the transformative potential of AZM in concert with synergistic combinations. Recognizing the multifaceted nature of inflammation, we strategically explored synergies with agents targeting diverse inflammatory pathways, presenting a holistic paradigm for optimal control and resolution of inflammatory responses. Our investigation unveils three strategic dimensions: augmenting AZM's potency through covalent warheads, synergizing with Pitavastatin (PIT), and pioneering HDAC3 inhibition for enhanced epigenetic modulation. Navigating the domain of irreversible covalent inhibitors, exemplified by Compound 10, our inquiry delved into acrolein-based derivatives. Experimental validation highlights the superior efficacy of Compound 10. Beyond unveiling heightened pharmacological potency, our findings dissect the intricate dynamics of acryloyl moieties' strategic placement, unveiling a delicate interplay between positioning, toxicity modulation, and anti-inflammatory efficacy. Experimental validation also showcases the increased potency of Compound M, a PIT-conjugated AZM derivative, in comparison to AZM. Our findings illuminate sophisticated strategies—covalent precision, synergistic augmentation, and selective HDAC3 modulation to enhance AZM's anti-inflammatory fitness. In Chapter 6, we delve into the realm of capuramycin-type nucleoside antibiotics as an independent endeavor, specifically focusing on glycorandomization of nucleosides using the OleD Loki variant glycosyl transferase enzyme. This deliberate approach serves as a promising strategy to enhance and broaden our nucleoside antibiotics platform. Moreover, by leveraging the OleD Loki variant glycosyl transferase enzyme, we aim to achieve glycorandomization of AZM, enabling the semisynthetic synthesis of a diverse library comprising aminosugar-conjugated AZM derivatives. This diverse library holds significance in our exploration of immunomodulation and proton trapping within macrophage lysosomes. In addition to the utility of the compounds developed herein, a useful framework is provided to further modification of macrolide antibiotic. Our findings contribute nuanced insights into AZM's anti-inflammatory properties, presenting a diverse toolkit for further exploration. This research, with its methodological rigor and innovative probes, holds potential for impactful contributions to the understanding of AZM's immunomodulatory potential and its promising role as a therapeutic agent in the realm of inflammatory diseases.

Influenza remains a major global health issue, demanding the need for more effective vaccine strategies. This thesis searches into a novel approach, employing cell-derived extracellular vesicles (EVs) to boost influenza vaccine effectiveness. It dives into the impact of EVs on the innate immune system and their role on a cellular basis. Using cell culture and in vitro immune assays, the composition and immunomodulatory traits of EVs from antigen-presenting cells are researched. The study systematically examines the interactions between these EVs and key components of the innate immune system, unveiling the mechanisms driving their immunostimulatory effects. This research yields crucial insights into EVs' potential as immunomodulators in influenza vaccines. Additionally, determining insights into the identification and validation of adjuvants that enhance EV-mediated immune responses marks a pivotal stride toward the advancement of novel influenza vaccines, increasing their efficacy and protection against diverse viral strains.

Dysregulated hyperinflammatory host immune response to underlying bacterial infections is a characteristic of sepsis. In sepsis, bacteria often trigger abnormal hyperinflammatory responses which can cause multiple organ failure and if sustained can lead to an immunosuppressive phase where the host is susceptible to secondary infections caused by opportunistic bacteria like Pseudomonas aeruginosa (P. aeruginosa). In our studies, we used a 2-hit model of cecal ligation and puncture (CLP) followed by P. aeruginosa secondary lung infection to investigate cellular and molecular mechanisms in the beneficial action of resolvin D2 (RvD2). Resolvins of the D-series are a group of fatty acids known as Specialized Pro-resolving Mediators (SPMs), synthesized endogenously from docosahexaenoic acid (DHA) by different immune cells including neutrophils and macrophages during infection and inflammation. In CLP sepsis, we observed that production of RvD2 in spleens had returned to basal levels by 48 hours after CLP surgery. Administering RvD2 (100 ng/mouse, i.v.) in CLP mice at this time point where endogenous RvD2 synthesis is low enabled CLP mice to clear blood bacteria more efficiently at 24 hours after administration compared to saline controls. There was no significant difference in plasma cytokine production. To further understand cellular and molecular mechanisms, we used flow cytometry to identify different immune cells in spleen and found that RvD2 treatment increased splenic neutrophils (Ly6G+) and myeloid-derived suppressor cells (MDSCs: CD11b+ Ly6G+ Ly6C+) compared to vehicle treated mice. RvD2 treatment increased oxidative burst in splenic neutrophils but not in splenic MDSCs compared to vehicle treated mice. These results provide evidence that RvD2 can expand splenic neutrophil and MDSC numbers to effect greater blood bacterial clearance even when given 48 hours after primary infection. When mice were challenged with P. aeruginosa (intranasally; 24 h after RvD2 treatment) we found that RvD2 increased bacterial clearance in lungs, increased alveolar macrophage numbers and reduced lung IL-23. These results suggest that late RvD2 administration boosted host defense to reduce infection and inflammation. This study provides insight into immunomodulatory effects of RvD2 in a 2-hit infection model of sepsis (Sundarasivarao et al., 2022).

Probiotics are nonpathogenic microorganisms that have been extensively studied for their ability to prevent various infectious, gastrointestinal, and autoimmune diseases. The mechanisms underlying these probiotic effects have not been elucidated. However, we and other researchers have evidence suggesting that probiotic bacteria secrete metabolites that are antimicrobial and anti-inflammatory. As such, we developed a methodology to collect the secreted metabolites from a probiotic bacterium, Lactobacillus acidophilus, and tested this cell free filtrate (CFF) both in vitro and in vivo. Using this CFF, we have demonstrated that L. acidophilus secretes a molecule(s) that has specific bactericidal activity against the opportunistic pathogen, Pseudomonas aeruginosa. Additionally, we have shown that the CFF inhibits P. aeruginosa biofilm formation, an important virulence factor that contributes to P. aeruginosa’s antibiotic resistance. Importantly, our data show that the CFF eradicates 20 h and 48 h established biofilm. We hypothesized that the CFF could modulate pro-inflammatory mediator secretion. To investigate the role of the CFF on tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) production, we measured TNF-α and IL-8 production from THP-1 monocytes and macrophages. Interestingly, our data show that the CFF could stimulate or reduce inflammatory mediator release, depending on the concentration of CFF and whether the cells had been prestimulated with LPS. To elucidate the mechanism underlying the immunomodulatory activity of the CFF, we conducted luciferase assays using a NF-κB reporter THP-1 cell line. Here, we provide evidence that the CFF modulates inflammatory mediators at least in part by regulating NF-κB activation. In addition, we tested the effects of the CFF in a mouse model of P. aeruginosa wound infection. The CFF cleared P. aeruginosa bacterial load and reduced plasma inflammatory mediator production by 5 days. To investigate the bioactive molecule within the CFF, we performed sequential fractionation steps and LCMS to identify and characterize the bioactive molecule, which has a similar mass to charge ratio (m/z) as known polyunsaturated fatty acids (PUFAs) and modified fatty acids. Taken together, our findings provide evidence that L. acidophilus secretes a bioactive molecule that has bactericidal, anti-biofilm, and immunomodulatory activities.

Parasitic nematodes are a global health concern and can infect a variety of organisms such as insects and mammals. As a result of infection, they can cause significant morbidity and mortality. Upon successful infection they can release excreted/ secreted proteins (ESPs) into the host, which enables them to evade or suppress host immunity and cause toxicity. Despite some characterization of ESPs in certain parasitic nematode species, very little is known about the mechanisms behind these interactions. Parasitic nematodes that infect insects called entomopathogenic nematodes (EPNs), have been employed to better understand molecular mechanism. Research with EPNs utilize insect model systems which enables circumvention of logistic and technical challenges encountered with using mammals. EPNs also have high homology with vertebrate parasitic nematodes which leads to the application of translational research. This work focuses on an ESP released by the EPN Steinernema carpocapsae, that displayed immunomodulatory effects in the model host Drosophila melanogaster. The ESP is a secreted phospholipase A2 (sPLA2) enzyme that I named Sc-sPLA2 and displayed immunosuppressive effects by reducing the number of hemocytes in the host, and likely by increasing circulation of an anti-inflammatory lipid. The sPLA2 operates enzymatically by cleaving fatty acids directly from the membrane which resulted in immunosuppressive effects on the humoral and cellular response. Analysis of fly hemolymph post injection of Sc-sPLA2 showed increase of eicosanoid and oxylipin precursors, and the increase of an anti-inflammatory fatty acid. In addition, the analysis of fly hemolymph post infection revealed several lipids that are depleted with the ability to rescue immunity upon treatment. This work also attempted to characterize endogenous lipid signaling mechanisms by identifying lipids, prostaglandins and endogenous sPLA2 enzymes that significantly improve the outcome of infection. Thus far this work has been able to establish that an eicosanoid precursor was able to stimulate the cellular response, furthering our understanding of how lipid signaling is immunomodulatory in D. melanogaster.

Multiple sclerosis (MS) is characterized by an immunological attack of the myelin sheath which leads to demyelination and axonal degradation. This debilitating disorder typically manifests as a progressive loss of motor function, with most cases involving relapsing flare-ups throughout disease progression. The underlying pathophysiology of adult-onset MS is similar to pediatric MS, however pediatric disease typically displays more acute inflammatory responses leading to degeneration. According to the MS society, in the US alone, there are 8,000-10,000 children diagnosed with MS that suffer from frequent relapses. Current therapies for MS reduce the incidence of flare-ups, but do not prevent progressive axonal and neurological degeneration. Cell-based therapies using mesenchymal stem/stromal cells (MSCs) have been under investigation in clinical trials for neurogenerative diseases including adult MS. The placenta has been suggested to be a unique source of MSCs that possess robust immunomodulatory properties and have been reported to be beneficial in graft verse host diseases mouse models. MSCs derived from the placenta (PMSCs) may be a more appropriate cell source for pediatric diseases, because during pregnancy the placenta demonstrates “fetomaternal tolerance”, which is attributed to the expression of human leukocyte antigen-G (HLA-G), a non-classical major histocompatibility complex (MHC) class I molecule that inhibits natural killer cell (NK) killing. Unlike bone marrow derived MSCs (BM-MSCs), PMSCs express HLA-G on their surface in response to interferon gamma (IFN), which is a key inflammatory mediator involved with the onset of MS. Therefore, the expression of HLA-G on PMSCs would make them a unique therapeutic cell source for the treatment of autoimmune diseases like pediatric MS. Currently, a clinical trial is underway using term placenta-derived PMSCs for adult MS and no paradoxical worsening of MS lesion counts was noted. Notably cell-based therapies are limited by potential immune rejection of donor cells and other safety concerns. Increasingly, studies have shown that MSC survival and integration within the host after transplantation are usually poor and that MSCs exert their therapeutic functions mainly via paracrine signaling mechanisms. Conditioned media of BM-MSCs is shown to protect neurons from apoptosis, activate macrophages and be pro-angiogenic. In particular, hepatocyte growth factor (HGF) secreted by MSCs into conditioned medium mediates recovery in a murine model of MS. Currently, the use of MSC conditioned media is limited in that the secreted protein factors are unstable, which creates technical difficulties for “off-the-shelf” clinical use. MSC derived extracellular vesicles (EVs) alternatively, are stable under long term storage conditions compared to freely secreted proteins and may serve as a superior source for cell-free therapy. MSC-derived EVs readily cross the blood brain barrier (BBB) and deliver therapeutic cargo to reduce the effects of neuropathologic diseases, such as MS. It has been demonstrated that MSC-derived EVs exhibit systemic immunomodulatory effects and can facilitate neurological recovery in vitro. PMSC-EVs contain numerous proangiogenic, immunomodulatory, and neuroprotective proteins, including HGF. HGF is a pleiotropic factor shown to have neuronal and oligodendrocyte protective properties. In an experimental autoimmune encephalomyelitis (EAE) rodent model of MS, overexpression of HGF by neurons conferred neuroprotection by reducing inflammation in the CNS and activation of Tregs. HGF is secreted both in soluble form from MSCs and is also contained in exosomes; however, the effects of each form could lead to different cellular responses. It has been suggested that MSCs secrete multiple categories of exosomes, which are involved in differing cellular processes. Therefore, deciphering the molecular mechanism by which MSC-derived EVs alleviate the effects of neurodegenerative diseases is warranted. The goal of the current research project is to demonstrate the therapeutic potential of PMSC-EVs utilizing translational models of MS as well as to and to examine the role of HGF. These studies will be crucial as pre- investigational new drug data moving towards novel and bioengineered cell-free therapeutic treatments for MS patients.

Graft rejection is an immunological response facilitated by the host T lymphocytes and this process could engender tissue destruction and alloantibody production. Allograft recipients must adhere to a rigorous immunosuppressant administration to avoid post-operative complications to the graft. Herein, an acellular drug-eluting vascular graft platform was proposed to alleviate the requirement for systemic drug administration via the targeted delivery of tacrolimus. The graft inner layer and drug-carrying matrix were electrospun with poly(L-lactide-co-caprolactone)/poly(D,L-lactic-co-glycolic acid) as the inner layer and poly(carbonate-urethane) as the outer layer. Mechanical characterization and degradation kinetics of the engineered graft were performed and the optimal material composition was determined. In vitro cytocompatibility assays utilizing endothelial cells, macrophages, and T-cells of primary sources were done and the optimal drug loading concentration was identified. The anastomosis of the drug-eluting vascular graft into the femoral artery of rats was performed to assess its in vivo tissue integration and therapeutic capability. This experimental approach was devised as a proof-of-concept study prior to an elaborate organ transplantation model. The graft maintained a high patency rate and a sustained concentration of tacrolimus after a month of implantation, therefore proving the feasibility of a novel biomaterial-mediated immunomodulation modality.

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients.

GM-CSF has been employed as an adjuvant to cancer immunotherapies in solid tumors due to its ability to drive immune stimulatory macrophages and other myeloid cells that promote anti-tumor responses. However, GM-CSF does not always exert immune stimulatory effects on myeloid cells, and sometimes even drives immune suppressive phenotypes. GM-CSF effects on myeloid cells and the tumor microenvironment (TME) appear to vary across dosages and even cancer types. However, an optimal dosing regimen for GM-CSF has yet to be determined to-date, and this is further complicated by the fact that GM-CSF is often administered via oncolytic viruses or tumor vaccines engineered to overexpress GM-CSF, which makes dosing difficult. One possibility is that GM-CSF effects on macrophages other than immunoregulatory properties has not been considered. Our laboratory previously showed a novel but under-appreciated function for GM-CSF in modulating tumor-associated macrophages (TAM)-mediated angiogenesis and oxygenation which may impact anti-tumor immunity. During times of hypoxia, TAMs, along with tumor cells, are normally producers of pro-angiogenic molecules such as vascular endothelial growth factor (VEGF) that drive dysfunctional tumor angiogenesis. We found that GM-CSF stimulated soluble VEGFR-1 (sVEGFR-1), an anti-angiogenic molecule that binds to and neutralizes VEGF from bioactivity on endothelial cells, from monocytes/macrophages in a dose-dependent manner. Intratumor (IT) injections of highdose GM-CSF ablated blood vessels and worsened hypoxia in orthotopic PyMT triple-negative breast cancer (TNBC) during the “anti-angiogenesis” era. However, it is now known that tumor hypoxia portends poor prognosis in breast and other solid tumors by driving immune suppression and therapeutic resistance. Current efforts in the field focus on reducing tumor hypoxia to improve therapeutic responses. Vessel normalization is an emerging strategy in the field which aims to repair dysfunctional tumor vasculature and enhance oxygen delivery to overcome hypoxia. Thus, we posited that low-dose GM-CSF may stimulate lower levels of TAM sVEGFR-1 that might bind excess VEGF to normalize the tumor vasculature and reduce hypoxia, while also driving immunostimulatory macrophages that promote anti-tumor immune responses for a novel “double-punch” effect. We also wondered whether high-dose GM-CSF may drive immune suppressive macrophage phenotype and reduced treatment efficacy due to exacerbation of hypoxia at this dose. Thus, the goal of this study was to assess and compare the oxygen-regulatory and immune-regulatory components of low-dose vs. high-dose GM-CSF in a mouse model of TNBC to better understand effects of GM-CSF and dosing on the tumor microenvironment. Using orthotopic PyMT tumors, we determined that low-dose IT GM-CSF did in fact reduce tumor hypoxia in vivo and normalized tumor vasculature by increasing pericyte coverage on the tumor vasculature. Priming of PyMT tumors with low-dose GM-CSF (hypoxia reduced) sensitized tumors to anti-programmed cell death-1 (anti-PD1) immune checkpoint blockade (ICB) immunotherapy. These effects were significant because PyMT tumors are notoriously immunologically “cold” (immune deserts) and ICB-resistant. Immunologically, while low-dose GM-CSF reduced hypoxic and inflammatory TAM transcriptional profiles, no phenotypic modulation of TAM or tumor-infiltrating lymphocytes (TIL) were observed by flow cytometry to suggest the primary effect of low-dose GM-CSF was reduction of hypoxia. On the other hand, high-dose GM-CSF primed PyMT tumors (hypoxia exacerbated) did not respond to anti-PD1. We also observed that high-dose GM-CSF priming increased infiltration of TAMs, however they lacked MHCIIhi phenotype or immune stimulatory marker expression despite the association of GM-CSF with driving immune stimulatory myeloid cells. Both the presence of TAM and hypoxia correlate with poor prognosis in breast cancer patients, and our results suggest that high dose GM-CSF drives increased TAM under hypoxia which may negatively affect immunotherapy. We next studied the effects of GM-CSF in the immunologically “hot” (considered “hot” vs. PyMT), anti-PD1-susceptible 4T1 model of TNBC to assess whether GM-CSF immunomodulation differed between tumor types. Interestingly, high-dose GM-CSF increased MHCIIhi TAM and immune stimulatory molecules in 4T1 tumors, suggesting disparate effects of high-dose GM-CSF across PyMT vs. 4T1 TNBC models which may contribute to confounding results across cancer types. These data demonstrate a novel role for low-dose GM-CSF in reducing tumor hypoxia for synergy with anti-PD1 and highlight that both oxygen-regulatory and immune-regulatory effects of GM-CSF require careful consideration in dosage and setting of GMCSF in cancer immunotherapy regimens. Lastly, IT injections of GM-CSF are not always clinically feasible, especially for less-accessible tumors. Here, we also formulated, characterized, and optimized GM-CSF-loaded polymeric nanoparticles intended for systemic but targeted delivery of dose-specific GM-CSF to tumors. Collectively, these data characterize novel effects and delivery modalities for GM-CSF that may improve use and efficacy in cancer immunotherapy.

The current work describes the capability of casein-chitosan microparticles to encapsulate Lactobacillus plantarum (CECT 220 and WCFS1 strains) and evaluates their ability to target the distal areas of the gut and to stimulate the immune system. Microparticles were prepared by complex coacervation, between sodium caseinate and chitosan in an aqueous suspension of the bacteria, and dried by spray-drying. In order to increase the survival rate of the loaded bacteria, microparticles were cross-linked with one of the following cross-linkers: tripolyphosphate, calcium salts or vanillin. Overall, microparticles displayed a mean size of about 7.5 μm with a bacteria loading of about 11 Log CFU/g, when cross-linked with vanillin (MP-LP-V). For conventional microparticles, the payload was 10.12 Log CFU/g. The storage stability study at 25 ◦C/60% RH, MP-LP-V offered the highest degree of protection without signif- icant modification of the payload in 260 days. Compared with control (aqueous suspension of bacteria), MP-LP-V also displayed a significantly higher degree of protection against probiotic inactivation in simulated gastric and intestinal fluids. In vivo results evidenced that microparticles, orally administered to rats, were able to reach the distal ileum and colon in about 4 h post-administration. Additionally, the effect of the daily administration of 107 CFU/mouse of MP-LP-V, for 3 weeks, induced an immunomodulatory effect characterized by an important enhancement of Th1 and Th17 responses. In conclusion, these microparticles seem to be a promising strategy for increasing survival and efficacy of probiotics, allowing the formulation of cost-effective and more stable and effective probiotic-based nutraceuticals.

Intrasynovial tendon injuries are common debilitating conditions characterized by persistent inflammation and tissue degeneration during healing, and are associated with poorer outcomes than extrasynovial tendon injuries. Changes in tendon structure and vascularity following injury facilitate circulating immune cell influx, of which, monocyte-derived macrophages dominate and persist throughout healing. Macrophages are emerging targets for modulating/manipulating tendon healing as they play a fundamental role by initiating and/or resolving inflammation and recruiting fibroblasts via secreted signals for extracellular matrix (ECM) synthesis. Macrophages respond to local stimuli, exhibit pro-inflammatory or anti-inflammatory/regulatory phenotypes, and exert differential effects at the tissue healing site. Therefore, the aim of this research was to investigate macrophage-derived inflammatory and regulatory signals, and subsequently examine their effects on intrasynovial flexor tendon cell bioactivity. Our specific objectives included (1) optimizing peripheral blood monocyte-derived macrophage in vitro differentiation protocols, and subsequently determining their inflammatory and regulatory cytokine secretion, and (2) investigating intrasynovial flexor tenocyte-macrophage interactions in direct (cell-to-cell contact) and transwell (non-contact mediated) in vitro co-culture model systems.Peripheral blood CD14+ monocytes were isolated from 5 horses (IACUC approved) via sequential density gradient centrifugation and magnetic bead-based positive selection. Forelimb intrasynovial deep digital flexor tendons opposing the distal sesamoid bone were harvested from 4 of those horses immediately following euthanasia, collagenase digested to obtain single cell suspensions, and monolayer passaged twice to isolate intrasynovial flexor tenocytes (CD90+105+27-45-). (1) Monocytes were maintained in basal medium (RPMI + 10% FBS + 1% penicillin-streptomycin) and ± 50ng/mL equine GM-CSF for 6 days for macrophage differentiation. Subsequently, regulatory or inflammatory phenotypes were induced via 24-hour priming with 20ng/mL IL-4 and 20ng/mL IL-10, or 50ng/mL IFNg and 100ng/mL LPS respectively. Macrophage morphology, proliferation (tetrazolium-based MTT assay), flow cytometry-based CD206 and 86 surface expression, and secretory profiles (ELISA quantification of TNFa, IL-1β, IL-1Ra, IL-6, and TGFβ-1) were assessed. (2) Donor-matched intrasynovial flexor tenocyte and inflammatory or regulatory macrophage co-cultures were established in direct and transwell systems. Tenocyte proliferation and migration were accessed in monolayer co-culture. Tenocytes were allowed to aggregate in ultra-low attachment plates for 72 hours prior to beginning co-culture to evaluate transcriptional and secretory changes. The co-cultures were maintained for 72 hours, and the tenocytes and co-culture media were stored in -80C for mRNA, qRT-PCR, and ELISA quantification. All data were assessed for normality (Shapiro-Wilk) and comparative differences between treatment groups were assessed with one-way analysis of variance or its non-parametric equivalent. Significance was set at p≤0.05.Monocytes cultured in basal medium with GM-CSF yielded macrophages with a rounded morphology. IL-4+IL-10 resulted in elongate, spindle-shaped macrophages, whereas LPS+IFNg gave rise to rounded, granular macrophages. These morphologies were not impacted by GM-CSF treatment. GM-CSF significantly (p=0.03) increased macrophage proliferation compared to those cultured in basal medium alone by 15-fold, with no differences between inflammatory or regulatory priming. CD86 expression increased during macrophage differentiation but was not statistically significant (p=0.6), and there was no difference in CD206 or 86 expression in regulatory or inflammatory macrophages. Macrophages cultured in basal medium alone secreted low levels of IL-1Ra, IL-10, TNFa, and IL-1β. Macrophages primed with LPS+IFNg showed a significant (p=0.012) increase in IL-1β secretion (median 136.1;100.4-198.3) pg/mL. Both regulatory and inflammatory priming increased TNFa (p=0.034) secretion. GM-CSF followed by LPS+IFNg and IL-4+IL-10 priming yielded a 6-fold increase in IL-1Ra secretion (p=0.029, p=0.014). IL-6 was not detected in macrophage differentiation culture medium irrespective of the treatment. (2) Tenocytes cultured with IL-4 + IL-10 (± GM-CSF) macrophage conditioned media exhibited a 1.5-fold increase in proliferation (p=0.003, p=0.02). Tenocytes co-cultured with IL-4+ IL-10 primed macrophages with GM-CSF pre-treatment had significantly (p=0.0005) increased migration compared to tenocytes cultured without macrophages. Direct co-culture with LPS+IFNg primed macrophages downregulated COL1A1 mRNA 5.2-fold (p=0.01), SOX-9 and ACAN mRNA by 3-fold (p=0.003, p=0.004) and COL2A1 mRNA by 15-fold (p=0.04). No significant differences were seen in transwell co-culture. MMP-3 mRNA was upregulated with both direct and transwell co-culture with IL-4 + IL-10 (+ GM-CSF) primed macrophages, and MMP-13 was upregulated 32-fold (p=0.01) in direct co-culture. IL-6 mRNA was upregulated 352-fold (p=0.04) with LPS+IFNg macrophage direct co-culture. IL-6 secretion was significantly increased in both direct and transwell co-culture whereas IL-6 was not quantified in the media of macrophage or tenocyte culture alone.Overall, when discussing our results specifically with respect to M1 and M2 macrophages differential effects of DDF tenocyte ECM gene expression, our results demonstrate that co-culture with M1 macrophages downregulates tenocyte matrix gene expression, whereas the matrix gene expression of DDF tenocytes under basal conditions and those co-cultured with M2 macrophages are largely equivalent. From a mechanistic standpoint, while elevated IL-6 concentrations during healing has been attributed to pro-resolving effects as well as towards tendon degeneration, the specific timeline for IL-6 impact on tenocyte ECM synthesis, and in turn, overall tendon healing outcomes are key areas of future work in the field of intrasynovial tendon immunomodulation.

Persistent inflammatory stimuli can result in chronic inflammation and ultimately disease states such as, rheumatoid arthritis and Alzheimer’s Disease. Typical therapeutic approaches have failed to resolve chronic inflammation in patients. Therefore, this proposal will investigate an alternative strategy of immunomodulatory biomaterials, specifically a deliverable collagen-mimetic peptide that has been engineered as a high affinity ligand for the inhibitory immune receptor LAIR-1, named Collagen-mimetic LAIR-1 Ligand Peptide (CLLP). The hypothesis of this research is that interactions between CLLP and LAIR-1 expressing immune cells, be it through functionalized biomaterials or particle delivery vehicles, will result in suppressed inflammatory activation, furthermore phagocytes seeded on CLLP surfaces will experience increased uptake (dictated in part by the extent of LAIR-1 expression). When investigating CLLP’s effect on inflammatory activation of macrophages and dendritic cells it was observed that LAIR-1 expressing cells were significantly inhibited in their inflammatory activation when seeded onto CLLP surfaces. In contrast, when investigating cells that had naturally low LAIR-1 expression or cells with significantly reduced LAIR-1 expression, via siRNA knockdown, CLLP failed to reduce inflammatory activation. Furthermore, both macrophages and dendritic cells experienced increases in uptake on CLLP functionalized surfaces. The results of these uptake experiments also strongly suggest that LAIR-1 is inhibitory to uptake and the observed CLLP induced increases in uptake are not due to LAIR-1 signaling. When dendritic cells are seeded on CLLP surfaces and stimulated with antigen functionalized nanoparticles, the cells exhibit increased antigen presentation. When CLLP was delivered to cells via nanoparticles it successfully inhibited macrophage activation. Interestingly functionalizing nanoparticles with CLLP significantly reduced the uptake of said particles by LAIR-1 expressing phagocytes. The immunomodulatory strategy of CLLP functionalization could potentially be broadly applied, for example toward the development of device coatings, anti-inflammatory particle-based therapeutics, and/or integrated biomaterials

Anecdotal evidence of beneficial behavioral and health effects of cannabidiol (CBD) use in companion animals has amplified the need to elucidate safety and potential impacts of CBD use. The purpose of this investigation was to determine the impact of industrial hemp-derived CBD administration on canine health and well-being. We hypothesized that CBD would produce beneficial effects on canine behavior without negatively impacting animal health. Dog treats were formulated to include CBD and shown to be palatable across a range of CBD inclusion levels. Dogs were supplemented with CBD treats and subjected to a noise-induced fear response test to assess potential anxiolytic effect of CBD. Behavioral response to the noise-induced fear response test was unaffected by CBD and did not support an anxiolytic effect of CBD when supplemented at 1.4 mg/kg BW/d. Next, triaxial accelerometers were fitted to dogs’ collars to assess the impact of CBD treats on voluntary daily activity. While voluntary daily activity of healthy adult dogs was unaffected by 1.8 and 4.5 mg CBD/kg BW/d, a reduction in daily scratching suggested a potential antipruritic effect of CBD. Potential impacts on canine health were also assessed by evaluating changes in the canine metabolome, hematology, and serum chemistry, and immune response upon exposure to a novel antigen. The canine metabolome, including amine/phenol, carbonyl, carboxyl, and hydroxyl metabolites, was altered with 4.5 mg CBD/kg BW/d and suggested an impact on glucose, amino acid, vitamin, and nucleotide metabolism. Hematological and clinical indices of health and serum immunoglobulins (IgG and IgM) were largely unaffected by 5 mg CBD/kg BW/d; however, elevated liver enzyme alkaline phosphatase may suggest altered liver function. These findings may be used to formulate recommendations for CBD use in a clinical environment, spur investigation into other potential therapeutic effects of CBD, and may serve as a foundation for the development of regulations on CBD use, all of which are essential to supporting the health and well-being of companion animals.

Advances in the field of immunology have created many avenues for the development of novel immunotherapies for conditions from autoimmune disorders and tissue defects to vaccine development. Immune system involvement in health maintenance is multifaceted and generally highly localized. Most of the recently developed treatments, however, are applied systemically which leads to hyperactivation or suppression of the immune system at non-target sites. Development of biomaterial scaffolds that can recruit relevant subclasses of the immune system cells and locally modify their phenotype in a controlled manner can reduce the side effects of immunotherapeutics and improve their efficacy.For biomaterials to be successful as local immunomodulatory scaffolds, they should be biodegradable and biocompatible, evading the foreign body response, yet encourage rapid immune cell infiltration, by providing immediate macroporosity. They should also synergize with other developing therapeutics by providing a large cargo capacity and mechanism for tunable controlled release. Finally, such biomaterials should be injectable and have a long shelf-life for ease of clinical application. Many biomaterials have been developed to possess these features; however, all fall short in one aspect or another. Our group has previously developed a novel biomaterial, microporous annealed particle gels, an injectable hydrogel scaffold, that provides immediate porosity to enable cell infiltration and improved wound closure. Here I introduce two novel immunomodulatory hydrogels based on the MAP technology with their applications in tissue regeneration and vaccination. For the first hydrogel technology, I present data on incorporating a minor change in MAP, by crosslinking hydrogels with unnatural D-chirality peptides (D-MAP) which made MAP immunogenic, resulting in type 2 response with IL33 producing macrophages, leading to regenerative healing of the skin. Notably, I show that this response was dependent on the adaptive immunity, without use of immunomodulatory agents or growth factors, highlighting utility of biomaterials in regenerative healing through activation of adaptive immunity. For the second hydrogel technology, I show that incorporation of antigens in the microfluidic fabrication of MAP (VaxMAP) can lead to a robust germinal center development and humoral response. Furthermore, by incorporation of polymeric nanoparticles carrying CpG ODN adjuvants in MAP, I was able to induce class switching of the antibodies to a T-helper 1 dependent isotype. Finally, I showed MAP’s unique capability of inducing different immune responses to two antigens within one vaccination. Together, these studies showcase the utility of MAP in immunomodulatory applications ranging from tissue regeneration to vaccination. At the other end of the spectrum for development of immunotherapeutics, is the in vitro study of immune cells for development of biologic immunotherapies, such as monoclonal antibodies. Phenotypic study of immune cells with single cell resolution is essential for development of such therapeutics. Microscale approaches to study antibody producing cells (APCs) with single cell resolution have been developed but none provide a widely accessible method to isolate target cells at high throughput, and generally require complicated assay formats for phenotypic studies. As such, labor intensive, time consuming, and costly techniques such as hybridoma technology remain the mainstream methods for development of therapeutics. Microgel technologies combined with microfluidic techniques hold promise to resolve these limitations. In the last chapter, I introduce the utility of a novel microparticle platform, nanovials, for performing plate-based secretion assays at single cell level. The nanovials are hydrogel-based cup shaped microparticles which play the role of nanoliter-sizes well plates and can be loaded with individual APCs to capture and assess antigen-specificity of their secreted antibodies. The nanovials are compatible with commercial fluorescence-assisted flowcytometry systems and can be used to sort APCs with a throughput of more than two million APCs in one day. The APCs are sorted alive and can be used for downstream work such as sequencing for antibody discovery, and further functional analysis for study of their biology.

CD5 and CD6 are closely related signal-transducing class I scavenger receptors mainly expressed on lymphocytes. Both receptors are involved in the modulation of the activation and differentiation cell processes triggered by clonotypic antigen-specific receptors present on T and B cells (TCR and BCR, respectively). To serve such a relevant immunomodulatory function, the extracellular region of CD5 and CD6 interacts with soluble and/or cell-bound endogenous counterreceptors but also microbial-associated molecular patterns (MAMPs). Evidence from genetically-modified mouse models indicates that the absence or blockade of CD5- and CD6-mediated signals results in dysregulated immune responses, which may be deleterious or advantageous in some pathological conditions, such as infection, cancer or autoimmunity. Bench to bedside translation from transgenic data is constrained by ethical concerns which can be overcome by exogenous administration of soluble proteins acting as decoy receptors and leading to transient “functional knockdown”. This review gathers information currently available on the therapeutic efficacy of soluble CD5 and CD6 receptor infusion in different experimental models of disease. The existing proof-of-concept warrants the interest of soluble CD5 and CD6 as safe and efficient immunotherapeutic agents in diverse and relevant pathological conditions.

Musculoskeletal tissue injuries affect around 1 in 3 Americans and 1.7 billion people worldwide. This is a huge economic burden, costing an estimated $120 billion in the US alone. With limited success from surgery or subcutaneous injections of medicine, where only temporary relief or complications can occur, alternative measures should be explored. Injectable biologically-loaded hydrogels are one avenue and act as drug delivery systems. They provide a minimally invasive approach to release biologics in a sustained and controlled manner to provide long-lasting relief without toxic effects and with less risk of surgical complications. In this study, the immunological application of a previously-developed nanofibrous PCL-interspersed collagen hydrogel, (PNCOL) was explored by loading PNCOL with the cytokine IL-4 and identifying its effect upon macrophages. Furthermore, the effect of a simulated digestive inflammatory environment (DIE) had upon protein release kinetics as well as scaffold integrity were characterized. Genipin crosslinking was then explored to improve scaffold resistance to degradation, and an optimal genipin concentration was identified to impart sufficient scaffold crosslinking, increased mechanical strength, and a prolonged release profile, with minimal cytotoxic effects. Lastly, the immunomodulatory effect of IL-4 released from crosslinked and uncrosslinked scaffolds were investigated through identifying the impact of IL-4 on macrophage differentiation. The IL-4 released from PNCOL polarized macrophages toward an anti-inflammatory, pro-healing state, while genipin crosslinking with and without IL-4's presence appeared to lower macrophage activity.

The delivery of disease-relevant antigens by nanoparticles is a promising strategy for selectively inhibiting pathogenic autoreactive cells while maintaining a protective immune repertoire. This process, known as antigen-specific immune tolerance, would be a curative treatment for patients with autoimmune disorders. Currently, symptoms are managed by broad-acting immunosuppressive therapies that increase the risk for opportunistic infection and cancer. This dissertation describes the mechanistic insights and engineered improvements made toward the application of polymeric nanoparticles (NPs) to the modulation of innate and adaptive immune responses. A new method for loading antigen into poly(lactide-co-glycolide) (PLG) particles was engineered using PLG-antigen polymer conjugates to incorporate antigen into the bulk of the particle’s polymer matrix. This method was a significant improvement compared to formulations that couple antigen to the particle surface or encapsulate antigen within the particle. By comparison, these antigen-polymer conjugate NPs significantly increased the range and precision of antigen-loading, eliminated uncontrolled payload release, and reduced the exposure of antigen on the particle surface. This loading strategy enabled the delivery of multiple immunodominant antigens within a single particle formulation, which induced tolerance to multiple antigens simultaneously, a significant advancement toward developing treatments for heterogeneous disease pathologies. The tolerogenic ability of these polymer conjugate particles was augmented by conjugating the anti-inflammatory cytokine transforming growth factor beta (TGF-β) to the particle surface. Both antigen and TGF-β were bioactively co-delivered and these TGF-β-coupled particles were able to enhance tolerance induction at decreased nanoparticle doses compared to antigen-only particles, demonstrating the therapeutic potential for influencing Ag presentation by co-delivering cytokines. Mechanistic studies demonstrated a significant role for the liver in particle-induced tolerance. Specifically, the Kupffer cells and liver sinusoidal endothelial cells contributed to tolerance induction by collectively presenting antigen, secreting inhibitory IL-10 and prostaglandin E2, and expressing inhibitory surface molecule PD-L1. Interestingly, Kupffer cells contributed to, but were not necessary for, particle-induced tolerance. The cellular mechanisms of particle-induced tolerance were evaluated in the context of antigen presentation to T cells. Dendritic cell transcription factor activity and T cell secretion of IL-2 and IFNγ were positively regulated by NP antigen loading. In mice, tolerance induction by antigen-containing particles was dependent on both particle dose and antigen loading. Specifically, tolerance induction required a minimal antigen loading of 8 μg/mg and could not be rescued at lower loadings by increasing the NP dose. A minimum NP dose of 0.5 mg was required for tolerance, even at the high antigen loading of 128 μg/mg. PLG and poly(lactic acid) (PLA) NPs were found to be inherently immunomodulatory and inhibited the macrophage production of inflammatory cytokines induced by bacterially-derived lipopolysaccharide (LPS). This inhibitory effect was influenced by polymer composition and surface charge. In the sepsis mouse model of lethal LPS-induced endotoxemia, prophylactic and therapeutic treatment with PLA NPs increased the survival rates to 63% and 14%, respectively. Collectively, this work has advanced the design, abilities, and understanding of antigen-loaded and cargo-free nanoparticles which have demonstrated the potential for clinically modulating pathological adaptive and innate immune responses.

Polysaccharides are complex biopolymers that play essential roles in the biological systems including energy storage, structural support, lubrication, and signal transduction. Despite their importance, the synthesis of polysaccharides has proven to be very challenging due to the presence of multiple hydroxyl groups and difficulty in controlling the stereochemical outcome of glycosylation reactions. As a conventional chemical method to synthesize polysaccharides, ring-opening polymerization of anhydrosugars enables the synthesis of stereoregular α-(1→6)-linked polysaccharides, but is less effective in preparing polysaccharides with other linkages. Enzymatic polymerizations have also been explored, however, these methods typically require expensive monomers, and suffer from a narrow scope of enzymes and small scale of reactions. The limited approaches to polysaccharides have inspired chemists to synthesize polysaccharide mimetics with achiral linkages that can be constructed efficiently. Poly-amido-saccharides (PASs) are a new type of saccharide polymers in which the O-glycosidic linkages in natural polysaccharides are replaced with (1→2)-amide linkages. With saccharide moieties inter-connected by amide bonds, PASs exhibit characteristics of both polysaccharides and polypeptides, such as possessing pyranose-backbones and lots of hydroxyl groups, and adopting a left-handed helical conformation. However, due to lack of sufficient terminal saccharide residues, previously synthesized glucose and galactose PASs display weak interactions with carbohydrate binding lectins and receptors, limiting their applications in biomedical and pharmaceutical fields. Herein, the design and synthesis of PASs with novel structures and properties is described. By pre-installing the stereochemistry in the monomer, Altrose PASs (Alt-PASs) with β-(1→2)-amide linkages are prepared via ring-opening polymerization of an altrose-based β-lactam followed by debenzylation. Circular dichroism shows that Alt-PASs adopt a right-handed helical conformation in aqueous solution. Via the polymerization of disaccharide-based β-lactams, two PASs with either 4-O-α-D-glucose branches (Mal-PASs) or 6-O-β-D-glucose branches (Gen-PASs) are obtained. Biological studies reveal that Mal-PASs are multivalent ligands to lectin Concanavalin A, while Gen-PASs activate RAW 264.7 macrophage cells by enhancing the secretion of TNF-α and NO. The anionic ring-opening polymerization of sugar-based β-lactams is a useful method to synthesize well-defined polysaccharide mimetics, and this method expands the current repertory of approaches available to complex saccharide polymers with biological activities.

A complex network of interactions exists between the immune, the olfactory, and the central nervous system (CNS). Inhalation of different fragrances can affect immunological reactions in response to an antigen but also may have effects on the CNS and cognitive activity. We performed an exploratory study of the immunomodulatory ability of a series of compounds representing each of the 10 odor categories or clusters described previously. We evaluated the impact of each particular odor on the immune response after immunization with the model antigen ovalbumin in combination with the TLR3 agonist poly I:C. We found that some odors behave as immunostimulatory agents, whereas others might be considered as potential immunosuppressant odors. Interestingly, the immunomodulatory capacity was, in some cases, strain-specific. In particular, one of the fragrances, carvone, was found to be immunostimulatory in BALB/c mice and immunosuppressive in C57BL/6J mice, facilitating or impairing viral clearance, respectively, in a model of a viral infection with a recombinant adenovirus. Importantly, inhalation of the odor improved the memory capacity in BALB/c mice in a fear-conditioning test, while it impaired this same capacity in C57BL/6J mice. The improvement in memory capacity in BALB/c was associated with higher CD3+ T cell infiltration into the hippocampus and increased local expression of mRNA coding for IL-1β, TNF-α, and IL-6 cytokines. In contrast, the memory impairment in C57BL/6 was associated with a reduction in CD3 numbers and an increase in IFN-γ. These data suggest an association between the immunomodulatory capacity of smells and their impact on the cognitive functions of the animals. These results highlight the potential of studying odors as therapeutic agents for CNS-related diseases.

Glioblastomas are the most common malignant brain tumor, with an annual incidence of 6 in 100,000 people. Conventional treatment modalities, including chemotherapy and radiation, are toxic and cause a decrease in quality of life as the disease progresses. Several investigators have turned to pharmacologically active derivatives of herbal remedies as less-toxic alternatives. Indirubin, a dark red isomer of indigo, is known to be effective in treating chronic inflammation and some forms of cancer, and the mechanism is, in part, due to activity as a ligand for the transcription factor aryl hydrocarbon receptor (AhR). As with most AhR ligands, indirubin induces the CYP1 family of metabolizing enzymes, alters cell cycling, and can be active through the GSK-3β pathway. In recent years, bioavailable derivatives have been synthesized, including indirubin-3'-(2,3 dihydroxy-propyl)-oximether (E804). Several lines of research show that E804 has anti-angiogenic and anti-proliferative properties, and we previously demonstrated anti-inflammatory properties of E804. In this study, T98G human glioblastoma cells were treated with E804 to determine the degree of AhR activation, the secretion of both immunosuppressive and pro-inflammatory cytokines by the tumor cells, and the effects of those tumor secretions on macrophage polarization. E804 is potent inducer of CYP1B1 in T98G cells, has modest effects on TGFβ2 secretion, and suppresses VEGF at high does, while enhancing VEGF secretion at low doses. The secretion of IL-6 by T98G cells was only modestly affected at the highest treatment levels. The effects of supernatants derived from E804-treated T98G cells on macrophage indicate that E804 may polarize macrophages to the pro-tumor M2 phenotype. Overall, the results of this study indicate that E804 concentrations would need to be carefully monitored in a clinical application. Future studies should include an in vivo model of brain tumors to test the potential of E804 as a standalone, or adjuvant treatment for glioblastoma.

Neuropathic pain occurring as a consequence of a disease or lesion in the somatosensory nervous system is devastating and refractory to current treatments. Neuropathic pain negatively affects the quality of life of patients and causes an economic burden to the sufferers and the global economy. Thus, there is an overwhelming need to investigate the underlying mechanisms and develop novel treatment approaches. Over the past decade, it has become clear that neuroinflammation plays a critical role in the pathogenesis of neuropathic pain. Indeed, the contribution of leukocytes such as T cells and macrophages, immune-like glial cells such as microglia and astrocytes, and inflammatory cytokines have been implicated in neuropathic pain caused by nervous system injury. Modification of such neuroinflammatory responses via immunomodulation is a potential novel therapeutic approach for the treatment of neuropathic pain. Myelin-derived altered peptide ligands (APLs) generated by transforming the amino acid sequence of native myelin peptides are known to modify immune and inflammatory responses, and are able to inhibit antigen-specific T cell function. APLs have demonstrated promising therapeutic effects in several in vivo and in vitro assays by changing the activation and functions of immune cells, and altering their cytokine profiles. In this thesis, we found that immunisation with the native myelin basic protein (MBP) peptide produced pain hypersensitivity and neuroinflammation in uninjured animals and that co-immunisation with the relevant APL reduced these effects. Next, we showed that immunisation with MBP-derived APL in animals with peripheral nerve injury significantly attenuated pain hypersensitivity and suppressed neuroinflammation, as well as altered some systemic immune responses. However, the same immunisation strategy failed to exert an analgesic effect following spinal cord injury. Lastly, the effects of gene therapy to introduce the anti-inflammatory cytokine interleukin (IL)-35 in the spinal cord were investigated in a mouse model of neuropathic pain. We found that intrathecal administration of a plasmid DNA encoding IL-35 significantly attenuated spinal microglia activation without ameliorating pain hypersensitivity in nerve-injured mice. These findings demonstrate the differential effects of various immunomodulatory approaches and provide important insights into the complexity of immunotherapy for the treatment of diverse neuropathic pain conditions.

Myeloid derived suppressor cells (MDSCs) are heterogeneous population of immature myeloid cells having immunosuppressive properties in tumor and sepsis conditions. MDSCs are well- characterized in mice and human disease conditions and also reported in dogs. So far pig MDSCs are not characterized but they might be playing an important role in immunosuppressive conditions like porcine reproductive and respiratory syndrome. In humans, treatment of peripheral blood mononuclear cells (PBMCs) with GM-CSF and IL-6 induces MDSCs phenotype and function. Thus, we made an attempt to characterize porcine MDSCs by treatment of magnetically sorted myeloid cells using porcine GM-CSF and IL-6. The presence of cell surface markers CD172, SLAII, CD80/86, CD11R3, CD11c and DC-SIGN on cytokines treated pig myeloid cells were analyzed by flow cytometry for phenotypic characterization of MDSCs. For their functional characterization, we performed autologous lymphocyte proliferation assay in coculture experiments and also measured induction of regulatory T cells and IL-10 cytokine production. Our results showed that, unlike human cells, pig myeloid cells treated with GM-CSF and IL-6 did not induce generation of typical identifiable population of porcine suppressor cells either phenotypically or functionally. However, this study has created interest and provided useful insights for future investigation of myeloid derived suppressor cells in pigs.

Diabetes mellitus, the third most common disease in the world, is a chronic metabolic disorder caused by a failure of insulin production and/or an inability to respond to insulin. Specifically, type 1 diabetes is a disorder characterized by targeted autoimmune-directed destruction of a patient's beta-cell population within the pancreatic islets of Langerhans. The current primary treatment for type 1 diabetes is daily multiple insulin injections. However, this treatment cannot provide sustained physiological release, and the insulin amount is not finely tuned to glycemia. Pancreatic transplants or islet transplants would be the preferred treatment method but the lack of donor tissue and immunoincompatibility has been shown to be a roadblock to their widespread use. The objective of this project is to develop an effective strategy for the treatment of type 1 diabetes using beta-cells based replacement therapy. To improve the viability of transplanted beta-cells, one novel approach is to transplant optimal size range of beta-cell spheroids rather than cell suspension. Uniform sized multicellular spheroids can be coated with a thin layer of non-degradable hydrogel for immunoisolation. In addition, the survival of spheroids of optimized size can be further improved with a novel coating of multiple layers of human mesenchymal stem cells (hMSCs), a cell type that has profound immunoregulatory effect, to prevent graft rejection. To prevent hMSC migrate away from spheroids, another layer of non-degradable hydrogel can be added. To further improve the viability and suppress the immune rejection, spheroids will be encapsulated with nanoparticles loaded with angiogenic and immune regulatory molecules. By this means the spheroid will passively evade the complications of stressors in addition to actively modulating the immune microenvironment for regulatory tolerance and long-term engraftment. Firstly, through optimizing our hydrogel systems based on poly (ethylene glycol) (PEG), we have created specific niche for beta-cells to form artificial islets in vitro. We have found that the optimal condition is the concentration of PEG at 5% and the ratios of 4-arm thiolated PEG to 4-arm PEG acrylate at 1:2. Conjugated with adhesive peptides, especially, RGD at 0.2 mM, can significantly promote the glucose stimulated insulin secretion of encapsulated beta-cells. Secondly, we have fabricated different sizes of uniformed beta-cells spheroids through our designed high-throughput automatic spheroids maker. Beta-cells in the spheroids of 200 μm exhibited largest insulin secretion based on glucose stimulus when compared to others with sizes of 100, 300, 400 and 500 μm. The novel core-shell structured spheroids-hMSCs complex was successfully achieved. Methylcellulose hydrogel was applied as physical barrier on the surface of beta-cells spheroids to inhibit invasion of hMSCs. Human MSCs prevented apoptosis of beta-cells spheroids and benefited insulin secretion when exposed to pro-inflammatory cytokines. Thirdly, immune regulatory molecules [leukemia inhibitory factor (LIF) and interleukin 10 (IL-10)] and angiogenic molecule [vascular endothelial growth factor (VEGF)] loaded poly (lactic-co-glycolic acid) (PLGA) nanoparticles have been successfully fabricated through solvent extraction/evaporation technique. These growth factors can be controlled release about 6 weeks. The bioactivity of released VEGF has been confirmed by the in vitro HAEC proliferation assay. Finally, beta-cells spheroids were transplanted under the kidney capsule to treat diabetic mice. Beta-cells spheroids kept the glucose level of diabetic mice constant. Co-transplanted hMSCs suppressed the host inflammation response, activated the regulatory T cells and also promoted angiogenesis at the transplantation site. The beta-cells spheroids/hMSCs/hydrogel complex initiated a mild inflammatory response. The LIF and IL-10, and VEGF loaded complex can further inhibited this response and promoted blood vessel network formation at the transplantation site. Our approach holds a great potential to treat type 1 diabetes.

Viruses such as influenza suppress host immune function by a variety of methods. This may result in a significant morbidity through several pathways, including facilitation of secondary bacterial pneumonia from pathogens such as Streptococcus pneumoniae. Lungresident alveolar macrophages (AMs) act as the first line of innate cellular immunity against respiratory bacterial pathogens, including pneumococcus. Therefore, they represent an attractive target for study Before investigating the impact of influenza infection on resident AMs, we first characterized different subsets of lung-resident macrophages in naïve mice using a novel in vivo labeling approach in conjunction with multicolor flow cytometric analysis and confocal microscopic examination. A stable fluorescent dye, PKH26-PCL, was administered intranasally to selectively label the lung-resident macrophages in a well-established murine model prior to influenza infection. We determined the turnover kinetics of the lung-resident macrophage subsets during the course of influenza infection. More than 90% of resident AMs were lost in the first week after influenza, while the remaining cells had a necrotic phenotype. To establish the impact of this innate immune defect, influenza-infected mice were challenged with a small dose of Streptococcus pneumoniae. Early AM-mediated bacterial clearance was significantly impaired during the AM depletion phase in influenza-infected mice – about 50% of the initial bacterial inoculum could be harvested from the alveolar airspaces 3 hours later. In mock-infected mice, by contrast, more than 95% of inocula up-to-50-fold higher was efficiently cleared. Co-infection during the AM depletion phase caused significant body weight loss and mortality. Two weeks after influenza, the AM population was fully replenished with successful re-establishment of the early innate host protection. Local GM-CSF treatment induced partial expansion of resident AMs during influenza infection. Thus, it led to partial restoring of the impaired early bacterial clearance with efficient protection against secondary pneumococcal pneumonia. We conclude that a novel immunosuppression mechanism occurs during influenza infection through the resident AM depletion. Among other potential effects, this establishes a niche for secondary pneumococcal infection by altering early cellular innate immunity in the lungs resulting in pneumococcal outgrowth and lethal pneumonia. This novel mechanism will inform development of novel therapeutic approaches to restore lung innate immunity against bacterial super-infections. Secondary bacterial pneumonia (SBP) is a leading cause of the increased hospitalizations and mortality during influenza epidemics and pandemics despite routine use of standard antibiotics. Antibiotic-induced immunopathology associated with bacterial cell wall lysis has been suggested to contribute to these poor outcomes. Using Streptococcus pneumoniae in a wellestablished murine model of SBP following influenza, we stratified disease severity based on the pneumococcal load in the lungs via in vivo bioluminescence imaging. Ampicillin treatment cured mice with mild pneumonia but was ineffective against severely pneumonic mice, despite effective bacterial killing. This treatment failure makes it crucial to explore immunmodulation approaches that can prevent the aggravated lung immunopathology during antibiotic treatment of severe SBP. Therefore, we tested the efficacy of the standard anti-inflammatory drug dexamethasone as an adjunctive corticosteroid therapy. Adjunctive dexamethasone treatment significantly improved ampicillin-induced immunopathology and survival outcomes in mice with severe SBP. However, early dexamethasone therapy during primary influenza infection impaired the adaptive immunity in the lungs as manifest by increased viral titers, with an associated loss of its protective functions in SBP. The clinical use of corticosteroids as an adjunctive therapy for treating pneumonia is still under debate. However, our findings support adjunctive clinical use of corticosteroids in severe cases of community-acquired pneumonia. Nonetheless, dexamethasone treatment has drawbacks implied by delayed body weight recovery in dexamethasone-treated mice, which may explain the published controversy on the corticosteroid efficacy in terms of disease morbidity. This relative success of our animal model of SBP to simulate the clinical therapeutic settings in humans will help explore novel immunomodulation approaches to improve the poor outcomes of antibiotic treatment of severe community-acquired pneumonia.

Influenza A virus (IAV) is generally associated with the seasonal malady that causes brief respiratory illness during the winter months, known simply as "the flu." Most otherwise healthy individuals will suffer from mild fever, congestion, headaches and myalgia that are resolved within 5-7 days of onset. However, there are nearly 500,000 influenza-related deaths that occur world-wide every year. Many of these casualties and patients hospitalized with influenza also test positive for bacterial pneumonia, the most common agent being Streptococcus pneumoniae. Although all individuals are subject to this viral:bacterial synergistic disease, the young, elderly, and immunocompromised are the most susceptible. Previous studies have shown that viral infection creates a prolonged hyper-responsive pro-inflammatory state in the lungs, which increases susceptibility to secondary bacterial infection. Lethality is due to detrimental pulmonary damage from a dysregulated host inflammatory response, known as the "cytokine storm." However, the nature of dual infection has not been well-studied in the elderly demographic. Therefore, we aim to better define this disease synergy in an aged mouse model and explore potential therapeutic alternatives that could be beneficial for the aged and other vulnerable populations. Sphingolipid modulation has emerged as a potential target to ameliorate the excessive inflammation (cytokine storm) elicited by highly pathogenic influenza. There is particular emphasis on sphingosine 1-phosphate (S1P) signaling, as well as control of intracellular S1P levels via sphingosine kinases (SK). Sphingolipids are involved in a multitude of cellular processes, and are tightly regulated by their metabolizing enzymes. We hypothesize that manipulation of sphingolipid signaling and alteration of the internal sphingolipid milieu will diminish the inflammatory response elicited by IAV infection. Using fluorescence-activated cell sorting (FACS), real-time PCR and cytometric bead array (CBA) analysis, we evaluated the immunomodulatory effects of systemic sphingosine analog treatment within the lung microenvironment under homeostatic and influenza-infected conditions. FTY720 treatment caused transient, but significant lymphopenia, influx of neutrophils and efflux of macrophages in the lungs, which was enhanced during a mild influenza infectionGene expression in the lungs was generally unaltered, but protein levels showed increases in specific influenza-induced cytokines, suggesting these treatments may have post-transcriptional effects on cytokine expression. To evaluate sphingolipid modulation in specific pulmonary cell types, we next observed the effects of these compounds and sphingosine kinase (SK) inhibitors in epithelial and alveolar macrophage-like cell lines. SK inhibitors and Enigmol demonstrated anti-viral effects in A549 cells, decreasing viral loads by up to 1.5 logs. Real-time PCR and CBA analysis further demonstrated that these effects were associated with alterations in key cytokine expression, including CCL2, CCL5, CXCL10, IL-6, and IL-8. Collectively, these findings indicate that therapeutic sphingolipid modulation has the potential for creating a protective microenvironment in the lungs that could alleviate or even prevent viral:bacterial synergistic disease.

The inflammatory process is a critical component of the repair and regeneration of skeletal muscle following injury. The influx of innate immune cells following injury is intricate, and temporal nature. Although required for proper repair and regeneration, the inflammatory process has been shown to exacerbate initial damage, prolonging the healing process. Complementary Alternative Medicine (CAM) treatments, such as massage therapy, are a promising substitute for pharmaceutical modulation of the inflammatory response, and recent studies into the efficacy of massage have begun to report the physiological benefits of massage application following injury. Nonetheless, there is a significant lack of sound mechanistic investigations into massage application and its effects on unperturbed tissue. To gain insight to its potential influences on healthy skeletal muscle, massage was applied at three different magnitudes of load in vivo. Using a custom fabricated device for cyclic compressive loading, Wistar rats receiving massage had an increased expression in genes associated with the immune response; a significant change in the macrophage populations within the muscle tissue; and demonstrated a systemic effect marked by the increase of immune cells in the non-massaged limb. Further elucidating the systemic and immunomodulatory effects of massage, Long Evans rats receiving non-constrained eccentric exercise followed by a single 30minute bout of massage, displayed a significant crossover effect just 6 hours post exercise through the modulation of inflammatory cells in the non-massaged limb. Together these investigations suggest that mechanotransductive properties of massage can promote modulation of the immune response absent of pharmaceuticals.

The threat from infectious diseases dates as far back as prehistoric times. Pathogens continue to pose serious challenges to human health. The emergence and spread of diseases such as HIV/AIDS, Severe Acute Respiratory Syndrome (SARS), avian influenza, and the threats of bioterrorism have made infectious diseases major public health concerns. Despite many successes in the discovery of anti-infective medications, the treatment of infectious diseases faces serious challenges, which include (i) the emergence and reemergence of infectious pathogens, (ii) the ability of pathogens to adapt and develop resistance to drugs, and (ii) a shortage in the development and discovery of new anti-infective drugs. Host-Oriented Broad-Spectrum (HOBS) treatments have the promising potential to alleviate these problems. The HOBS treatment paradigm focuses on finding drug targets in human host that are simultaneously effective against a wide variety of infectious agents and toxins. In this dissertation, we present a computational approach to predict HOBS treatments by integrative analysis of three types of data, namely, (a) gene expression data representing host responses upon infection by a pathogen, (b) annotations of genes to pre-defined biological pathways and processes, and (iii) genes that are targets of known drugs. Our methods combine gene set-level enrichment with biclustering. We applied our approach to a compendium of gene expression data sets derived from host cells exposed to bacterial or to fungal pathogens, to functional annotation data from multiple databases, and to drug targets from DrugBank. We present putative host drug targets and drugs with extensive support in the literature for their potential to treat multiple bacterial and fungal infections. These results showcase the potential of our computational approach to predict HOBS drug targets that may be effective against two or more pathogens. Our study takes a clean-slate approach that promises to yield unsuspected or unknown associations between pathogens and biological processes, and thus discern candidate gene/proteins to be further probed as HOBS targets. Furthermore, by focusing on host responses to pathogens as captured by transcriptional data, our proposed approach stimulates host-oriented drug target identification, which has potential to alleviate the problem of drug resistance.

BACKGROUND: Adeno-associated vectors (rAAV) have been used to attain long-term liver gene expression. In humans, the cellular immune response poses a serious obstacle for transgene persistence while neutralizing humoral immunity curtails re-administration. Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria) benefits from liver gene transfer in mouse models and clinical trials are about to begin. In this study, we sought to study in non-human primates the feasibility of repeated gene-transfer with intravenous administration of rAAV5 vectors under the effects of an intensive immunosuppressive regimen and to analyze its ability to circumvent T-cell immunity and thereby prolong transgene expression. METHODS: Three female Macaca fascicularis were intravenously injected with 1x1013 genome copies/kg of rAAV5 encoding the human PBGD. Mycophenolate mofetil (MMF), anti-thymocyte immunoglobulin, methylprednisolone, tacrolimus and rituximab were given in combination during 12 weeks to block T- and B-cell mediated adaptive immune responses in two macaques. Immunodeficient and immunocompetent mice were intravenously injected with 5x1012 genome copies/kg of rAAV5-encoding luciferase protein. Forty days later MMF, tacrolimus and rituximab were daily administrated to ascertain whether the immunosuppressants or their metabolites could interfere with transgene expression. RESULTS: Macaques given a rAAV5 vector encoding human PBGD developed cellular and humoral immunity against viral capsids but not towards the transgene. Anti-AAV humoral responses were attenuated during 12 weeks but intensely rebounded following cessation of the immunosuppressants. Accordingly, subsequent gene transfer with a rAAV5 vector encoding green fluorescent protein was impossible. One macaque showed enhanced PBGD expression 25 weeks after rAAV5-pbgd administration but overexpression had not been detected while the animal was under immunosuppression. As a potential explanation, MMF decreases transgene expression in mouse livers that had been successfully transduced by a rAAV5 several weeks before MMF onset. Such a silencing effect was independent of AAV complementary strand synthesis and requires an adaptive immune system. CONCLUSIONS: These results indicate that our transient and intensive pharmacological immunosuppression fails to improve AAV5-based liver gene transfer in non-human primates. The reasons include an incomplete restraint of humoral immune responses to viral capsids that interfere with repeated gene transfer in addition to an intriguing MMF-dependent drug-mediated interference with liver transgene expression.

In the last 150 years, tremendous headway has been made in our understanding of the human immune system. Pioneers in the field such as Paul Ehrlich, Elie Metchnikoff, Louis Pasteur, Robert Koch and Walter Reed carried out seminal studies that established the groundwork for our understanding of humoral and cellular immunity in humans. However, this direct line of evidence into human immunology was diverted in the mid-20th century with the adoption of a model which allowed for investigators to use a reductionist-approach with the promise to resolve immunity at a molecular level. This revolutionary model was the scientific commercialization of various inbred strains of mice. It seems inconceivable how a four-legged nocturnal rodent managed to become the focus of billions of dollars of research to improve our understanding of human immunity. Nevertheless, this strange surrogate for human immunity did provide us with major conceptual advances in areas, such as identification of dendritic cell population heterogeneity, T cell help for B cell antibody production, MHC-restricted recognition of virusinfected cells, and even the discovery of cell types like NKT cells. However, these prior advances have now been prefaced with decades-worth of disappointing, non-translational findings. The best examples of such disappointments are in murine models of autoimmunity, cancer immunotherapy, and vaccinology where numerous studies have revealed promising outcomes in mice but were met with failure or limited success upon translation into humans. We do not look at this as a failure of the murine model; rather we consider it a call to arms to innovate in vitro surrogates to examine human immunity when otherwise bound by ethical limitation from working directly in humans. To overcome these challenges, we developed a system to interrogate novel immunogens that begins by generating human dendritic cells (DCs), a cell type necessary to mounting a protective immune response. DCs for research and clinical applications are typically derived from purified blood monocytes that are cultured in a cocktail of cytokines for a week or more. Because it has been iv suggested that these cytokine-derived DCs may be deficient in some important immunological functions and might not accurately represent antigen presenting cell (APC) populations found under normal conditions in vivo, there is an interest in developing methods that permit the derivation of DCs in a more physiologically relevant manner in vitro. Here, we describe a simple and reliable technique for generating large numbers of highly purified DCs that is based on a one-way migration of blood monocytes through a layer of human umbilical vein endothelial cells (HUVECs) that are cultured to confluency in the upper chamber of a Transwell device. The resultant APCs, harvested from the lower Transwell chamber, resemble other cultured DC populations in their expression of major histocompatibility (MHC) and costimulatory molecules, ability to phagocytose protein antigens and capacity to trigger primary antigen-specific T cell responses. This technique offers several advantages over the standard method of in vitro cytokine-driven DC development, including: (1) the rapidity of this approach, as DC differentiation occurs in only 2 days, (2) the differentiation process itself, which is more akin to the development of DCs under physiologic conditions and (3) the cost effectiveness of the system, since no monocyte pre-selection is required and DC development occurs in the absence of expensive recombinant cytokines. Taken together, this approach allows for the exploration of novel immunogens utilizing a physiologically representative population of APCs enriched from circulating blood. The outbreak of the swine-origin H1N1 influenza in the spring of 2009 took epidemiologists, immunologists, and vaccinologists by surprise and galvanized a massive worldwide effort to produce millions of vaccine doses to protect against this single virus strain. Of particular concern was the apparent lack of pre-existing antibody capable of eliciting cross-protective immunity against this novel virus, which fueled fears this strain would trigger a particularly far-reaching and lethal pandemic. Given that disease caused by the swine-origin virus was far less severe than expected, we hypothesized v cellular immunity to cross-conserved T cell epitopes might have played a significant role in protecting against the pandemic H1N1 in the absence of cross-reactive humoral immunity. We collaborated with bioinformaticians to develop an immunoinformatics approach to predict CD4+ T cell epitopes conserved between the 2008-2009 seasonal H1N1 vaccine strain and pandemic H1N1 (A/California/04/2009) hemagglutinin proteins that could act as novel immunogens and function as potential vaccine candidates or compliments to current vaccine formulations. We examined these peptides using T cells from human donors not exposed to the pandemic virus demonstrating that pre-existing CD4+ T cells can elicit cross-reactive effector responses against the pandemic H1N1 virus. As well, we showed the computational tools created by our collaborators were 80-90% accurate in predicting CD4+ T cell epitopes and their HLA-DRB1-dependent response profiles in donors that were chosen at random for HLA haplotype. Combined, these results confirm the power of coupling immunoinformatics to define broadly reactive CD4+ T cell epitopes with a highly sensitive in vitro model to verify these in silico predictions as a means to understand human cellular immunity, including cross-protective responses, and to define CD4+ T cell epitopes for potential vaccination efforts against future influenza viruses and other pathogens. It is thought that utilizing highly conserved peptides as novel immunogens of cellular immunity for future vaccination strategies may require an adjuvant for efficacy. However, the FDA has approved the use of only two adjuvant compounds (Alum or MPL®) which may not be compatible or offer effective immune enhancement in novel vaccine preparations, thereby soliciting the need for novel adjuvants. Nanoparticles have since been a topic of adjuvant potential. Nanoparticles harbor great potential because they possess unique physicochemical properties compared to their larger counter parts as a result of quantum-size effects and their inherent large surface area to volume ratio. These physicochemical properties govern how a nanoparticle will behave in its environment. However, vi researchers have only just begun to catalogue the biological effect these properties illicit. Moreover, little is known about the interaction between the immune system and NPs. However, in light of the recent development in new adjuvants that involves composites and coatings of polymers, lipids, ligands, TLR agonist, the ability of a simple metal oxide nanopowder to effectively induce or couple immunomodulation would provide researchers a basic alternative to costly and complex adjuvant development. Considering the evidence suggesting NPs can act as immunopotentiators, we questioned whether these materials can act not only as innate adjuvants, but as novel immunogens to cellular immunity. To accomplish this, we under took a set of studies to investigate any nanoparticle size-induced effects using TiO2, one of the most widely manufactured nanoparticles, as a model. We explored titanium dioxide synthesized into its three most commonly nanoarchitectures: anatase (7-10 nm), rutile (15-20 nm), and nanotube (10-15 nm diameters, 70-150 nm length) in comparison to a micron-sized formulation. We used the fully human autologous MIMIC® immunological construct has been utilized as a predictive, nonanimal alternative to diagnose nanoparticle immunogenicity. Cumulatively, treatment with titanium dioxide nanoparticles in the MIMIC® system led to elevated levels of proinflammatory cytokines and increased maturation and expression of costimulatory molecules on dendritic cells. Additionally, these treatments effectively primed activation and proliferation of naïve CD4+ T cells in comparison to dendritic cells treated with titanium dioxide microspheres, characteristic of an in vivo inflammatory response, providing evidence of a size induced difference between the nano-sized and micron-sized material, revealing novel immune cell recognition and activation by a crystalline nanomaterial in a size-dependent manner. Having identified nanomaterial size as a contributing feature of nanoparticle induced immunopotentiation, we became interested if additional physicochemical properties such as surface vii reactivity or catalytic behavior could also be immunostimulatory. Moreover, because we witnessed a stimulatory effect to dendritic cells following nanoparticle treatment, we were curious how these nanoparticle-touched dendritic cells would impact adaptive immunity. Since TiO2 acts as an oxidant we chose an antioxidant nanoparticle, CeO2, as a counterpart to explore how divergent nanoparticle surface reactivity impacts innate and adaptive immunity. We focused on the effect these nanoparticles had on human dendritic cells and TH cells as a strategy towards defining their impact to cellular immunity. Combined, we report that TiO2 nanoparticles potentiate DC maturation inducing the secretion of IL- 12p70 and IL-1B, while treatment with CeO2 nanoparticles induced IL-10, a hallmark of suppression. When delivered to T cells alone TiO2 nanoparticles induced stronger proliferation in comparison to CeO2 which also stimulated TReg differentiation. When co-cultured in allogeneic T cell assays, the materials directed alternate TH polarization whereby TiO2 drives largely a TH1 dominate response, whereas CeO2 drove a TH2 bias. Combined, we report a novel immunomodulatory capacity of nanomaterials with catalytic activity. While unintentional exposure to these nanomaterials could pose a serious health risk, development and targeted use of such immunomodulatory nanoparticles could provide researchers with new tools for novel adjuvant strategies or therapeutics.

Abstract: Poxviruses are renowned for encoding proteins that modulate virtually every aspect of the host immune system. One effective barrier against virus infection is apoptosis, a form of programmed cell death. Apoptosis is controlled at the mitochondria by pro- and anti-apoptotic members of the highly conserved Bcl-2 family of proteins, and two members in particular, Bak and Bax, are absolutely critical to the induction of cell death. Although poxviruses encode an array of effective inhibitors of apoptosis, only members of the Avipoxvirus genus, of which fowlpox virus is the prototypical member, encode proteins with obvious, albeit limited, sequence identity to cellular Bcl-2 proteins. Fowlpox virus, the prototypical avipoxvirus, encodes FPV039, a protein that possesses two of the four highly conserved Bcl-2 homology (BH) domains that characterise the Bcl-2 family. Here we demonstrate that, like cellular Bcl-2 proteins, FPV039 localised to the mitochondria where it prevented apoptosis induced by a variety of cytotoxic stimuli, including virus infection itself. FPV039 inhibited apoptosis induced by Bak and Bax through an interaction with Bak and activated Bax. FPV039 also interacted with a discrete subset of BH3-only proteins, the upstream activators of Bak and Bax, to prevent Bax activation in the first place. Additionally, we have characterised the function and mechanism of action of a novel deerpox virus protein, DPV022. Intriguingly, DPV022 lacks obvious homology to cellular Bcl-2 proteins but shares limited regions of amino acid identity with two other poxviral inhibitors of apoptosis, vaccinia virus F1L and myxoma virus M11L, which are themselves unrelated. Here we demonstrate that DPV022 localised to the mitochondria where it interacted directly with Bak and Bax to inhibit apoptosis, even in the absence all cellular anti-apoptotic Bcl-2 proteins. We have also embarked on a preliminary analysis of the apical events that initially trigger apoptosis during infection with vaccinia virus, the prototypical poxvirus. Accordingly, we demonstrate that the BH3-only protein Noxa contributed to the vaccinia virus-induced apoptotic response, possibly through an involvement with dsRNA. Together, this study represents a comprehensive analysis of the ways in which poxviruses manipulate the cellular Bcl-2 family of proteins, the arbiters of cell death.

Immunotherapy, as a strategy to trigger immunity and eradicate a variety of chronic infectious diseases and cancers, has been explored for several decades with significant success in animal models. However, effective translation of these strategies into human clinical settings has proven elusive. Several cell-based anti-tumor therapies have progressed to clinical trials where antigen presenting dendritic cells (DCs) are isolated from patients, loaded with viral or tumor antigens and infused back in the patients. These ex-vivo “trained” DCs then present antigens to naïve T cells (adoptive therapy). However there exist several major limitations to this approach, including morbidity associated with patient cell isolation, high cost of ex vivo cell manipulation, time lag in “training” the immune cells, regulatory concerns, as well as the fact that ~ 90% of transplanted DCs die before they even home to lymph nodes. On the other hand, current immunotherapy approaches using recombinant proteins, synthetic peptides or nucleic acids, which "train" the immune cells in vivo to mount an immune response, have failed to address the tremendous challenge in generating efficient, sustained and protective immunity. There are two major challenges that must be overcome, (a) there exist relatively fewer numbers of immune cells at the sites of vaccine administration and given that these antigens themselves are weakly immunogenic, vaccine formulations must be tailored to attract large number of DCs to the immunization site and (b) immunologically, conventional nucleic acid or protein/peptide based vaccines do not elicit the required T helper type (Th) immunity along with a strong Cytotoxic T Lymphocyte (CTL) response against viral or tumor antigens and therefore new formulations must be able to “direct” the immune response towards a specific Th-type. Our goal was to design polymer-based sustained release formulations to addresses these challenges. Specifically, we have designed and developed delivery systems that can carry multiple biomolecules (nucleic acids, proteins, peptides, and chemoattractants) in a single injectable formulation. The delivery system promoted efficient migration of a large number of DCs to the site of injection and successful delivery of antigen resulting in activation of DCs. The multi-modal delivery system has the ability to bias or switch the immune response to the desired phenotype (e.g. Th1 or Th2) in a controlled manner. Using an infectious disease model against hepatitis B we have shown that co-encapsulation of Interleukin-10 (IL10) cytokine targeted siRNA within polymeric, surface-functionalized microparticles can further enhance DC activation and T cell proliferation in vitro as well as switch the hepatitis-specific immune response towards a strong Th1 phenotype in vivo. Further, in a weakly immunogenic A20 B cell lymphoma mouse model, a combination of microparticles and chemokine releasing in situ crosslinkable hydrogel provided significant Th1 type cellular immune response and delayed the onset of tumor development. Thus, the in situ crosslinkable hydrogel co-delivering microparticles and DC attracting chemokines creates an immune priming center with broad applications in a variety of disease models.

Pseudomonas aeruginosa is a significant pathogen of immunocompromised individuals, and the main mechanism by which it mediates virulence is through the coordination of gene expression by an intricate quorum sensing system. One of the signalling molecules of this system, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to have immunomodulatory capabilities, discrete to its quorum sensing role. While the general focus of research in this area is on the physiological outcomes of this interaction on cell function, there is currently little concrete evidence identifying the receptor(s) for OdDHL in mammalian cells, and hence the biochemical mechanism behind the immunomodulation caused by OdDHL remains largely unknown. This study identifies peroxisome proliferator-activated receptor γ (PPARγ) as a mammalian target for OdDHL. PPARγ is a mammalian transcription factor involved in fatty acid metabolism and is heavily involved in the inflammatory response, being a negative regulator of inflammation. It is shown here that OdDHL is able to instigate signalling through PPARγ by activation of the ligand binding domain (LBD), suggesting that OdDHL may act as a PPARγ agonist. OdDHL is able to compete with the PPARγ agonist, rosiglitazone, causing a relative antagonism of PPARγ activity when given in tandem with the agonist. The bacterial signalling molecule is unable to displace the irreversible PPARγ antagonist GW9662. This effect on PPARγ is specific to OdDHL, as the smaller P. aeruginosa signalling molecule, N-butyryl-L-homoserine lactone had no significant effect on PPARγ activation. In order to confirm PPARγ as a putative receptor for OdDHL in mammalian cells, initial experiments were undertaken to optimise conditions to produce PPARγ LBD protein for binding interaction studies. The fidelity of the protein sequence was established and expression of the protein in an appropriate vector was confirmed. The protein produced was insoluble and hence not functional for binding studies, suggesting that additional optimisation of expression conditions, or manipulation and refolding of the protein is necessary before further experimentation can take place. The identification of PPARγ as a receptor for OdDHL in mammalian cells is an important step in understanding the nature and scope of the interaction between OdDHL and host cell physiology, especially the significance of this interaction during P. aeruginosa infection. Continuation of this research, in particular completion of protein-ligand binding studies between OdDHL and PPARγ has the potential to clarify the significance of the immunomodulation caused by OdDHL, while providing us with a platform from which we may exploit it.

In Pseudomonas aeruginosa, which causes severe secondary infections in immunocompromised patients, virulence factor expression is regulated by quorum sensing signal molecules known as acyl homoserine lactones (AHLs). One of the major AHLs produced by P. aeruginosa, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL), has also been shown to alter the function of a range of mammalian cells. The goals of experiments reported in this thesis were to use murine models to investigate the effects of in vivo exposure to OdDHL on TH responses, define the direct effects of OdDHL on TH cells and to explore the mechanism by which OdDHL alters the function of TH cells. It was found that in vivo exposure to OdDHL led to changes in cytokine and antibody subclass production indicative of a shift towards the underlying TH bias of the mouse strain studied. Such shifts may play a role in infections with P. aeruginosa, as strong TH1 or TH2 responses have been associated with worsening prognosis for the host, while more balanced responses have been associated with decreases in both infection and pathology. These results suggest that treatments targeting the immunomodulatory activities of OdDHL may be of benefit in the clinical setting in the future. Direct analysis of TH cells in defined in vitro systems revealed that exposure to OdDHL led to uniform decreases in cytokine production and proliferation. These decreases in cytokine production were found to be the result of OdDHL acting on both TH cells and the antigen presenting cells (APCs) that activate them, and only occurred when cells were exposed to OdDHL within 4 hours of stimulation. These findings suggest that OdDHL is acting on a molecular target common to several cells types, and that in TH cells and APCs, this target is involved in the early stages of TH cell activation. Experiments in which T cells were activated with mitogens that bypass the cell membrane revealed that OdDHL is not acting on the cell membrane or membrane-associated activation factors, suggesting that OdDHL is instead inhibiting TH cell function through interactions with one or more intracellular signalling molecules.

The vaccinia virus complement control protein (VCP) is involved in the modulation of the host inflammatory response during vaccinia virus infection. It possesses the ability to inhibit both classical and alternative pathways of complement activation, as well as bind to heparin or heparan sulfate proteoglycans, making it a unique multifunctional protein with therapeutic potential. VCP is able to block complement activity through its ability to bind C3b and C4b. Other very novel activities arise from VCP's ability to bind heparin and heparan sulfate proteoglycans, allowing the protein to attach itself to cell surfaces. The goal of this research was to fully characterize the structure and various functions of VCP. The structural basis for VCP's ability to bind heparin was investigated using heparin affinity chromatography, surface plasmon resonance analysis, and homology modeling with well-characterized heparin binding proteins. VCP was found to possess two regions involved in heparin binding, one involved in weak binding located at the junction of complement control protein (CCP) modules 1 and 2, and a second involved in strong binding located at the extreme C-terminal tip of the protein. Additional functions, involved in blocking molecular interactions with cells, were also identified and determined to be a result of VCP's ability to bind heparin. The structural basis for VCP's ability to bind complement was investigated using the hemolysis assay, surface plasmon resonance analysis, and homology modeling with well-mapped complement regulatory proteins. VCP was found to be the smallest functional unit able to bind and cause factor I cleavage of C3b/C4b, thus inhibiting complement activity. VCP was also shown to possess the ability to simultaneously bind and inhibit complement activation, using its entire exposed surface, while remaining attached to heparin by its C-terminal heparin binding site. The structure of VCP and its structural and functional stability were determined using X-ray crystallography, NMR, DSC, and the hemolysis assay. VCP was found to be an elongated filamentous protein, with defined CCP modular regions with limited intermodular interfacing, existing as a monomer in solution. VCP was found to possess the ability to structurally withstand temperatures in excess of 90oC. In addition, VCP was able to retain functional integrity following exposure to many adverse physical conditions, which would result in irreversible denaturation of most other proteins. Lastly, the in vivo pharmacokinetics of VCP was investigated in rats. VCP was injected as a bolus at different routes and using various concentrations. Continuous and intermittent intravenous injection strategies were also investigated. In all cases, the serum CH50 was monitored over time to determine the half-life of VCP activity in the serum. Traces of intact VCP was detected in urine by SDS-PAGE analysis, giving clues to its excretion and elimination from the body. This research has provided greater understanding of the architectural features of a protein that contribute to heparin and complement binding. In addition, this research may prove important for the future use of VCP as a novel immunomodulating agent, as well as future protein-engineering efforts to design a better treatment for many complement mediated diseases, such as: Alzheimer's disease, central nervous system (CNS) injury, systemic lupus erythematosus, and xenograft transplant rejections.