Atherosclerosis is a chronic inflammatory disorder of medium and large vessels. Immune signaling and dyslipidemia are two of several processes which influence lesion development in atherosclerosis. Cannabinoids, such as those found in marijuana, exert their effects through two cannabinoid receptors, CB1 and CB2. Recent studies using CB2 knockout mice and CB2-selective ligands have shed light on a protective role of CB2 in early stages of atherosclerosis. However, the role of CB2 in advanced stages of atherosclerosis remains unclear. To determine if CB2 plays a role in advanced atherosclerotic lesion composition and progression, we investigated the effects of systemic CB2 gene deletion on advanced atherogenesis in Ldlr-null mice fed an atherogenic high fat diet (HFD) for 20-24 weeks. CB2 deficiency did not significantly affect aortic root lesion area, however, CB2-/- mice had a significant increase (~1.9 fold) in the percentage of abdominal aorta surface occupied by lesion. CB2-/- mice also displayed increased lesional macrophage content (~2.3 fold) and an unstable phenotype characterized by significantly reduced smooth muscle cell/macrophage ratio and increased matrix metalloproteinase-9 activity and mineralization. These results suggest that although CB2 does not affect the size of atherosclerotic lesions, it does modulate the cellular and extracellular matrix composition and promotes a stable phenotype. CB2+/+ and CB2-/- mice were also subjected to treatments with either CB2-selective agonist, JWH-015, or antagonist, SR144528, over the last four weeks of a 24 week atherogenic diet to identify the effects of CB2 stimulation on calcification of advanced lesions. No change was observed in body weight or cholesterol in response to either treatment. SR144528 reduced triglycerides and mineralization of aortic root lesions in CB2+/+ mice only. Aortic Runx2 and osteopontin were increased in response to JWH-015 by a CB2-dependent mechanism. Administration of synthetic cannabinoids in an ex vivo organ culture of CB2+/+ aortas revealed increased vascular calcification in response to CB2 blockade and decreased vascular calcification in response to CB2 activation. All together, these results support a protective role for CB2 in late stages of atherosclerosis and suggests that drugs targeting CB2 may be beneficial in the treatment of advanced atherosclerosis by affecting osteogenic mechanisms implicated in the mineralization of lesions.

Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors of the immunoglobin family expressed by epithelium and macrophages. RAGE expression increases following ligand binding and when diverse cells are exposed to a variety of insults including cigarette smoke extract (CSE). The current research sought to characterize the pro-inflammatory contributions of RAGE expressed by alveolar macrophages (AMs) following CSE exposure. Acute exposure of mice to CSE via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer AMs in RAGE null mice compared to controls. Primary AMs were obtained from BAL, exposed to CSE in vitro, and RNA, DNA, and protein were analyzed. CSE significantly increased RAGE expression by wild type AMs. Employing ELISAs, wild type AMs exposed to CSE had increased levels of active Ras, a small GTPase that perpetuates pro-inflammatory signaling. Conversely, RAGE null AMs had less Ras activation compared to wild type AMs after exposure to CSE. In RAGE null AMs, assessment of p38 MAPK and NF-κB, important intracellular signaling intermediates induced during an inflammatory response, revealed CSE-induced inflammation occurs at least in part via RAGE signaling. For example, activated p38 was diminished in RAGE null AMs compared to controls and assessment of phosphorylated NF-κB in CSE exposed RAGE null AMs suggest lessened nuclear translocation of NF-κB compared to wild type AMs exposed to CSE. Importantly, quantitative RT-PCR revealed that mRNA expression of pro-inflammatory cytokines including TNF-α and IL-1β were detectably decreased and analysis of secreted proteins by ELISA displayed diminished IL-1β in RAGE null AMs exposed to CSE compared to CSE-exposed wild type AMs. These results reveal that primary AMs orchestrate CSE-induced inflammation, at least in part, via RAGE-mediated mechanisms.

Peripheral artery disease (PAD) is characterized by the development of atherosclerotic plaques on arterial walls, leading to the narrowing of blood vessels, resulting in ischemia in the downstream tissue. In the United States, 12% of the adult population is affected by PAD and its related symptoms. Current surgical revascularization techniques can be effective in part of the patient population, but there is a need for other options. Alternatively, collateral blood vessels, or natural bypass arteries, enlarge to increase blood flow to the ischemic tissue in a process called arteriogenesis, which has been studied as a therapeutic option. Cell-based therapies, such as BM-MNCs, have been investigated as means to enhance arteriogenesis, but have largely failed in clinical trials. An alternative cell-based therapy candidate are myoblasts, or muscle progenitor cells. Myoblasts increase arteriogenesis in murine models and are known to interact with macrophages, which are immune cells that are primary regulators of arteriogenesis. Macrophages can polarize to inflammatory (M1) and regenerative (M2) phenotypes, with the M2 phenotype promoting enhanced arteriogenesis. This interaction suggests that myoblasts may be signaling macrophage polarization to enhance arteriogenesis. The purpose of this study was to determine if myoblasts in vitro can affect macrophage polarization into inflammatory (M1) or regenerative (M2) phenotypes. Protocols for macrophage culture and polarization were implemented, and then macrophages were co-cultured with myoblasts for 24 hours to assess the effects in vitro. Concentrations of known inflammatory (TNF-a) and regenerative (IL-10) cytokines released by macrophages were measured after co-culture with myoblasts. Surprisingly, macrophages co-cultured with myoblasts showed a decrease in both TNF-a and IL-10 compared to macrophages cultured alone. Morphology changes of macrophages were also measured after co-culture, with, surprisingly, little difference in the groups co-cultured with myoblasts. Pilot experiments suggest there may be an initial lag time greater than 24 hours for myoblasts to affect macrophage phenotype. Future work ideally will include longer time points and optimizing viability and proliferation of myoblasts in co-culture settings.

Functions and phenotypes change in macrophages (Mφ) during tumor growth. Although analyzing functional and phenotypic changes are important in understanding the mechanism of tumor-induced immunosuppression, it is necessary to look beneath the surface and expose the mechanisms behind these changes. Flow cytometrically isolated Mac-1⁺, -2⁺, or -3⁺ Mφ showed that although both normal host and tumor-bearing host (TBH) Mac-2⁺ Mφ were the primary source of prostaglandin E2, no specific TBH suppressor Mφ could be identified. To determine if normal host and TBH Mφ respond to in vitro activating agents differently, normal host and TBH Mø, were treated with lipopolysaccharide. Functional, phenotypic, and molecular changes were observed in the lipopolysaccharide-treated M4J. Three- and 24-h lipopolysaccharide treatment reduced TBH Mφ-mediated suppression, while only 24-h lipopolysaccharide treatment reduced it in normal host Mφ. Prolonged adherence, which induces Mφ differentiation, increases the number of Mac-2⁺ TBH Mφ. Tumor growth causes an increase of Mφ in the S and G₂/M phases of the cell cycle. Lipopolysaccharide and adherence increase the number of normal host Mφ in Sand G₂/M; however, these same treatments reduce the number of TBH Mφ in these same phases. Earlier work showed an increase in the number of TBH Mφ that did not express class II major histocompatibility complex molecules and that there was an increase in the suppression mediated by these Mφ. TBH Mφ have a decreased response to interferon-γ-induced class II mRNA expression and a decrease in its mRNA stability. TBH Mφ have an increase in lipopolysaccharide- and prostaglandin E₂-mediated suppression of class II mRNA. Tumor necrosis factor-α can induce class II mRNA expression in TBH Mφ, but suppresses it in normal host Mφ. The effects of tumor necrosis factor-α on class II mRNA is due, in part, to the maturation stage of the Md. To examine further mechanisms that regulate Mφ maturation, intracellular Mac-2 expression was examined. The expression of nuclear Mac-2 increases during tumor growth and after 24-h adherence. This increase in Mac-2 protein parallels the increase in Mac-2 mRNA expression. Because there is a change in TBH Mφ cell-cycle kinetics and maturation, proto-oncogene expression was examined in normal host and TBH Mφ. The proto-oncogenes c-myb, c-myc, c-fos, and c-fms are constitutively expressed in TBH Mφ, while normal host M@ express c-fos and c-fms but at lower levels. Adherence suppresses the expression of the proto-oncogenes c-myb, c-myc, and c-fms in TBH Mφ, while inducing c-fos and c-fms in the normal host. Lipopolysaccharide induces c-myc, c-fos and c-fms in both normal and TBH Mφ, but suppresses c-myb expression in TBH Mφ. The results suggest that tumor growth causes a shift in Mφ maturity, and that this shift is responsible for alterations in Mφ function and phenotype. It is possible, however, to activate Mφ by lipopolysaccharide in the absence of triggering differentiation, and to trigger differentiation by adhesion without activating Mφ. TBH Mφ are more suppressive than normal host Mφ, but TBH Mφ can respond to activating signals. / Ph. D.

Tumors evade immune system tumor-controlling functions. T cells critical to antitumor immunity are tolerogenic in tumor-burdened animals, and fail to lyse neoplastic cells. Our goal was to investigate the kinetics of immune dysfunction related to tumor-burdened host (TBH) memory T cell responses (or the lack thereof). We demonstrate tumor growth impairs T cell activation by modulating CD4+ T cell infiltration and systemic CD44 and CD62L activation marker expression, and by downregulating TBH TH1 cytokine production by splenic CD4+ T cells. Since chemotherapeutic treatments have potent cytostatic effects, we posited they enhance T cell dysfunctionality; which leads to limited therapeutic efficacy. Paclitaxel is a potent chemotherapeutic agent currently being administered in Stage III clinical trials; however, it reduces T cell proliferative capacity and interferon-γ (IFN-γ) production. In contrast, our data suggest that administration of low dose paclitaxel prolongs adaptive immunity in a limited capacity. We show paclitaxel enhances CD4highCD62Llow cell populations that drive TH1 cytokine production and prolongs the production of interleukin-2 (IL-2) in TBHs. We hypothesize that the initiation and maintenance of activated TH1 cell populations in patients during therapy serves as a reliable prognostic indicator of a favorable therapeutic response. Paclitaxel's limited therapeutic effects are due, in part, to its suppression of T cell activities; but the administration of low dose chemotherapy in combination with immunotherapeutic agents temporally takes advantage of paclitaxel's immunostimulatory capabilities. Our work will enhance current understanding of immune dysregulation during cancer development, and promote advances in the monitoring and development of combinatorial cancer treatments. / Master of Science

L'endométriose est une maladie gynécologique, touchant les femmes en âge de procréer. Cette pathologie est caractérisée par la présence de tissu endométrial ectopique, c'est-à-dire en dehors de la cavité utérine. Des dysfonctions du système immunitaire sont de plus en plus souvent suspectées comme étant un des éléments responsables de la pathogenèse de cette maladie. L’objectif général de ce projet a donc été d’étudier les mécanismes cellulaires de molécules pro-inflammatoires aux propriétés variées et à l'expression anormalement élevée dans cette pathologie, que sont MIF et les prostaglandines PGE2 et PGF2α, dans les anomalies inflammatoires et invasives en cause dans cette pathologie. La première partie de nos travaux a porté sur l’étude d’un modèle murin de l’endométriose déficient du gène MIF. Le nombre et le volume des lésions collectées à partir des souris déficientes pour le gène MIF sont significativement inférieurs à ceux mesurés dans des souris sauvages utilisées comme contrôle. L’analyse par PCR des cellules isolées des lésions de souris déficientes du gène MIF a révélé une expression réprimée des protéines d’adhésion, d’inflammation et d’angiogenèse. Ces données démontrent pour la première fois que le MIF agit directement sur la croissance et la progression de lésions d’endométriose in vivo. Une partie de nos travaux a porté sur les molécules nécessaires au métabolisme de PGE2 et PGF2α dans l'endomètre eutopique des femmes normales et l'endomètre eutopique et ectopique des femmes atteintes d'endométriose. Selon nos données, l'expression de certains de ces facteurs est perturbée durant cette maladie, ce qui peut avoir des effets délétères sur la physiologie de la procréation. La stimulation des cellules ectopiques par PGF2α entraîne une libération accrue de VEGF et CXCL-8, ceci via l'induction de COX-2 et des deux variants d’épissage du récepteur FP. De plus, la PKC joue un rôle dans ce phénomène, dépendamment et indépendamment de la PLC. Par son effet inducteur sur la libération de VEGF et CXCL-8, PGF2α pourrait favoriser l'aspect inflammatoire et le développement ectopique des lésions d'endométriose, notamment par des phénomènes d’angiogenèse et de prolifération cellulaire accrus. L'effet de PGF2α sur la libération de VEGF et CXCL-8 par les cellules endométriales ectopiques pourrait également expliquer les quantités élevées de ces cytokines dans le liquide péritonéal des femmes atteintes d'endométriose, un phénomène suspecté dans l'infertilité et les douleurs associées à cette maladie. Nos derniers résultats obtenus à partir du liquide péritonéal montrent un profil cytokinique en faveur de l’angiogenèse et la prolifération des lésions d’endométriose, avec une forte augmentation des facteurs suivants : EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC et Rantes, confortant nos observations préalables redéfinissant la maladie comme étant d’origine angio-inflammatoire. L'endométriose et ses symptômes sont des phénomènes complexes ayant probablement plus qu'une seule origine. Parmi les nombreux facteurs à l'expression altérée dans l'endométriose, notre étude montre que MIF, PGE2 et PGF2α, ainsi qu’une pléthore de facteurs pro-angiogéniques pourraient être de ceux jouant un rôle dans l'infertilité et les douleurs reliées à cette maladie. / Endometriosis is a menstrual disorders, is mainly diagnosed in the peritoneal cavity by the presence of lesions, which are thought to originate from the endometrium. Little is known about the causes of endometriosis and no targeted treatment is available. Our studies were the first to show functional defects involving the immune system in the endometrium, i.e. before this tissue migrates and grows into abnormal locations. Our current main hypothesis is that endometriosis development requires a combination of immune dysfunction involving factors, such as MIF, PGE2 and PGF2α. Using a mouse model where MIF-knock out (KO) mice received intra-peritoneal injection of endometrial tissue from MIF-KO or syngenic wild type (WT) mice and vice versa, we first revealed that MIF genetic depletion resulted in a marked reduction ectopic endometrial tissue growth, a disrupted tissue structure and a significant downregulation of the expression of major inflammatory, cell adhesion, survival and angiogenic factors relevant to endometriosis pathogenesis, whereas MIF add-back to MIF-KO mice significantly restored endometriosis-like lesions number and size. This study provides compelling evidence for the role of MIF in endometriosis development and its possible interest for a targeted treatment of endometriosis. We then revealed for the first time multiple defects in PG biosynthesis and receptivity pathways, which differ between eutopic intrauterine and ectopic endometrial tissues and may, owing to the wide spectrum of PG properties, contribute to the initial steps of endometrial tissue growth and development and have an important role to play in the pathogenesis and symptoms of this disease. Afterward, we focussed on PGF2α which markedly up-regulated PGE2, CXCL-8 and VEGF secretion in endometriotic cells, through COX-2 activation. Such an effect was abolished by AL8810, a specific FP antagonist, and significantly down-regulated after specific inhibition of FP different variants signalling pathways. PGF2α enhanced angiogenesis through endothelial tubal formation and proliferation processes. These results show for the first time that PGF2α exerts an indirect angiogenic effects by interacting with ectopic stromal cells and induces the secretion of major angiogenic factors via FP signalling pathways. This study provides evidence for a new mechanism underlying endometriosis development and pathophysiology. As our last data showed distinct patterns of peritoneal fluid cytokine concentrations in endometriotic women most notably a marked increase in EGF, FGF-2, IL-1α, MIP-1β, TGFα, PDGF-AA, PDGF-BB, MCP-3, sCD40L, Gro Pan, IL-17α, MDC and Rantes. These changes may exacerbate the local peritoneal angiogenic and proliferative reaction observed in women with endometriosis, and contributes to its pathophysiology. Inflammation is a major hallmark of endometriosis and angiogenesis is crucial to ectopic endometrial tissue growth. Once viewed as archetypical mediators of inflammation and pain, prostaglandins and angiogenic cytokines should be now regarded major promoters of endometriotic lesions growth.

Indiana University-Purdue University Indianapolis (IUPUI) / Among the cytokines regulating immunity, interleukin 9 (IL-9) has gained considerable attention for its role in inflammation, immune tolerance, and tumor immunity. IL-9 has a broad array of functions and acts on multiple cell types to regulate immune responses. IL-9 receptor is expressed on both non-hematopoietic cells and hematopoietic cells in the innate and adaptive immune system. IL-9 demonstrates a remarkable degree of tissue-specific functionality that varies by tissue site and the context of the inflammatory milieu. In this dissertation, we investigate the biological activities of IL-9 and identify distinct IL-9-responsive cell type in the immune pathogenesis of disease models including allergic airway disease, lung cancer, and urinary tract infection. When examining airway hyperreactivity, we found IL-9-dependent mast cell function was critical. Using adoptive transfer models and newly generated mice with an inactivation of the Il9 gene restricted to T cells generated by CD4-cre/LoxP-mediated targeting, we demonstrate that T cell secreted IL-9 promotes mast cell progenitor proliferation and CCR2-dependent mast cell migration during allergic airway inflammation. In IL-9-mediated pro-tumor responses, interstitial macrophages, but not mast cells, respond to T cell IL-9 to enhance B16 metastatic tumor growth. In the context of urinary tract infection, IL-9 contributes to protection against E. coli bladder infection potentially by enhancing CCL20 production in epithelial cells to recruit macrophages and neutrophils. Altogether, IL-9 can exert cell type-specific effects that identify its roles in immunity and disease. This perspective will be important in defining the diseases where targeting IL-9 as a therapeutic strategy would be beneficial, and where it has the potential to complicate clinical outcomes.

T-cell lymphomas (TCLs) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas derived from post-thymic mature T- and NK-cells broadly classified peripheral (PTCL) or cutaneous (CTCL), indicating either a nodal or skin-homing disease, respectively. One of the main clinical challenges contributing to dismal outcomes for TCL patients is both the lack of curative treatment strategies and the high rate of relapse for currently approved therapies, underscoring the need for identification of novel targeted therapies for the treatment of TCL. While combination therapeutic strategies have been proposed and show great promise in pre-clinical and clinical trials for PTCL and CTCL, none are yet approved. Additional contributing factors toward the difficulty in studying TCL and the high rate of therapeutic failure is the highly heterogeneous genetic and molecular mechanisms driving TCL as well as the poorly understood role of non-tumor microenvironment cells in the pathogenesis of TCL. Indeed, while several studies have suggested that tumor associated macrophages play both a significant functional role in supporting tumor maintenance and are therapeutically targetable, less is known about potential tumor supporting roles of other cell microenvironment populations. Here, I used an unbiased and high-throughput approach to discover novel drug combinations in CTCL and to characterize at the single-cell level relevant molecular mechanisms driving T-cell lymphomagenesis. First, I demonstrate that the combination of romidepsin, a selective class I HDAC inhibitor, with afatinib, an inhibitor of the epidermal growth factor receptor (EGFR) family, produces strongly synergistic antitumor effects in CTCL models, both in vitro and in vivo, using mechanisms of action that involve down-regulation of the JAK-STAT signaling pathway. This result suggests a potential therapeutic role for the combination of HDAC inhibitors with afatinib in the treatment of CTCL that had not been previously recognized. Second, we developed single-nuclei analysis on a cohort of 30 TCL (PTCL-NOS, AITL, epstein barr virus positive PTCL) and 6 normal patient samples to identify and deconvolute genomic and functional mechanisms contributing to T-cell lymphomagenesis. Here, I implemented a comprehensive bioinformatics pipeline for the analysis of sparse single-nuclei transcriptomic data and characterized heterogeneous molecular mechanisms driving T-cell lymphomagenesis, such as the upregulation of the PI3K-AKT-mTOR and WNT signaling pathways in a subset of 5/9 PTCL-NOS and 7/19 AITL samples, respectively. Additionally, I identified the enrichment of both the macrophage compartment in PTCL-NOS and AITL, and the specific enrichment of CD8+ T cells in AITL. These results suggest a correlation between patient-specific characteristics, such as mutational status, and possibly therapeutically targetable molecular mechanisms driving neoplastic cell growth that warrants further investigation.

Psoriasis is an immune mediated inflammatory disease which affects 2-3% of the world’s population. Over the last decade, psoriasis has been acknowledged as an independent risk factor for atherosclerosis. The precise mechanism or mechanisms of the heightened risk is widely speculated. Endothelial cells and macrophages are central players in the immunopathological development of both diseases. Interleukin-36 cytokines (IL-36) have been heavily implicated in psoriasis immunopathology. Significant upregulation of epidermal IL-36 is a recognised characteristic of psoriatic skin inflammation. IL-36 induces inflammatory responses in dendritic cells, fibroblasts and epithelial cells. While vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in dermal inflammation, the effects of IL-36 on endothelial cells have not been defined. We report that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated following IL-36γ stimulation, and this is reversed in the presence of the endogenous IL-36 receptor antagonist. IL-36γ-stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T-cells following IL-36γ stimulation of endothelial cells. Both resident and infiltrating inflammatory myeloid cells contribute to the immunopathology of psoriasis by promoting the IL-23/IL-17 axis. We show that IL-36γ induces the production of psoriasis-associated cytokines from macrophages (IL-23, TNFα) and that this response is enhanced in macrophages from psoriasis patients. This effect is specific for IL-36γ and could not be mimicked by other IL-1 family cytokines such as IL-1α. Furthermore, IL-36γ stimulated macrophages potently activated endothelial cells as illustrated by ICAM-1(CD54) upregulation, and led to increased adherence of monocytes, effects that were markedly more pronounced for psoriatic macrophages. Interestingly, regardless of stimulus, monocytes isolated from psoriasis patients showed increased adherence to both the stimulated and unstimulated endothelium when compared to monocytes from healthy individuals. Collectively, these findings add to the growing evidence for IL-36γ having roles in psoriatic responses, by enhancing endothelium directed leukocyte infiltration into the skin and strengthening the IL-23/IL-17 pathway. Our findings also point to a cellular response which could potentially support cardiovascular comorbidities in psoriasis.

No description available.