Chronic Liver Disease (CLD) and Acute Liver Failure (ALF) are serious medical syndromes. Current therapeutic options consist of managing complications, and liver transplant. Even if liver transplantation is thought to be suitable for CLD or ALF patients, there are not enough organs available and thus increasingly more deaths occur on the transplant waiting list. Therefore, there is a pressing need to develop additional therapies. This thesis firstly systematically reviews trials in autologous cell therapies as possible treatments for patients with cirrhosis. The published literature is imperfect and the difference in trial design means it has not been possible to conduct a meta-analysis. Regardless of these shortcomings, cell therapy is a potentially positive prospect. In ALF and CLD monocyte-macrophages have diverse roles within the liver. Monocyte and immune cell changes in ALF are investigated and it is demonstrated for the first time that patients with paracetamol induced ALF have a significantly altered blood compartment and that these changes correlate with patient outcome. It is possible that these results may help stratify which patients may spontaneously survive and which patients may require an emergency liver transplant. Furthermore, modulation of these changes may improve outcomes for patients. The thesis also examines monocyte-macrophages in cirrhotic patients and demonstrates the feasibility of differentiating cirrhotic patients’ monocytes into functional macrophages, comparable to healthy volunteers in a Good Manufacturing Practice (GMP) environment. A first in-man trial using macrophages infused to patients with cirrhosis as a potential new treatment is also detailed. Finally, this thesis outlines developmental work for cell therapy in patients with cirrhosis in the multi-centre REALISTIC trial. Patients were randomly assigned to receive; standard medical care, Granulocyte Stimulating Factor (GCSF) injections alone or GCSF combined with repeated stem cell infusion.

Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

Vascular inflammation and associated leukocyte influx is a hallmark in the pathogenesis of atherosclerosis. In both animal models and human subjects, inhibiting monocyte recruitment is beneficial in preventing atherosclerosis and its clinical manifestations. The trafficking signals that recruit cells to areas of inflammation are provided by small secreted proteins called chemokines. Chemokines play a major role in the pathogenesis of inflammation, and redundancy among the chemokine signaling pathways means that blocking one pathway could result in another assuming its function. Therefore, we aim to block a cell’s response to a range of chemokine-induced directional migration signals. Slit2 treatment inhibits monocyte migration in vitro using transwell migration assays, and in vivo, using a murine peritonitis model of inflammatory cell influx. This inhibition is shown to be dose- and time- dependent. Furthermore, Slit2 inhibits monocyte adhesion to activated endothelial cell monolayers. These data may suggest a therapeutic role for Slit2 in atherosclerosis.

Adjuvants are compounds that enhance immune responses to antigens present in a vaccine. They are particularly important in subunit vaccines; without adjuvants, these vaccines are often poorly immunogenic. A novel adjuvant platform developed at VIDO-InterVac is comprised of CpG-ODN or poly I:C, innate defense regulator peptides, and a new class of adjuvant called polyphosphazene. The polyphosphazenes have demonstrated a great potential as a safe and effective adjuvant. In particular, the polysphosphazenes poly[di(carboxylatophenoxy)-phosphazene] PCPP and poly[di(sodium carboxylatoethylpehnoxy)-phosphazene](PCEP) have been used in numerous animal studies where they not only have been shown to enhance the quality and quantity of the adaptive immune response, but also were shown to induce parenteral and mucosal immune responses with many different antigens, demonstrating their versatility. However the mechanisms by which the polyphosphazenes stimulate the innate immune response are only partially understood. Antigen presenting cells (APCs) are capable of facilitating the uptake of antigen and directing the immune response. Based upon the proposed mechanism of action of another adjuvant, we chose to investigate whether porcine monocytes could be induced to secrete pro-inflammatory cytokines IL-1 and IL-18 in response to stimulation with polyphosphazenes PCEP and PCPP. The release of these cytokines is thought to be mediated by the Nod-Like Receptors (NLRs), which are cytosolic pattern recognition receptors expressed in APCs. It is suggested that these receptors act in conjunction with TLR transcription pathways to control caspase-1 and release associated pro-inflammatory cytokines IL-1and IL-18 (Kawai and Akira, 2011). We first investigated the relative gene expression of three Nod-like receptor genes: nod1, nod2 and nlrp3 in various populations of porcine peripheral blood mononuclear cells (PBMCs) and found that monocytes, dendritic cells and B cells express increased relative levels of these receptors as compared to T cells. Subsequently, we evaluated the relative NLR expression in several porcine mucosal and lymphoid tissues and observed genes to be most significantly expressed in nasal mucosa, bronchial mucosa, and lung while limited in tissues associated with Peyer’s patches, jejunal wall. Both the mesenteric lymph node and bronchial lymph node exhibited similar patterns and levels of expression of nod1, nod2 and nlrp3. To characterize the activation of NOD1, NOD2 and NLRP3 receptors in response to stimulation with polyphosphazenes, porcine monocytes were stimulated with PCEP or PCPP in both the presence and absence of a second signal (poly I:C and CpG-ODN, TLR-7 and -9 agonists respectively). We found that PCEP and PCPP alone did not significantly upregulate nod1, nod2 and nlrp3, nor genes for cell activation markers such as CD80 and CD86. However monocytes cultured with the combination of CpG-ODN, Poly I:C and PCPP appeared to moderately express IL-18, CD80 and CD86. The secretion of pro-inflammatory cytokines from cultured monocytes was determined with Enzyme Linked Immunosorbent Assays (ELISA). It was found that IL-1was secreted in significantly higher quantities in the supernatant of cells stimulated with both polyphosphazene and TLR ligands, as opposed to those cultured with polyphosphazene alone. Assays for IL-18, IL-6, IL-10 and IL-12 did not detect a significant presence of these proteins in the supernatant. Furthermore, we found that a soluble caspase inhibitor did not significantly reduce the production of IL-1by monocytes, and was likely attributable to cell death at high concentrations. Taken together, these results suggest that porcine monocytes, B cells and dendritic cells express elevated levels of the NLRs as compared to T cells. Additionally, areas of the respiratory tract appear to express increased levels of these receptors relative to mucosal and lymphoid tissues of the gastrointestinal tract. Neither PCPP or PCEP alone were capable of inducing significant production of IL-1 or IL-18 by cultured monocytes, however stimulation of these cells with a combination of CpG-ODN, poly I:C and polyphosphazene resulted in the secretion of IL-1.

Thesis advisor: Kenneth C. Williams / HIV infection elicits dysregulation of the immune system and is associated with a number of comorbidities. A common link among HIV-associated comorbidities is monocyte and macrophage activation, accumulation, and high turnover. Anti-retroviral therapy (ART), while useful in preventing HIV infection from progressing to immune dysfunction characterized by AIDS, does not eliminate infection. Even with ART, individuals retain a low level of virus that is able to reseed infection and a higher rate of comorbidities than uninfected people. Prior research has revealed an inflammatory monocyte/macrophage cell population that is uniquely in tissues with infection in an HIV model that uses simian immunodeficiency viral (SIV) infection of rhesus macaques. This cell type is characterized by expression of the inflammatory marker calprotectin. Through measurements of soluble calprotectin present in the plasma of SIV-infected rhesus macaques, I found that calprotectin levels remained low within the first two weeks of infection, sharply increased around three weeks post-infection, typically increased to a maximum during late stage chronic disease, and positively correlated with plasma viral load. Initial calprotectin levels suggests a trend that high pre-infection levels are associated with not progressing to AIDS or SIV encephalitis. Through immunostaining monocytes and flow cytometry, I found that calprotectin expression on classical, intermediate, and non-classical monocytes initially decreases with SIV infection, rebounds for most of infection, and sharply decreases again with late-stage chronic disease. Flow cytometry further showed that the calprotectin-expressing monocyte expresses CD163, CD169, and CCR2, but lacks expression for CX3CR1 and CCR5. Analysis of RNAseq data illustrates trends that suggest an increase in gene expression of genes involved in antiviral/antibacterial and chemotactic functions during conditions when calprotectin gene expression is also increasing. In summary, the data presented in this thesis suggest that the calprotectin-expressing monocyte/macrophage may come from an intermediate monocyte and play a role in inflammation through calprotectin secretion, activation, and increased chemotactic function. / Thesis (MS) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Inflammatory diseases often involve complex and dynamic responses of monocytes, crucial cells of the innate immune system. Understanding these responses, particularly to lipopolysaccharide (LPS), a key inflammatory stimulus, is vital yet remains challenging due to their heterogeneity and plasticity. Upon analyzing available single-cell RNA sequencing data sets, we defined key patterns of monocyte inflammatory responses challenged with varying LPS dosages. We found that high-dose LPS induced the generation of exhausted monocytes with elevated expression of genes associated with pathogenic inflammation and immune suppression.. In contrast, super-low-dose LPS led to a state of low-grade inflammation, characterized by enhanced chemotaxis; immune-enhancement; and adhesion.. Pseudo-time analysis revealed a potential bifurcation of monocytes, starting from a proliferative, less-differentiated and premature state into either the exhausted state (under prolonged high dose LPS challenge) or the low-grade inflammatory state (under the prolonged super-low dose LPS treatment). Complementing our analyses with in vitro cultured murine monocytes, we observed similar exhaustion of monocytes collected from septic murine hearts published in an independent study. Furthermore, we analyzed publicly available scRNAseq datasets regarding monocytes from septic and severe COVID human patients and revealed a similar exhaustion phenotype as we documented in murine exhausted monocytes. In contrast, our analyses of newly published scRNAseq data regarding monocytes from chronic autoimmune patients reveal key distinct low-grade inflammation features. With translational potential, we analyzed the scRNAseq datasets of monocytes trained with 4-PBA, a potent anti-inflammatory compound, and observed that 4-PBA can effectively arrest monocytes in an anti-inflammatory state. Together, our comparative analyses reveal a systems landscape of monocyte memory dynamics with distinct dosage and history of LPS challenges, and offer novel insights for potential therapeutic strategies for modulating both acute sepsis and chronic inflammatory diseases. Our studies also provide a foundation for guiding future mechanistic and translational studies regarding monocyte dynamics and their involvements in health and disease pathogenesis. / Doctor of Philosophy / Inflammation is the body's natural response to injury or infection. A key player in this process is a type of immune cell called monocyte. Monocytes are our body's first line of defense, rushing to the site of injury or infection. However, the way these cells respond can vary greatly, depending on the dosage and duration of external challenges. In our research, we analyzed data collected through an advanced technique called single-cell RNA sequencing, in order to take a detailed look at how an individual monocyte responds to different amounts of LPS, a key substance found in most bacteria. We found that when exposed to a prolonged challenge of higher dose LPS, monocytes become exhausted with pathogenic inflammation and immune suppression, as seen in sepsis. However, when the LPS dose is low, these cells enter a state of low-grade inflammation, responsible for chronic inflammation as seen in autoimmune diseases, atherosclerosis and other chronic diseases. We found that this paradigm of exhaustion and low-grade inflammation can be seen in data analyzed from either patients with severe infections such as sepsis or severe COVID-19, or patients with long-term autoimmune diseases. In simpler terms, our study provides a detailed road map regarding how our body's first responders, namely the monocytes, react under different levels of threat, and how we might be able to guide their responses toward a beneficial direction. Understanding these processes more clearly may lead to new ways to treat a range of infectious or inflammatory diseases.

Most studies of the induction of cytokine synthesis in monocytes have employed an exogenous triggering agent such as lipopolysaccharide. However, in nonseptic inflammatory responses (e.g. rheumatoid arthritis) monocyte activation occurs as a result of T cell‐generated signals. In previous reports, we and others have demonstrated that contact‐dependent T cell‐generated signals are capable of contributing to macrophage activation. We have shown that plasma membranes from anti‐CD3 activated purified peripheral CD4+ T cells (TmA) but not from resting CD4+ cells (TmR) induce monocytes to synthesize interleukin (IL)‐1 in the absence of co‐stimulatory cytokines. Studies to determine the expression kinetics of the molecule(s) unique to activated CD4+ T cells which interact with monocytes to induce IL‐1 revealed that optimal expression occurred at 6 h post activation. This matched the previously reported kinetics of expression of CD40 ligand (CD40L) on activated peripheral T cells, implicating the CD40‐CD40L interaction as a candidate for the initiator of the IL‐1 signaling event. The ability of TmA to induce IL‐1 synthesis in resting monocytes could be markedly reduced by addition of a monoclonal anti‐CD40L antibody, 5c8. In addition, a monoclonal anti‐CD40 IgM (BL‐C4) proved dramatic in its ability to induce resting monocytes to synthesize IL‐1. In summary, these results demonstrate that the CD40‐CD40L interaction provides a critical component of CD4+ T cell contact‐dependent activation of monocyte IL‐1 synthesis.

La MAP kinase p38 régule la transduction du signal en réponse à un stress environnemental. Les inhibiteurs spécifiques de p38, qui sont connus pour bloquer la production de cytokines pro-inflammatoires, mais peuvent également intervenir sur le phénomène d'apoptose. C'est dans cette dernière optique que de nouvelles structures d'inhibiteurs, ont été synthétisées. Ces structures, après caractérisation par RMN, ont été ensuite évaluées en termes de mécanisme d'action et de disponibilité biologique. Outre les méthodes classiques cette évaluation a finalement fait appel aux techniques HR-MAS. Les sept molécules ainsi synthétisées ont été regroupées en trois famille (1a, 2a-c, 3a-c). Quoique les propriétés physico-chimique de ces motifs soient très différentes, il en ressort une potentialité d'action commune en tant qu'inhibiteur de la MAP kinase p38. / P38 MAP Kinase regulates the signal's transduction in response to an environmental stress. The specific p38 inhibitors, which are known to bloc the pro inflammatory (cytokines) production, can intervene on the (apoptosis) phenomenon too. This is in this last perspective that news inhibitors' structures have been synthesized. These structures, after RMN characterization, have been estimated in term of the mechanics of an action and biological ability. As well as the classical methods, this evaluation finally requires HR-MAS technics. The 7 molecules synthesized in this manner have been in 3 families regrouped (1a, 2a-c, 3a-c). Although the physic- chemical properties of these motives are very different, it stands out a common potentiality of action as an MAP Kinase p38 inhibitor

L’athérosclérose est une pathologie chronique inflammatoire qui résulte du dérèglement d’une réaction inflammatoire non résolue ayant pour but initial d’éliminer l’accumulation excessive de lipides au niveau de l’intima. Cette élimination est exercée par les monocytes/macrophages, dont l’infiltration et l’accumulation au niveau des lésions contribue à l’inflammation chronique locale.SR-BI est un récepteur scavenger multi-fonction capable de reconnaître un large spectre de ligands allant des lipoprotéines natives et modifiées jusqu’aux endotoxines. Outre de jouer un rôle crucial dans l’homéostasie du cholestérol dans le foie, est considéré comme un PRR capable d’être impliqué dans l’immunité inné. Un nombre croissant de données suggère un rôle athéro-protecteur de SR-BI dans les cellules dérivées de la moelle osseuse et notamment dans les macrophages. La contribution de SR-BI dans les macrophages au cours de l’athérosclérose et l’identification des mécanismes sous-jacents ne sont pas élucidées. Nous avons démontré que la délétion de SR-BI dans les macrophages entraîne une accélération du développement de l’athérosclérose et une augmentation de la cellularité au sein des lésions en absence d’effet sur la cholestérolémie. Ces effets athéro-protecteurs peuvent être attribués à une diminution de l’apoptose et à une augmentation de la prolifération cellulaire au sein des plaques.Nos données suggèrent également que la diminution de la susceptibilité à l’apoptose des macrophages déficients en SR-BI pourrait impliquer la voie d’activation P38. En parallèle de cette étude, le rôle de SR-BI dans la réponse inflammatoire a été exploré dans des conditions d’endotoxémie. / Atherosclerosis is a chronic inflammatory pathology which results from an uncontrolled inflammatory reaction secondary to an abnormal accumulation of lipids in the intima. The lipid clearance is performed by monocytes/macrophages. Their infiltration and accumulation in lesions contribute/ enhance the chronic local inflammation. SR-BI is a multifunction scavenger receptor capable of recognizing and binding a large spectrum of ligands from native and modified lipoproteins to endotoxins. Besides its crucial role in cholesterol homeostasis in the liver, SR-BI is also described as a PRR. An increasing number of data suggests that SR-BI exerts an atheroprotective role in bone marrow-derived cells and in particular macrophages.The specific contribution of SR-BI in macrophages in atherosclerosis development and the identification of the underlying mechanisms have yet to be elucidated.We have demonstrated that SR-BI deletion in macrophages increases atherosclerosis development and lesion cellularity without affecting cholesterolemia. These atheroprotective effects could be explained by decreased apoptosis and increased cell proliferation in plaques.Our data also suggest that the decrease in apoptosis sensitivity in SR-BI deficient macrophages could involve the P38 MAPK and STAT1 signaling pathways.In parallel to this study, the role of SR-BI in the inflammatory response has also been explored in endotoxemia and sepsis.

The renin-angiotensin system (RAS) has been implicated in vascular inflammation and atherosclerosis. Angiotensin II via the ATR1 can activate monocytes to produce inflammatory factors and increase adhesion. ATR1 expression is partly regulated by alternate splicing of the ATR1 gene. The RAS may also regulate immune function as part of the stress response: a model is proposed. ATR1 expression in two monocyte cell lines (U937 and THP-1) compared to a human microvascular endothelial cell line (HMEC1) was investigated. Western blot showed ATR1 protein expression in all cell types. PCR protocols targeted to the terminal protein-coding exon common to all transcript variants confirmed mRNA expression of the ATR1 gene in EC and monocytes. The 5 known splice variants were not identified in monocytes. 5-RLM RACE was used to identify the 5 untranslated ATR1 exons in monocytes. These data suggest a novel monocyte-specific splice variant, which may function in the cardiovascular disease process.