Les cellules tumorales requièrent un approvisionnement continu de nutriments pour produire de l’énergie. Cependant le micro-environnement autour de la tumeur en fournit une quantité insuffisante. Nous avons donc émis l'hypothèse qu'il existe une symbiose nutritionnelle entre les cellules stromales et les cellules tumorales, qui contribue à la tumorigenèse et, nous nous sommes concentrés sur les macrophages. Typiquement, ils sont classés comme classiques (M1-like) ou alternatifs (M2-like) et dans le stroma, les macrophages ont tendance à avoir un phénotype M2-like. Concernant leur profil métabolique, les macrophages M1-like ont un métabolisme glycolytique. Toutefois, les caractéristiques métaboliques des macrophages M2-like ne sont toujours pas clairement définies. Dans nos travaux, en utilisant la lignée cellulaire murine de macrophage, les RAW 264.7, nous avons confirmé que les macrophages M1-like sont exclusivement glycolytiques alors que les M2-like ont plutôt un profil oxydatif. Nous avons démontré qu’il existe une certaine plasticité métabolique des cellules M2-likes car elles sont capables de basculer vers la glycolyse quand la respiration mitochondriale est inhibée. De plus, un blocage de la glycolyse n'a révélé aucune adaptation métabolique des macrophages M1-like mais influe sur les cellules M2, en réduisant leur capacité respiratoire. Finalement, nous avons observé que la polarisation fonctionnelle des macrophages M1-like pourrait être affectée par des changements dans le métabolisme cellulaire. Ces données suggèrent que le métabolisme est un facteur déterminant du phénotype fonctionnel des macrophages. / Tumor cells require a constant supply of nutrients and yet, their tumor microenvironment supplies insufficient amount of nutrients. We therefore hypothesize that it exists a nutritional symbiosis between stromal cells and tumor cells which contribute to tumorigenesis. Of these stromal cells, we focused on macrophages. Typically, they are classified as inflammatory (M1-like) or alternative (M2-like) and within the stroma, macrophages tend to exhibit an M2-like phenotype. Concerning their metabolic profile, M1-like macrophages have been described to exhibit a glycolytic metabolism. However, the metabolic features of M2-like macrophages remain pretty unclear. In our work, using the mouse monocyte macrophage cell line RAW 264.7, we have confirmed that M1-like cells are exclusively glycolytic whereas M2-like macrophages appear as mainly oxidative. We were able to demonstrate some metabolic plasticity for M2 cells that shift to glycolysis when mitochondrial respiration is inhibited. The targeting of glycolysis through the blockade of downstream lactic acid export catalyzed by monocarboxylate transporters, did not reveal any metabolic adaptation of M1-like macrophages but impacted on M2 cells, reducing their respiratory rate. By manipulating metabolic features of M1- and M2-like macrophages (i.e. glycolysis vs oxidative phosphorylation), we also investigated whether metabolic pathways themselves could impact on macrophage polarization phenotype. We observed that functional M1 polarization of macrophages could be affected by changes in cell metabolism. Taken together, these data suggest that metabolism is a crucial determinant of macrophage functional phenotype.

The immunomodulatory effects of Trichinella spiralis or Trichinella pseudospiralis soluble antigen extracts were examined in an effort to characterize the differences in immune responses seen during these Trichinella infections. The newborn larvae extracts of either parasite exhibited similar potency for stimulating macrophage PGE production; however, the muscle larvae extracts of T. pseudospiralis stimulated greater levels of PGE than did the muscle larvae extracts of T. spiralis. These data clearly indicate that Trichinella antigens possess immunomodulatory capabilities.

Development of chronic inflammation or autoimmunity may be related to deregulated mechanisms orchestrating successful resolution of inflammation, especially apoptosis of inflammatory cells and their subsequent clearance by macrophages (Mφ). Chronically inflamed sites are characterised by an excess of the key pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) and importantly, TNF-α inhibitors, widely used in the clinical setting for the treatment of rheumatoid arthritis (RA), inflammatory bowel disease and psoriasis, significantly delay disease progression. TNF-α therefore may affect processes implicated in resolution of inflammation. Although TNF-α and pro-inflammatory bacterial products such as lipopolysaccharide (LPS) influence rates of inflammatory cell apoptosis, little is known about their effects on Mφ phagocytosis of apoptotic cells (efferocytosis). In this PhD thesis, the effects of several pro-inflammatory agents (i.e., LPS, lipoteichoic acid (LTA), peptidoglycan (PGN) and TNF-α) on efferocytosis by human blood monocytederived Mφ (MDMφ) have been investigated. LPS, LTA and PGN all inhibited MDMφ efferocytosis in a concentration- and time-dependent manner; however, LPS did not inhibit the uptake of immunoglobulin-G (IgG)-opsonized erythrocytes. Moreover, although TNF-α did inhibit efferocytosis, phagocytosis of IgG-opsonized erythrocytes was not inhibited. Furthermore, the LPS effect was attenuated by dimeric soluble human recombinant TNF receptor-1 (sTNFR1/ Fc), indicating a critical role of TNF-α. Concomitant treatments with monomeric soluble human recombinant TNF receptor-1 (sTNF-R1) or the TNF-α Converting Enzyme (TACE) inhibitor, TOPI-0, only partially reversed the inhibitory effect of LPS. Even though TNF-α release takes place within the first few hours following LPS stimulation, the LPS-induced inhibitory effect occurred only if treatment was performed for 96 hours or longer. Analysis of supernatants obtained from LPS-treated MDMφ revealed that there appears to be interplay between concentrations of TNF-α and interleukin-10 (IL-10) and that these cytokines exert opposing actions on efferocytosis. IL-10 per se increased MDMφ efferocytosis and addition of exogenous IL-10 to LPS-treated samples rescued phagocytosis. The latter effect was associated with the IL-10-induced, concentration-dependent inhibition of TNF-α release. Interestingly, when IL-10 was added to TNF-α-treated MDMφ, only slight augmentation of phagocytosis was observed. Furthermore, when IL-10-mediated effects were blocked by concomitant treatment with anti-human IL-10 receptor 1 antibody (anti-IL-10- R1Ab), the LPS inhibitory effect on phagocytosis was much greater and occurred at 24 hours after treatment. The role of IL-10 on efferocytosis was also investigated using IL-10 deficient murine bone marrow-derived Mφ (BMDMφ). IL-10 deficient BMDMφ, when compared to wild-type, were characterised by a much lower ability to phagocytose apoptotic neutrophils and this effect was independent of culture conditions (control samples and LPS or TNF-α treatments). Finally, effects of the synthetic steroid (dexamethasone) and nonsteroidal anti-inflammatory drugs (NSAID) on MDMφ phagocytosis were examined. Dexamethasone, like IL-10, augmented MDMφ efferocytosis, reversed the inhibitory effects of both LPS and TNF-α, and suppressed LPS-induced production of TNF-α. In contrast NSAID did not increase MDMφ efferocytosis per se. However, preliminary data suggest that aspirin blocks the inhibitory effect of TNF-α on phagocytosis. In summary, it has been determined that prolonged treatment with proinflammatory agents such as LPS, LTA and PGN inhibits MDMφ efferocytosis which may potentially postpone the resolution of inflammation in vivo. I have shown that TNF-α is a key mediator in this process and that IL-10 exerts an important regulatory effect on TNF-α production and consequently on efferocytosis. Furthermore, several approaches have been unveiled to successfully reverse LPS-mediated inhibition of efferocytosis by decreasing either TNF-α production or its inhibitory effect with sTNF-RI/Fc, exogenous IL- 10 or dexamethasone. These findings indicate that TNF-α and other agents which influence efferocytosis may have significance in the resolution phase of inflammation. In addition, presented findings provide important mechanistic information into the potential mode of action of anti-TNF-α agents and steroids and may help to explain their clinical success in the treatment of chronic inflammatory diseases.

No description available.

published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

No description available.

No description available.

No description available.

Disruption of <i>N-</i>mannosylation and <i>O</i>-mannosylation on the <i>C. albicans</i> outer cell wall increased the rate by which <i>C. albicans</i> is ingested by macrophages. Conversely, disruption of phosphomannosylation reduced the rate of <i>C. albicans</i> is phagocytosis. Alterations to the outer cell wall and genetic or chemical inhibition of hyphal morphogenesis in <i>C. albicans</i> resulted in significantly abrogated macrophage killing <i>in vitro</i>. Disruption of <i>C. albicans </i>ability to tolerate oxidative stresses also perturbed its ability to escape from and kill macrophages. The engagement of specific receptors on the macrophage surface is an essential component of <i>C. albicans</i> recognition and clearance. In the presence of serum, blocking pattern recognition receptors associated with specific fungal cell wall epitopes (Mannose Receptor, Dectin 1 and CD16/32) resulted in an initial decrease in phagocytosis and decreased macrophage killing. Blocking macrophage pattern recognition receptors using soluble components of the<i> C. albicans</i> cell wall resulted in decreased phagocytosis under serum free conditions of <i>O-</i>linked mannans only, and reduced macrophage killing for macrophages pre-exposed to <i>N-</i>mannan and laminarin. The presence of serum increased the rate of uptake for macrophages pre-exposed to <i>N-</i>mannan and laminarin, and had no affect upon macrophage killing. The interaction of <i>C. albicans</i> cell wall epitopes with macrophage pattern recognition receptors, coupled with <i>C. albicans</i> ability to respond to stresses encountered after ingestion are critical determinants of the macrophage’s ability to ingest and process <i>C. albicans.</i>

Macrophages (M?) are widely distributed immune cells and can be phenotypically polarised by microenvironment to mount specific functional programs. M? polarised in vitro can be broadly classified in two main groups: classically activated (or Ml), and alternatively activated (or M2) M?. M1 exhibit potent microbicidal properties and induce pro-inflammatory responses, whilst M2 play a role in resolution of inflammation and reduce pro-inflammatory responses.