Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGF- β, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.

Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here, we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e., increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance, implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals the essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions—trained immunity or immune tolerance—appears to be shaped by pathogen dose.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)±epinephrine, norepinephrine, and dopamine at 5×10-6M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by β-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that β-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.

Previous studies have demonstrated that bacterial lipopolysaccharide (LPS) and heat killed Staphylococcus aureus (SA) activation of inflammatory cells depended in part upon activation of heterotrimeric Gi proteins. It has also been shown that (1→3) beta-D-glucan can suppress inflammatory cell activation by microbial products although the cellular mechanism of the glucan effect remains to be clearly defined. We hypothesized that Gi proteins function as a common convergent signaling pathway for both LPS and SA leading to monocyte mediator production. Additionally, we hypothesized that soluble glucan suppresses LPS and SA induced cytokine production via Gi protein coupled signaling. Human THP-1 promonocytic cells were pretreated with pertussis toxin (PTx, 100ng/ml or 1 microgram/ml) 6 hours prior to stimulation with LPS (10 microgram/ml) and SA (10 microgram/ml) and/or soluble glucan (10 microgram/ml). Both LPS and SA significantly (p<0.05) induced cytokine production IL-6 >TNF alpha >IL-1 beta >GM-CSF >IL-10 >IFN gamma. The induction of these cytokines was significantly (p<0.05) suppressed by PTx. Glucan treatment alone had no effect on cytokine production but suppressed (P<0.05) LPS and SA induced cytokines. PTx further augmented (p<0.05) the inhibitory effect of glucan on the LPS and SA induced cytokine expression. The data support the hypothesis that Gi proteins function as a common signaling protein for both LPS and SA induction of pro-and anti-inflammatory cytokines and that soluble glucan effectively suppresses cytokine production to the microbial stimuli. In contrast, the effect of soluble glucan on inhibiting cellular activation by LPS and SA is Gi protein independent.

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Helicobacter pylori infection is a leading cause of gastric cancer, which is the second-most common cancer-related death in the world. The chronic inflammatory environment in the gastric mucosal epithelia during H. pylori infection stimulates intracellular signaling pathways, namely inflammatory signals, which may lead to the promotion and progression of cancer cells. We herein report two important signal transduction pathways, the LPS-TLR4 and CagA-MET pathways. Upon H. pylori stimulation, lipopolysaccharide (LPS) binds to toll-like receptor 4 (TLR4) mainly on macrophages and gastric epithelial cells. This induces an inflammatory response in the gastric epithelia to upregulate transcription factors, such as NF-κB, AP-1, and IRFs, all of which contribute to the initiation and progression of gastric cancer cells. Compared with other bacterial LPSs, H. pylori LPS has a unique function of inhibiting the mononuclear cell (MNC)-based production of IL-12 and IFN-γ. While this mechanism reduces the degree of inflammatory reaction of immune cells, it also promotes the survival of gastric cancer cells. The HGF/SF-MET signaling plays a major role in promoting cellular proliferation, motility, migration, survival, and angiogenesis, all of which are essential factors for cancer progression. H. pylori infection may facilitate MET downstream signaling in gastric cancer cells through its CagA protein via phosphorylation-dependent and/or phosphorylation-independent pathways. Other signaling pathways involved in H. pylori infection include EGFR, FAK, and Wnt/β-Catenin. These pathways function in the inflammatory process of gastric epithelial mucosa, as well as the progression of gastric cancer cells. Thus, H. pylori infection-mediated chronic inflammation plays an important role in the development and progression of gastric cancer.

No description available.

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