[French below]TLRs are the sentinels of our cells, they are located at the cell surface and alert the whole immune system of the presence of viruses or bacteria. They detect pathogens by recognizing their molecular patterns; this recognition is specific in order to avoid self-recognition, but they need some degree of promiscuity to remedy to pathogen heterogeneity or mutations. Promiscuity is generally defined as an indiscriminate association with molecules regardless their structure and is the contrary of specificity proper of the classic paradigm of key-lock receptor activation. My thesis results demonstrate that TLR4 and TLR2 are more promiscuous than what was believed and that this promiscuity leads to the recognition of cationic lipids and cardiolipins.Cationic lipids lipopolyamines are synthetic molecules nucleic acid nanocarriers proposed to be used for gene therapy, which consists in replacing a gene that is functioning improperly. This thesis demonstrates that lipopolyamines activate TLR2 by forming conserved and/or alternative H-bonds with TLR residues, simulating the recognition of bacterial lipopeptides and inducing pro-inflammatory cytokines secretion; which is deleterious when we aim to use these nanocarriers in the context of gene therapy. We propose the use of unsaturated cationic lipids to avoid TLR2 recognition. TLR activation could be useful instead to prepare one-component vaccine adjuvants, for which both antigen carrier and TLR activation are needed to turn on the immune system and produce antibodies. The second chapter of this thesis investigates the pro-inflammatory properties of other cationic lipids and describes new lipopolyamines able to activate both TLR2 and TLR4. The study of their adjuvanticity properties showed that they are as efficient as the aluminium salts in stimulating antibodies production.The promiscuity of TLR2 and TLR4 raised questions about the presence of endogenous TLR modulators, often countered by contamination concerns. In the third chapter, we investigate the pro- and anti-inflammatory properties of cardiolipin (CL). CL is a tetra-acylated diphosphatidylglycerol located in the inner mitochondrial membrane. Its fatty acid chain length and degree of unsaturation vary depending on species, tissue and pathological conditions. Here we show that unsaturated cardiolipin acts as a competitive TLR4 antagonist by occupying the binding site of LPS and that unsaturations discriminate between TLR4 antagonistic and agonistic activity. Under physiological conditions, mammalian CL chains are unsaturated, whereas there is an increase of saturated CL in patients affected by the Barth Syndrome. Hence we suggest that unsaturated CL could negatively regulate the inflammation during cell damage or death, via TLR4 inhibition. By contrast, saturated CLs may be involved in the inflammatory state associated with the disease. Finally, our study provides new understandings of the mechanism of TLR4 regulation and extends the library of TLR4 agonists and antagonists to molecules of easier synthesis, lower price and higher biocompatibility compared to LPS-based structures. / Resumé en français Les récepteurs Toll-like (TLRs) sont des protéines transmembranaires qui constituent la première barrière de notre système immunitaire inné. Ils détectent la présence de bactéries et virus et alertent l’organisme via la sécrétion de molécules pro-inflammatoires appelés cytokines. Parmi les TLRs, TLR2 and TLR4 reconnaissent respectivement des lipides spécifiques aux bactéries, les lipopeptides et les lipopolysaccharides bactériens LPS. La reconnaissance de motifs moléculaires spécifiques aux pathogènes et absents dans notre organisme est essentiel afin d’éviter une réponse immunitaire venant du soi. Le but de notre thèse était de démontrer que les récepteurs Toll-like possèdent une certaine plasticité et peuvent reconnaître des ligands non identifiés jusqu’ici tels les lipides cationiques et la cardiolipine. Les lipides cationiques sont des molécules synthétiques utilisées comme agents de transfection. Notre travail démontre que les lipides cationiques dont la tête polaire est constituée par des polyamines peuvent mimer les propriétés des ligands naturels et induire la sécrétion de cytokines pro-inflammatoire via l’activation des TLRs. Cette interaction implique des interactions entre la chaine principale de la protéine et les lipides sans intervention des chaines latérales. Cette réaction inflammatoire est contre-indiquée en thérapie génique et nous proposons donc de remplacer les chaines acylées saturées par des chaines insaturées pour la synthèse des nouveaux agents de transfection non-immunogénique. D’autre part, l’activation des TLRs par des agents de transfections active le système immunitaire inné, ce qui permet l’activation du système adaptatif et la production d’anticorps. Nous avons étudié une large gamme des lipides cationiques et identifié des nouveaux activateurs á la fois de TLR2 et de TLR4. L’étude de leurs propriétés adjuvantes a démontré que les lipides cationiques sont des adjuvants comparables aux sels d’aluminium en terme de production d’anticorps. La cardiolipine est un lipide localisé dans la membrane des mitochondries et des bactéries. Le domaine hydrophobe est constitué de quatre chaines acylées qui chez les mammifères sont insaturées. Il a été démontré que la cardiolipine extracellulaire inhibe la sécrétion de cytokines induite par LPS. Notre travail de thèse démontre que cet effet inhibiteur est du à la capacité de la cardiolipine à bloquer le site de liaison du LPS. Le travail démontre aussi que lorsque les chaines acylées sont saturées, c’est le cas dans le Syndrome de Barth, la cardiolipine devient un activateur de TLR4 en interagissant avec TLR4 de façon similaire à LPS. Ce dernier résultat pourrait expliquer l’aspect inflammatoire du Syndrome de Barth et élargie la librairie de ligands de TLR4 á des molécules de structure plus simple et plus aisées à synthétiser que les dérivés de LPS et qui pourraient être utilisés comme adjuvants ou anti-inflammatoires. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Doctor of Philosophy / Department of Animal Sciences and Industry / J. Ernest Minton / Toll-like receptors (TLR) are instrumental in discriminating between pathogenic and commensal bacteria and act as mediators, along with downstream chemokines, of subsequent innate and adaptive immune responses. However, little is known about the expression and regulation of TLR or chemokines in swine. The objectives of the experiments described herein were to characterize the expression of porcine TLR and to identify regulatory patterns in these receptors in the presence of live Salmonella enterica serovar Typhimurium (ST) or Choleraesuis (SC). The first two experiments evaluated the in vivo and in vitro expression of TLR2, 4, 5 and 9. Our results indicate that TLR2, 4, 5 and 9 are constitutively expressed in vitro in a porcine jejunal epithelial cell line (IPEC-J2), porcine mononuclear phagocytes (pMPs) and in vivo in the distal ileum. In IPEC-J2 cells, ST elicited an increase in TLR2 mRNA (P < 0.05), and both ST and SC increased TLR2 mRNA in pMPs (P < 0.05). In vivo, oral challenge with ST increased (P < 0.05) both TLR2 and TLR4 mRNA in the distal ileum. In addition, the second experiment evaluated interleukin 8 (IL8) and CC chemokine ligand 20 (CCL20) expression in IPEC-J2 cells in response to ST or purified bacterial flagellin (Flag). TLR5 was constitutively expressed in the ileum and in IPEC-J2 and pMP cells. Interestingly, IL8 and CCL20 mRNA and protein were increased (P < 0.05) by ST and Flag, even in the absence of changes in TLR5. In the third experiment, the expression of TLR and chemoattractive mediators were evaluated in a panel of tissues obtained from pigs challenged with ST and SC. All genes of interest were constitutively expressed; however, the effects of treatment were limited to isolated tissues and genes. Taken together, the data indicate that TLR and chemoattractive mediators are expressed in porcine tissues and cells and that the observations described represent novel evidence that pig pathogens may regulate TLR expression and activate chemokine secretion.

Probiotics have been defined as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”. The beneficial effects of probiotics in the gut are well described and roles including immunomodulation and colonisation resistance have been documented. Recent reports suggest that topical use of probiotic bacteria may be an effective strategy to promote skin health or inhibit disease. Therefore, in this thesis the potential of probiotics to protect skin from pathogenic bacteria was assessed using primary keratinocytes as a model system, and the skin pathogen, Staphylococcus aureus. The ability of three probiotics, L. reuteri ATCC 55730, L. rhamnosus AC413 and L. salivarius UCC118 to inhibit the growth of S. aureus was tested using well-diffusion assays and spot on the lawn assays. All three probiotics inhibited the growth of S. aureus in well-diffusion assays, though this property was dependent on growth medium. Inhibition of S. aureus growth was principally via the production of organic acids rather than bacteriocin production. Next, to determine whether probiotics could protect keratinocytes, confluent normal human epidermal keratinocytes (NHEK) were infected with S. aureus (106 CFU/ml) in the presence or absence of the probiotic (108 CFU/ml). NHEK viability was measured using trypan blue exclusion assays. L. reuteri had a significant protective effect on NHEK when applied 1h prior to (P=0.0003), or simultaneously with S. aureus (P=0.002). L. reuteri did not however protect NHEK when applied 1h after S. aureus addition. There was no change in the number of viable S. aureus in cell culture assays. To determine whether the protective effect was due to the inhibition of adhesion, NHEK were either pre-exposed to the probiotic for 1h, simultaneously exposed to the probiotic and S. aureus for 1h, or exposed to the probiotic 30 minutes after S. aureus addition for 1h. Pre-exposure of NHEK to L. reuteri (exclusion) and simultaneous exposure to L. reuteri and S. aureus (competition) resulted in significantly less staphylococci adhering to NHEK (P=0.03 and P=0.008 respectively). However when L. reuteri was added after S. aureus (displacement), the number of adherent staphylococci was not reduced. The necessity of S. aureus adherence for the inactivation of NHEK was demonstrated using a α5β1 integrin blocking antibody. Finally, to compare the innate response of NHEK to probiotics with S. capitis and S. aureus, TLR-2, antimicrobial peptide (AMP) expression and IL-8 production were measured. TLR-2 protein (but not mRNA) expression was reduced in the presence of S. aureus (P=0.018). NHEK pre-exposed to S. capitis prior to S. aureus infection however, exhibited elevated TLR-2 protein and mRNA expression (P<0.0001 and P=0.009 respectively). NHEK pre-exposed to L. reuteri prior to S. aureus had no significant change in TLR-2 expression compared to untreated controls. ELISAs demonstrated that IL-8 production was significantly increased in NHEK pre-exposed to L. reuteri prior to S. aureus infection (P=0.0001). In conclusion, L. reuteri protected NHEK from the toxic effects of S. aureus at least partly through competitive exclusion of binding sites on NHEK. Finally, NHEK innate responses to probiotic bacteria were akin to those to the skin commensal, S. capitis. L. reuteri induced expression of a neutrophil chemoattractant, suggesting it could be of importance in priming the innate immune response against S. aureus infections. Taken together, these results suggest that probiotic bacteria could be used prophylactically within skin creams and soaps to prevent S. aureus colonisation and infection in skin.

Hematopoiesis is a vital process in which red blood cells and cells of the immune system are formed. It is initiated during early embryonic development when we find hematopoietic progenitors in separate anatomical sites. Embryonic hematopoiesis comprises three successive and partly overlapping waves of progenitors with a different hematopoietic potential. The primary anatomical place where hematopoiesis takes place shortly before the birth is the bone marrow (BM). Since at this time point of development BM is already populated by hematopoietic stem cell (HSCs) progenitors, it becomes also the site of hematopoiesis in adulthood. However, the bone marrow is not the only place where hematopoietic progenitors emerge and develop. The Yolk sac (YS) and the Aorta-Gonad-Mesonephros (AGM) region are the initial sites of the appearance of the three waves of progenitors in the early embryogenesis. These progenitors and their descendants play an indispensable role during the development of an individual. Because there are no specific markers that would unambiguously characterize progenitors of these individual waves, their physical separation and hence also functional characterization is still incomplete. Recent studies have shown that Toll-like receptors (TLRs) are expressed on adult HSCs. The stimulation of...

Introduction: Diabetes mellitus is a polygene disease and on its manifestation have influence also enviromental factors. We have studied the role of extrapituitary prolactin (PRL) and toll-like receptors (TLR) 2 and 4 in the etiopathogenesis of autoimmune diabetes. PRL is mainly produced by hypophysis, but in small concentrations also in the periphery, where it participates in the immune reactions. Therefore, we investigated the influence of the levels of monocytic PRL mRNA on the development of diabetes, and also the influence of G allele of the -1149 G/T polymorphism in the extrapituitary promotor, which has already been associated with other autoimmune diseases. TLRs are receptors of the immune cells that recognize patogenes entering into the body. They play an important role in the iniciation of the immune response. We aimed to find out their function in the pathogenesis of the autoimmune diabetes by the detection of their mRNA levels and protein levels expressed on the cell surface of the monocytes. Material and methods: In this study we included 30 T1D and 21 LADA patients. Three control groups consisted of 23 T2D patients, 23 patients with a nondiabetic disease (neDM) and 60 healthy blood donors (TO). Blood samples have been taken from the individuals. From these blood samples we isolated...

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

AimsWe have reported that either toll-like receptor 4 deficiency (TLR4 -/-) or TLR2 modulation protects against myocardial ischaemia/reperfusion (I/R) injury. The mechanisms involve attenuation of I/R-induced nuclear factor KappaB (NF-κB) activation. MicroRNA-146a (miR-146a) has been reported to target interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), resulting in inhibiting NF-κB activation. This study examined the role of microRNA-146a in myocardial I/R injury.Methods and resultsWe constructed lentivirus expressing miR-146a (LmiR-146a). LmiR-146a was transfected into mouse hearts through the right common carotid artery. The lentivirus vector (LmiR-Con) served as vector control. Untransfected mice served as I/R control. Sham operation served as sham control. Seven days after transfection, the hearts were subjected to ischaemia (60 min) followed by reperfusion (4 h). Myocardial infarct size was analysed by triphenyltetrazolium chloride (TTC) staining. In separate experiments, the hearts were subjected to ischaemia (60 min) followed by reperfusion for up to 7 days. Cardiac function was measured by echocardiography prior to I/R, 3 and 7 days after myocardial I/R. LmiR-146a transfection significantly decreased I/R-induced myocardial infarct size by 55% and prevented I/R-induced decreases in ejection fraction (EF%) and fractional shortening (%FS). LmiR-146a transfection attenuated I/R-induced myocardial apoptosis and caspase-3/7 and-8 activities. LmiR-146a transfection suppresses IRAK1 and TRAF6 expression in the myocardium. In addition, transfection of LmiR-146a prevented I/R-induced NF-κB activation and inflammatory cytokine production.ConclusionsMicroRNA-146a protects the myocardium from I/R injury. The mechanisms may involve attenuation of NF-κB activation and inflammatory cytokine production by suppressing IRAK1 and TRAF6.

Aims Toll-like receptor (TLR)-mediated signalling pathways have been implicated in myocardial ischaemia/reperfusion (I/R) injury. Activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway protects the myocardium from ischaemic injury. We hypothesized that the modulation of TLR2 would induce cardioprotection against I/R injury via activation of the PI3K/Akt signalling. Methods and results Mice were treated with TLR2 ligands, peptidoglycan (PGN) or Pam3CSK4, respectively, 1 h before the hearts were subjected to ischaemia (1 h), followed by reperfusion (4 h). Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac function and haemodynamic performance were evaluated. Infarct size was significantly reduced in PGN-or Pam3CSK4-treated mice compared with untreated I/R mice. Administration of TLR2 ligands improved cardiac function following I/R. PGN treatment increased the levels of phospho-Akt and phospho-GSK-3β (glycogen synthase kinase-3β), compared with untreated I/R hearts. PGN stimulation increased TLR2 tyrosine phosphorylation and association of the p85 subunit of PI3K with TLR2. To investigate the role of PI3K/Akt signalling in PGN-induced cardioprotection, we administered the PI3K inhibitor, Wortmannin, to the mice 15 min before PGN treatment. We also administered PGN to kinase-deficient Akt (kdAkt) transgenic mice 1 h before myocardial I/R. Both PI3K inhibition and kdAkt mice abolished the cardioprotection induced by PGN. To examine the role of TLR2 in PGN-induced cardioprotection, we administrated PGN to TLR2 knockout mice 1 h before the hearts were subjected to I/R. PGN-induced cardioprotection was lost in TLR2-deficient mice. Conclusion These results demonstrate that TLR2 ligands induced cardioprotection, which is mediated through a TLR2/PI3K/Akt-dependent mechanism.

Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 μg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca 2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca 2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca 2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (If), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 μg/ml) reduced If in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated If. LPS reduced the slope conductance of the tail currents from 498 ± 140 pS/pF to 223 ± 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on If, whereas Pam3CSK4 had no effect on If. We conclude that LPS inhibits activation of I f, enhances its deactivation, and impairs regulation of [Ca 2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.