Les dommages cérébraux survenant durant la période périnatale sont associés à plusieurs pathologies neurodéveloppementales, dont les encéphalopathies néonatales. Ces lésions résultent d’agressions hypoxiques-ischémiques (HI) et infectieuses survenant durant une étape cruciale du développement cérébral. Le seul traitement disponible pour diminuer l’impact de ces agressions sur le neurodéveloppement de l'enfant est l’hypothermie. Les deux types d’agressions ont un facteur commun, l’inflammation, et surtout la production de cytokines pro-inflammatoires telle que l’interleukine (IL)-1. Bien que l'association entre l'expression d'IL-1 et la modulation du développement cérébral soit connue, le lien de cause à effet entre les deux est encore méconnue. Ceci est dû, entre autres, à l'utilisation de nombreux modèles expérimentaux manquant souvent de pertinence avec ce qui est observé en clinique. Dans le but de comprendre le rôle de l’IL-1 dans les dommages cérébraux périnataux du nouveau-né à terme, nous avons fait la caractérisation anatomique et fonctionnelle d’un modèle animal combinant les deux types d’agressions les plus fréquemment rencontrées chez l’humain, afin d’en établir la concordance par rapport à la pathologie humaine. À l’aide de ce modèle, nous avons ensuite démontré que le système de l’IL-1 était au cœur de la pathophysiologie. De plus, nous avons montré que le stade de développement cérébral correspondant au nouveau-né à terme comporte des différences par rapport à l’adulte et au nouveau-né prématuré dans les composantes inflammatoires exprimées et par le type cellulaire exprimant ces composantes. En inhibant le système de l’IL-1, nous avons démontré le lien causal entre l’expression de l’IL-1 et la survenue de dommages cérébraux ainsi que son impact sur la motricité des animaux via l'administration de l'antagoniste du récepteur de l'IL-1 (IL-1Ra). Nous avons démontré le potentiel thérapeutique post-natal de l’IL-1ra administré directement aux animaux lors de l’agression. En conclusion, les travaux présentés dans cette thèse démontrent le rôle central de l’IL-1 dans la genèse des lésions cérébrales périnatales suite à une agression de type HI et infectieuse/inflammatoire ainsi que le potentiel thérapeutique de l'administration post-natal de l'IL-1Ra dans un modèle animal correspondant au nouveau-né à terme.

Le syndrome métabolique (SM) augmente le risque de diabète et de maladies cardiovasculaires. Les composantes de cet état sont l’obésité, l’hypertension, le profil lipidique, la résistance à l’insuline, l’inflammation et l’atteinte hépatique. L’accumulation excessive de la masse grasse, surtout abdominale, entraîne une inflammation systémique de faible niveau. En effet, le tissu adipeux secrète des adipokines (leptine, visfatine, IL-6 et TNF-a) qui sont impliquées dans l’inflammation. Notre but a été d’étudier : 1) les associations de l’évolution de 5 ans de l’indice de masse corporelle (IMC) et de la leptine circulante avec les facteurs du SM et 2) l’expression de certaines adipokines dans les lymphocytes. Pour ces études, la cohorte STANISLAS a été un outil parfait (recueil des données sur les facteurs du SM, bio-banques : lymphocytes, sérum, plasma, ADN). L’étude longitudinale a démontré que certains de facteurs du SM évoluent ensemble au fil du temps et que ces groupes de facteurs sont liés à l’IMC en fonction du sexe. Ensuite, nous avons constaté des associations de la leptine avec des facteurs de risque du SM, différentes en fonction du sexe, indépendamment de la masse grasse. Enfin, nous avons détecté l’ARNm de nos adipokines et sa quantification a démontré des associations de la visfatine avec l’IMC et l’expression du TNF-a, et de la leptine avec la pression artérielle. L’ensemble de nos résultats souligne le rôle-médiateur de la leptine et l’impact du sexe sur les actions de cette hormone et ouvre de nouvelles perspectives pour étudier les adipokines dans le contexte de l’inflammation, en proposant un modèle d’étude : les lymphocytes. / The metabolic syndrome (MS) raises the risk for developing diabetes and cardiovascular disease. The components of this state are obesity, hypertension, disturbed lipid profile, insulin resistance, inflammation and hepatic steatosis. The excessive accumulation of fat mass, particularly in the abdomen, leads to low-grade systemic inflammation. Indeed, the adipose tissue produces adipokines such as leptin, visfatin, IL-6 and TNF-a that are implicated in the inflammatory processes. Our goal was to study: 1) the associations between 5 year-changes of the body mass index (BMI) and circulating leptin concentrations with the factors of the MS and 2) the expression of some adipokines in the lymphocytes. The STANISLAS cohort has enabled us to fulfil our research work (collection of a great number of data concerning MS factors, bio-banks: lymphocytes, serum, plasma, DNA). The longitudinal study on BMI changes and the factors associated with MS has shown that some of these factors evolve together (clusters) and that these groups of factors are associated with BMI changes in a sex dependent manner. We described also, associations of circulating leptin with MS factors, dependent to sex and independently of fat mass. Finally, we were able to detect and quantify mRNA of adipokines in lymphocytes. The quantification of the mRNA has shown associations of visfatin with BMI and gene expression of TNF-a and of leptin with blood pressure. The results obtained by this research work underline the mediating role of leptin and the impact of sex on the actions of this hormone and offers new perspectives for studying adipokines in the context of inflammation, by proposing a new model: PBMCs.

Thesis advisor: Welkin Johnson / HIV associated cardiovascular disease is likely due to multiple factors ranging from accelerated aging, the direct effects of HIV proteins, and increased inflammation and immune activation. Monocytes/macrophages play roles in the development and progression of HIV and cardiovascular disease. Increased monocyte/macrophage inflammation and immune activation associated with HIV infection likely contributes to the increased risk of cardiovascular disease development associated with HIV infection. To further understand the role of monocytes/macrophages in the development of HIV-associated cardiovascular disease we: 1) assessed monocyte activation longitudinally to determine if they correlate with and can be predictive of cardiac fibrosis and inflammation; 2) we examined cardiac tissues from the SIV-infected CD8+ T-lymphocyte depleted animals to determine the effects of monocyte/macrophage inflammation on cardiac fibrosis; 3) in parallel we examined cardiovascular tissues from HIV+ individuals on durable cART to determine if aortic and cardiac inflammation persists with infection and if soluble factors (sCD163) correlated with intima-media thickness and fibrosis; 4) we next examined the effects of blocking leukocytes trafficking to the heart on SIV-associated cardiac inflammation and fibrosis; 5) and finally we examined if targeting monocyte/macrophage activation (as opposed to traffic) directly using MGBG decreases SIV-associated cardiovascular pathology, inflammation and fibrosis. We found that early increased monocyte activation was predictive of animals that developed cardiac fibrosis and SIV encephalitis (SIVE). Animals with both cardiac fibrosis and SIVE had increased macrophage inflammation in the heart, suggesting that there is a link between cardiac and CNS inflammation seen with HIV infection (Chapter 2). We found in a SIV-infected CD8+ T-lymphocyte depletion model of rapid AIDS increased prevalence of cardiac disease compared to nondepleted animals, and increased cardiac inflammation that correlated with cardiac fibrosis. Monocyte/macrophage traffic to the heart occurred later with SIV infection, possibly with the development of AIDS (Chapter 3). In post-mortem human tissues studies we found that inflammation in aorta and heart correlated with increased soluble CD163, and correlated with aortic intima-media thickness and cardiac fibrosis with HIV infection (Chapter 4). Blocking leukocyte traffic to the heart using an anti-α4 antibody decreased macrophage inflammation in the heart that correlated with decreased cardiac fibrosis (Chapter 5). Using MGBG, a polyamine biosynthesis inhibitor that directly targets monocyte/macrophage activation, we found decreased inflammation in the carotid artery and heart correlated with decreased carotid artery intima-media thickness and cardiac fibrosis (Chapter 6). Overall these studies provide evidence for ongoing monocyte/macrophage cardiovascular inflammation with HIV and SIV infection. Macrophage inflammation correlates with markers of cardiovascular disease (fibrosis and intima-media thickness, cardiomyocyte damage). Directly targeting monocyte/macrophage traffic (anti-α4 antibody) and activation (MGBG) decreased cardiovascular pathology, inflammation, fibrosis, and intima-media thickness. Taken together, the data in this thesis indicate that targeting monocytes/macrophages in conjunction with combination anti-retroviral therapy could alleviate cardiovascular disease in HIV-infected individuals. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

University of Macau / Institute of Chinese Medical Sciences

Galectins-1, -3 and -9, are endowed with many immune-regulatory properties, with galectins-1 and -9 largely regarded as anti-inflammatory and galectin-3 as pro-inflammatory. Expression levels increase in activated adaptive immune cells, with peak expression often correlating with peak inflammation. Galectin actions are not only determined by their expression levels but also target tissue permissibility to galectin binding, which is in turn determined by the profile of specific carbohydrate residues, namely N-acetyllactosamine, recognised by these lectins. How expression levels and actions are modulated in innate immune cells during inflammation has not been systematically characterised. This study therefore set out to delineate the effects of inflammation on neutrophil glycophenotype, as well as elucidate the temporal and spatial modulation of galectins during resolving inflammation. The neutrophil glycophenotype was modulated during trafficking with decreased levels of all terminal glycan residues assessed. However, this did not correlate with galectin binding permissibility suggesting this is not a useful indicator in this model. The overall change in glycosylation may theoretically be a consequence of rapid modulation of cell surface glycoproteins by activated neutrophils (i.e. CD62L shedding) rather than the actions of specific glycosylation enzymes as demonstrated in T- and endothelial cells. Assessment of galectin levels in leukocytes over a 96h zymosan-induced resolving peritonitis demonstrated alterations both spatially and temporally with increased galectin-3 expression in neutrophils at the inflammatory site compared to the periphery and a peak expression at 24h adding supporting evidence that modulation of galectin expression allows delineation of galectin responses by neutrophils. This study also demonstrated a novel pro-resolution effect of galectin-3 with defective resolution observed in galectin-3 null mice. In conclusion this work demonstrated that neutrophil permissibility for galectins-1, -3 and -9 binding is more likely a consequence of the exposure to galectins at specific time points in the resolving inflammatory response rather than due to a modulation of the glycophenotype upon activation. This study also 3 demonstrated that as well as an important role in the induction of an inflammatory response galectin-3 is involved in resolution, a novel finding which may lead to a better understanding of the resolution process.

Females are relatively protected from inflammatory diseases, particularly conditions that are characterised by excessive tissue infiltration of neutrophils (PMNs), such as ischaemia/reperfusion (I/R) injury. Therefore, understanding sex-differences is very important particularly for appropriate treatment of inflammatory disorders in men and women. Unfortunately, efforts to exploit sex-differences therapeutically have been unsuccessful since the precise mechanisms that confer the protective advantage in females over males are unclear. Many fundamental aspects of the nature of sex-differences have not been investigated, particularly in the regulation of PMN mobilisation from the bone marrow during acute inflammation. The aim of this thesis is to determine the nature and mechanism of leukocyte activation and recruitment in males and females, particularly in I/R. This thesis demonstrates for the first time that regulation of PMN mobilisation during acute inflammation is distinct in females. In comparison to males, females demonstrate reduced expression of mediators that cause the release of PMNs, including GCSF, CXCR2 and CXCL5, and increased expression of CXCR4 and CXCL12, which mediate PMN retention in the bone marrow. Reduced granulopoiesis, PMN mobilisation and recruitment into tissues in response to inflammogens protect females from collateral damage incurred by PMN-derived mediators that contribute to tissue injury and loss of function. This thesis has also revealed a novel, and possibly predominant, role for the ELR+ CXC chemokine CXCL5 in mobilisation of tissue-damaging PMNs from the bone marrow, whereby CXCL5 production, PMN mobilisation and tissue infiltration was profoundly greater in males during acute inflammation. CXCL5 appears to stimulate PMN mobilisation by 1) upregulating CXCR2 v expression on bone marrow cells and simultaneously downregulating CXCR4 expression; 2) inducing its own expression; 3) stimulating GCSF expression; and 4) increasing PMN expression of β2 integrin. Thus, the thesis proposes that reduced CXCL5 expression, and increased CXCR4/CXCL12 expression, in females during acute inflammation may be a novel mechanism underlying the protection against tissue injury. The fact that these sex-differences were apparent in different species (rat, mouse), tissues (mesenteric, lung, kidney, heart), in response to different stimuli (mesenteric, renal, myocardial I/R and carrageenan-induced pleurisy) and shown both in vivo and in vitro, suggests that this is a fundamental, and more generalised, phenomenon in males and females following acute inflammation. The inherent differences between the sexes have important clinical implications in that they demonstrate the need of considering sex-differences in research. This thesis demonstrates that sex-differences must be taken into account when developing such therapies for specific inflammatory diseases such as I/R injury as there are major differences in the temporal profile of chemokines and the extent of PMN infiltration.

Cytokine based therapies can be targeted to the sites of active inflammation by modifying a given cytokine as a LAP-cytokine. IL-17A has been shown to directly contribute to pathogenesis of rheumatoid arthritis (RA). IL-17F, another member of the IL-17 cytokines family shares structural homology, receptor binding and biological properties with IL-17A but is 30-100 times less potent than IL-17A. (H161R) IL-17F mutant, a natural variant of IL-17F was shown to be protective against asthma in Japanese population. In vitro, IL-17F mutant competitively inhibited wild-type IL-17F and lacked the ability to activate downstream signaling pathways. I hypothesized that (H161R) IL-17F mutant is an additional inhibitor of IL-17A and if modified as LAP-IL-17F mutant, would be an effective targeted therapy for RA. (H161R) IL-17F mutant was created by substituting nucleotide A at position 485 in the wild type IL-17F by G. In vitro assays showed that the IL-17F mutant could bind to IL-17RC but lacked the ability to stimulate IL-6 secretion in HFFF2, 3T3 and HeLa cells and phosphorylate ERK1/2 in HeLa cells. IL-17F mutant also inhibited IL-17A induced secretion of IL-6 in all these cell lines. In order to assess in vivo therapeutic efficacy of LAP-IL-17F mutant in collagen induced arthritis mice, three mouse analogues of human IL-17F mutant were developed. Of these, (Q158R) IL-17F mutant displayed IL-17 agonistic properties, (H157R) IL-17F mutant could not be expressed in vitro and the truncated IL-17F mutant could not bind to mouse IL-17RC. Investigation of in vivo expression and pharmacokinetics of intravenous hydrodynamically delivered human full-length and LAP-IL-17 plasmid DNAs in naïve SCID and C57BL/6 mice showed that human IL-17 transgene expression was detectable in mouse serum at 48 hours post-delivery. The transgene expression however declined rapidly over the next two weeks. The local expression of transgene in C57BL/6 airpouch lavage fluid was less than 5% of its systemic levels. Taken together, the findings of the study warrant an investigation of in vivo therapeutic efficacy of human (H161F) IL-17F mutant in a suitable preclinical RA model, such as RA synovium/SCID mice.

Macrophages are key orchestrators of the innate immune response with a dynamic role in the promotion and resolution of inflammation. Macrophage polarization to a pro-inflammatory or anti-inflammatory phenotype must be tightly controlled to maintain appropriate responses to stimuli as well as to maintain tissue homeostasis. The nutrient and energy sensor Mammalian Target of Rapamycin (mTOR) integrates upstream signals from the PI3K/Akt pathway to orchestrate cellular protein, lipid, and glucose metabolism. This key metabolic pathway has been implicated in T-helper cell skewing and in the innate immune regulation. The mechanisms of innate immune regulation by mTOR are currently unclear as most studies use pharmacological inhibitors with potential off target effects. In this study, we use a novel model of TSC1 deficiency in myeloid lineage cells to elucidate a role for mTOR in macrophage polarization. We show, for the first time, that Tsc1-deficiency and constitutive mTORC1 activity in macrophages leads to a marked defect in M2 polarization when stimulated with the Th2 cytokine IL-4. Tsc1-deficient macrophages display attenuated Akt signaling in response to IL-4 consistent with negative feedback of mTORC1 on upstream IRS2/PI3K signaling, and we demonstrate that this parallel signaling pathway is critical for induction of a subset of M2 markers. Tsc1-deficient macrophages fail to upregulate the M2 genes Pgc-1!, Arg-1, Fizz-1, and Mgl1 in addition to other M2 markers despite normal STAT6 signaling in response to IL-4. Consistent with downregulation of Pgc-1!, Tsc1-deficient macrophages also display defects in fatty acid metabolism and mitchochondrial biogenesis. Furthermore, LPS stimulation in Tsc-1 deficient macrophages leads to an enhanced inflammatory response with increased production of pro-inflammatory cytokines. We believe that Tsc1-deficient macrophages are a model of constitutive mTORC1 activity akin to obesity, where chronic nutrient excess leads to increases in mTORC1 activity, attenuation of IRS/PI3K/Akt signaling, and defective M2 polarization of macrophages in metabolic tissues.

Inflammation is implicated in a wide array of diseases and is associated with the ageing process: 'inflammageing' is the low-grade inflammation that occurs as an organism ages. I was particularly interested in age-related inflammation of the brain, thought to be mediated by microglia, the immune cells of the central nervous system (CNS). To this end, I carried out RNA sequencing of microglia from young (6 months) and aged mice (23 months). I found that microglia from aged mice have a very distinct transcriptome signature. Interestingly, pathways associated with mTOR signalling and inflammation were upregulated. Given the evidence that mTOR is a key modulator of ageing, I investigated its role in inflammation in microglia. Using a mouse model in which Rheb, a positive regulator of mTORC1, was knocked out in Csf1r-expressing cells (microglia and macrophages), I found that in vivo LPS stimulation caused a significant increase in the transcription of inflammatory genes in microglia from mTORC1-deficient mice compared to controls. The effect was further exaggerated in mTORC1-deficient aged mice, suggesting a role for mTORC1 in the priming of aged microglia. However, these transcriptome changes were not translated into protein; indeed, Csf1r-Cre; Rheb f/f mice showed reduced overall inflammation, as measured by sickness behaviour in the openfield test and by plasma cytokine levels. On the other hand, long-term treatment with rapamycin in vivo showed a very distinct phenotype, with reduced inflammation following LPS stimulation in young mice but no effect in aged ones. This PhD thesis sheds new light on pathways regulating microglia in ageing and has clinical implications for pathologies in which inflammation plays a major role.

The central thesis of this project is that damage caused by reactive nitrogen species, e.g. 3-nitrotyrosine (Tyr-NO2), constitutes a marker of disease progression/severity. A new sensitive electrochemiluminescence ELISA was optimised and validated for Tyr-NO2 measurement, giving a lower limit of quantification of 0.04 nM BSA-NO2, intra- and inter-assay CVs of 6.5% and 11.3%, an average recovery of 106 ± 3% and average linearity 0.998 ± 0.001. Nitrative stress, carbonyl stress and C-reactive protein (CRP) concentrations were measured before and after major elective surgery. CRP measurements confirmed the induction of an inflammatory response. Median serum Tyr-NO2 levels increased post-surgery to a median (inter-quartile range) value of 0.97 (0 – 1.7) fmol nitrated BSA (BSA-NO2) equivalents/mg protein compared with a pre-surgery level of 0.59 (0 – 1.3) fmol BSA-NO2 equivalents/mg protein (p<0.05). Oxidative damage was confirmed by serum protein carbonyl levels (p<0.05). In a second pre-/post- surgery study, patients who developed sepsis postoperatively had significantly higher serum Tyr-NO2 levels one day prior to diagnosis (median (IQR) 4.5 (1.65 – 8.21) fmol BSA-NO2 equivalents/mg protein) compared to patients without sepsis (1.2 (0.74 – 5.97) fmol BSA-NO2 equivalents/mg protein; p<0.05). Tyr-NO2 levels have not previously been measured before clinical diagnosis. However, Tyr-NO2 did not improve upon CRP as a diagnostic marker (area under the curve: Tyr-NO2 0.69 versus CRP 0.88). Nitrate (NO3¯) supplementation in healthy smokers was also studied. Plasma Tyr-NO2 levels were unaltered by supplementation or smoking status. Salivary nitration was unaffected by smoking and decreased with NO3¯ supplementation: the median (IQR) pre-supplementation was 0.67 (0.31-1.14) and post-supplementation was 0.43 (0.12-0.61) pmol BSA-NO2 equivalents/mg protein. Ozone-based chemiluminescence was utilised for nitrite (NO2¯) and NO3¯ measurement as indicators of ˙NO production. Plasma and salivary NO2¯ and NO3¯ concentrations increased significantly with NO3¯ supplementation (p<0.05). In contrast to published studies, brain frontal lobe Tyr-NO2 levels were not higher in dementia: the median (IQR) levels in dementia were 0.29 (0.19-0.57) and in non-dementia controls were 0.3 (0.22-0.55) pmol BSA-NO2 equivalents/mg protein. However, the median brain tissue NO2¯ concentration was significantly higher in the Alzheimer’s disease group (p<0.05). Western blotting revealed that nitration was predominantly in a few select proteins, with TOF-MS/MS analysis suggesting haemoglobin is one of these proteins. Measurement of nitrative stress using ozone-based chemiluminescence and an electrochemiluminescence-based-ELISA overcomes earlier methodological flaws, such as low sensitivity. Detection of total Tyr-NO2 in different inflammatory states indicates that its measurement could have potential as a marker of disease, but measurement of nitration in specific proteins may be more informative than total Tyr-NO2.