Le projet Milieu Intérieur a pour but d'identifier les facteurs génétiques et environnementaux ayant un impact sur la variabilité immunitaire naturelle. Cette analyse multiparamétrique requière néanmoins d'utiliser des outils standardisés. Afin d'étudier la réponse immune induite, nous avons utilisé un système optimisé prêt à l'emploi de stimulation du sang et développé un protocole unique de quantification de l'ARN afin d'étudier la signature transcriptionnelle en réponse à des immuno-modulateurs. Testant l'hypothèse que la réponse à des composés complexes peut être définie par la signature ARN de cytokines clefs et utilisant une méthode statistique robuste, nous avons identifié 44 gènes capables d'optimiser la capture de la réponse à des stimulations plus complexes aidant à la réduction dimensionnelle pour la décomposition de réponses innées. Dans une seconde étude, l'analyse semi-automatisée par cytomètrie en flux des cellules du sang a été associée à l'analyse épidémiologique et génotypique pour les 1,000 donneurs inclus dans la cohorte. Nous avons observé que le tabac, l'âge, le genre et l'infection latente par le cytomégalovirus sont les facteurs impactant le plus la variabilité immunitaire. Cette étude a montré que les paramètres des cellules innées sont contrôlés par des facteurs génétiques alors que ceux des cellules adaptatives le sont plutôt par des expositions environnementales tout au long de la vie. Des outils interactifs incluant ces données de référence accompagnent ces études. Ces analyses montrent que nous avons développé des outils performants pour une étude intégrative du modèle humain constituant une approche innovante vers une médecine personnalisée. / The project Milieu Intérieur aims to study the genetic and environmental factors that can have a major impact on occurring immunological variance in healthy human population. This characterization requires the use of standardized immunophenotyping technologies for integrating diverse, complex datasets. With this goal in mind, we used an optimized suite of standardized whole-blood stimulation systems to study the human induced immune response in physiological condition and developed a unique standardized protocol to analyze the ARN signatures upon whole-blood stimulation to test the hypothesis that responses to complex stimuli can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, which captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. This provides new strategies for dimension reduction of large datasets and for deconvolution of innate immune responses, applicable for characterizing novel immunomodulatory molecules.In a second related study, we aimed to identify the environmental and genetic factors driving innate and adaptive immune cell parameters in homeostatic conditions. To do so, we combined semi-automated flow cytometric analysis of blood leukocytes and genome-wide DNA genotyping in the 1,000 healthy donors included in the collection. We show that smoking, age, gender and latent cytomegalovirus infection, are main drivers of human variation (i.e. numbers of Treg and MAIT cells). These results demonstrated that innate cell parameters are strongly controlled by genetic factors, whereas adaptive cells are driven by life-long environmental exposures. In addition, to help on the public data mining, we developed interactive R-Shiny application including healthy donor reference values for both studies.All together, these results indicate that we developed powerful tools for human system biology approaches to support personalized medecine.

It is now clear that human and animal exposure to estrogenic compound occurs through several sources. This include: i) naturally occurring endogenous estrogens, ii) exogenous or intentional estrogens for prophylactic (e.g. oral contraceptive) and therapeutic (e.g. as replacement therapy for ovulation in nulliparous women and in menopausal women, and in some men suffering from prostate cancer) purposes, iii) accidental via estrogenic chemical exposure (e.g. pesticides, industrial byproducts) and phytoestrogens (e.g. soybeans). It has long been recognized that estrogen, a female sex hormone, functions not only on the reproductive system, but also on various other systems including the immune system. Estrogens are thought to be of both physiologic and pathologic importance. Female in general, have better immune capabilities than males, a phenomenon attributed to the action of sex hormones on the immune system. There is also a female-gender bias in susceptibility to autoimmune diseases. Estrogens have been linked either directly or indirectly to the etiology and pathogenesis of various female-predominant autoimmune diseases. Estrogens have also been linked to the onset of cancer, and conditions where the immune system often malfunctions. Estrogen affects the functions of both B and T cells, possibly by regulating such factors as lymphokine gene expression and/or cellular death by apoptosis. However, the functioning of both B and T cells under the influence of long-term exposure to estrogen has not been fully understood. The primary aim of this thesis was to investigate the effect of long-term exposure to 17β-estradiol on lymphokine and bcl-2/fas (proto-oncogenes) mRNA expression. We evaluated the effects of estrogen on the expression of genes for lymphokines, which are essential for the immune response. It is hypothesized that estrogen may regulate the immune system by modifying the expression of lymphokine genes and/or genes that regulate apoptosis. The results demonstrated that long-term 17β-estradiol exposure reduced the viability of lymphocytes when compared to lymphocytes from placebo-treated mice. IL-2 and IFN-g mRNA was consistently higher in ConA-stimulated lymphocytes from estrogen-treated mice (P < 0.05). The mRNA for TGF-β₁ lymphokine was also increased but was not consistent at all time points of incubation. The expression of IL-4 mRNA was not noticeably affected by estrogen treatment of mice. Long-term exposure to 17β-estradiol appear to have some influence on the mRNA expression of proto-oncogenes fas and bcl-2 in splenic and thymic T lymphocytes. There was a trend of increased bcl-2 mRNA expression in estrogen-treated mice compared to placebo-treated mice, whereas the mRNA expression of fas gene appeared to be lower compared to controls. Overall, these findings suggest that 17β-estradiol may selectively influence lymphokine and proto-oncogene mRNA expression. These results suggest that the one mode of modulation of the immune response by 17β-estradiol may be through alterations in the lymphokine and proto-oncogene expression. Since estrogen-treatment markedly induces atrophy of the thymus and diminishes the cellularity of the lymphoid organs (e.g. Spleen), it became necessary to perform multiple assays on the same cells, particularly lymphokine and apoptosis gene expression. A secondary objective of this thesis was to investigate whether lymphocytes, which have undergone proliferation in Lympho-Pro™ assay (Alamar Blue assay), could be utilized for further analysis. In this regard, we found that a non-radioactive assay that utilizes Alamar Blue had significant advantages over the conventional ³H-thymidine incorporation assay. By using cells from estrogen and placebo-treated mice in the Alamar Blue assay, we found that this assay not only allowed determination of lymphocyte proliferation, but also the assessment of mRNA expression, cytogenetics, apoptosis and immunophenotyping of the same lymphocytes. / Master of Science

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Der Systemische Lupus Erythematodes (SLE) ist eine meist schwer verlaufende Autoimmunerkrankung, die jedes Organ betreffen kann. Trotz zahlreicher und intensiver Anstrengungen die Pathophysiologie des SLE aufzuklären, wird sie aktuell nur in ihren Grundzügen verstanden. Eine Vielzahl zellulärer und molekularer Auffälligkeiten wurden in verschiedenen Immunzellen von Patienten mit SLE beschrieben, wobei die gesteigerte Aktivierung von T und B Zellen ist ein Schlüsselmerkmal ist. Verschiedene Auffälligkeiten der T Zell Funktion wurden in den vergangenen Jahren berichtet, unter anderem die gesteigerte Expression und Aktivierung verschiedener Transkriptionsfaktoren, darunter cAMP Responsive Element Modulator (CREM)α und Signal Transducer and Activator of Transcription (Stat)3. Eine Rolle von CREMα bei der Entstehung von Effektor T Zell Phänotypen bei Patienten mit SLE wurde in Studien belegt. Die gesteigerte Expression von CREMα ist (zumindest teilweise) für die gesteigerte Expression von IL-17A und die reduzierte Expression von IL-2 verantwortlich, welche für die Pathogenese und die Entstehung von Gewebeschäden mitverantwortlich sind. Neben gut charakterisierten CD4+ Effektor T Zellen, spielen TCR+CD3+CD4-CD8-, sogenannte „doppelt negative“ (DN) T Zellen, eine Rolle in der Pathophysiologie des SLE. Die Zahl DN T Zellen ist im peripheren Blut von SLE Patienten gesteigert. DN T Zellen infiltrieren entzündete Gewebe, insbesondere die Nieren, wo sie IL-17A exprimieren und zu Gewebeschäden beitragen können. Da DN T Zellen durch den Verlust der Oberflächenexpression des CD8 Co-Rezeptors aus CD8+ T Lymphozyten hervorgehen können, stellte wir die Frage, ob CREMα an diesem Prozess beteiligt ist. In den vorliegenden Studien konnten wir zeigen, dass CREMα an hochkonservierte nichtkodierende Sequenzen des CD8 Gen Clusters bindet und den CD8B Promoter trans-reprimiert. CREMα stellt damit den ersten berichteten Transkriptionsfaktor dar, der zu trans-Repression von CD8 führt. Zudem co-rekrutiert CREMα die DNA Methyltransferase DNMT3a und die Histon Methyltransferase G9a an hochkonservierte nichtkodierende Elemente des CD8 Gen Clusters in CD8+ T Zellen. Hierdurch trägt CREMα zur Chromatinkondensation, folglich reduzierter CD8 Expression und letztendlich der Generierung von DN T Zellen bei. Da die Expression von CREMα sowohl in T Zellen von SLE Patienten als auch in T Zellen von MRL.lrp Mäusen gesteigert ist, könnten die beschriebenen Effekte auf die CD8 Expression eine Rolle für eine Reihe von Autoimmunerkrankungen spielen, die mit einer erhöhten Zahl von DN T Zellen einhergehen (z.B. Patienten mit Autoimmune Lymphoproliferative Syndrome; ALPS). Proteine der Stat Transkriptionsfaktor Familie spielen eine Rolle während der Differenzierung und Aktivierung von Effektor T Zellen. Speziell die Transkriptionsfaktoren Stat3 und Stat5 scheinen für das Gleichgewicht zwischen Th17 Effektor Phänotypen (Stat3) und regulatorischen T Zellen (Stat5) von Bedeutung zu sein. Stat3 spielt eine wichtige Rolle bei der Differenzierung von IL-17A produzierenden CD4+ Th17 Helferzellen, welche eine pathophysiologische Rolle beim SLE spielen. Durch die Aktivierung der Expression weiterer Zytokine (z.B. IL-6, IL-10, und IL-21) in verschiedenen T Lymphozytenpopulationen, sind die Effekte von Stat3 nicht auf die genannten T Helferzellpopulationen beschränkt. Interleukin-10 ist ein immunregulatorisches Zytokin, welches neben seinen anti-inflammatorischen Effekten auch zur Aktivierung von B Lymphozyten und Antikörperproduktion beiträgt. Eine mögliche Pathophysiologische Rolle von IL-10 beim SLE ergibt sich aus gesteigerten IL-10 Serumspiegeln in SLE Patienten und nicht zuletzt aus einer kleinen Kohorte von SLE Patienten, die klinische Besserung nach therapeutischer Blockade von IL-10 erfahren hatte. Wie IL17A, wird auch IL10 durch Transkriptionsfaktoren der Stat Familie kontrolliert. Da die Expression und Aktivierung von Stat3 in T Zellen von SLE Patienten gesteigert ist, untersuchten wir am Beispiel des IL10 Gens Effekte von fehlregulierter Stat Aktivierung. Wir konnten zeigen, dass Stat3 und Stat5 das IL10 Gen durch trans-Aktivierung und die Induktion von epigenetischen Remodeling durch die Co-Rekrutierung von p300 regulieren. Der transkriptionelle Co-Aktivator p300 besitzt Histon Azetyltransferase Aktivität und induziert die „Öffnung“ des IL10 Gens. In T Zellen von SLE Patienten ist die Rekrutierung von Stat3 durch reduzierte DNA Methylierung am proximalen Promoter und einem intronischen Enhancer (I-SRE) erleichtert. Zudem verdrängt Stat3 den Transkriptionsfaktor Stat5 von einem Bindungselement im 4. Intron (I-SRE) des IL10 Gens. Zusammen führen diese Ereignisse zu gesteigerter Expression von IL-10 in T Zellen von SLE Patienten. Da die Aktivierung von Stat3 zu gesteigerter Expression einer Reihe von Zytokinen beträgt und die Stat3 Aktivierung sowohl beim SLE als auch bei anderen Autoimmunerkrankungen gesteigert ist, könnten die beschriebenen Effekte nicht nur auf die Expression von IL-10 in T Zellen von SLE Patienten beschränkt sein. Unsere Beobachtungen unterstreichen das Potenzial fehregulierte Transkriptionsfaktoren, speziell CREMα und Stat3, als Biomarker und/oder therapeutische Ziele beim SLE zu nutzen. Es bleibt jedoch an dieser Stelle noch zu klären, ob CREMα und/oder Stat3 auch Chromatin Remodeling während der physiologischen Generierung von DN oder CD4+ T Helfer Zellen kontrollieren oder ob sie ausschließlich oder zumindest in gesteigertem Maße an der pathologischen Generierung von Effektor T Zellen bei Autoimmunerkrankungen beteiligt sein. Die translationale Bedeutung unserer Beobachtungen wird durch den neuerdings begonnenen Einsatz von JAK/Stat Inhibitoren in der Therapie verschiedener Autoimmunerkrankungen unterstrichen. / Systemic lupus erythematosus (SLE) is a severe autoimmune disease that can affect any organ of the human body. Despite intense efforts towards a better understanding, the pathophysiology of SLE remains largely unknown. A number of cellular and molecular anomalies have been reported in immune cells from patients with SLE, and increased activation of B and T lymphocytes are considered hallmarks of the disease. Several alterations to T cell function and phenotypes have been reported, including the increased expression of the transcription factors cAMP response element modulatorα (CREM α) and signal transducer and activator of transcription 3 (Stat3). A role of CREMα in the generation of effector T cells has been demonstrated. Enhanced expression of CREMα is (at least partially) responsible for increased expression of IL-17A and reduced expression of IL-2 from effector T cells in SLE patients; and altered cytokine expression contributes to the pathophysiology and tissue damage. In addition to well-characterized effector CD4+ T cells, TCR+CD3+CD4-CD8-, so-called “double negative” (DN) T cells, also play a role in the pathophysiology of SLE. Increased numbers of DN T cells in the peripheral blood of SLE patients invade inflamed tissues, including the kidneys, where they produce IL-17A and contribute to tissue damage. Double negative T cells can derive from CD8+ T cells through the down-regulation of CD8 co-receptor expression. Thus, we asked whether CREMα may be involved in this process. In the studies presented here, we demonstrate that CREMα recruits to highly conserved non-coding sequences of the CD8 gene cluster and trans-represses the CD8B promoter. Thus, CREMα is the first reported transcription factor that negatively regulates CD8 expression. Furthermore, CREMα co-recruits DNA methyltransferase (DNMT)3a and histone methyltransferase G9a to highly conserved regions within the CD8 cluster in CD8+ T cells. Through these interactions, CREMα induces chromatin condensation, reduced CD8 expression, and the generation of DN T cells. Since CREMα expression is greater in T cells from SLE patients and lupus-prone MRL.lpr mice, the reported effects may play a role in several autoimmune disorders that are characterized by increased numbers of DN T cells (such as autoimmune lymphoproliferative syndrome; ALPS). Stat family transcription factors play a role during the differentiation and activation of T cells. Particularly Stat3 and Stat5 appear to be of central importance to the balance between effector Th17 phenotypes (Stat3) and regulatory T cells (Stat5). Stat3 is involved in the generation of IL-17A producing CD4+ Th17 cells, which contribute to tissue damage. Through the induction of cytokines other than IL-17A (e.g. IL-6, IL-10, IL-21), effects of Stat3 are not limited to individual T helper cell populations. Interleukin-10 is an immune-regulatory cytokine. In addition to anti-inflammatory effects, IL-10 is involved in the activation of B lymphocytes and induces immunoglobulin production. Increased IL-10 serum levels in SLE patients and a cohort of SLE patients that clinically responded to therapeutic blockade of IL-10 suggest a pathophysiological role for IL-10 in the disease. As with IL17A, the IL10 gene is regulated by Stat transcription factors. Since expression and activation of Stat3 are increased in T cells from patients with SLE, we investigated effects of dysbalanced Stat activation on the IL10 gene. In the presented study, we demonstrate that Stat3 and Stat5 trans-activate IL10 and induce epigenetic remodeling through co-recruitment of p300. The transcriptional co-activator p300 functions as histone acetyltransferase and induced epigenetic “opening” of the IL10 gene. In T cells from SLE patients, recruitment of Stat3 is enhanced by reduced levels of DNA methylation of the proximal promoter and an intronic enhancer, harboring a Stat responsive element (I-SRE). Stat3 replaces the transcription factor Stat5 at I-SRE in a potentially competitive manner. Altogether, these effects result in increased expression of IL-10 in T cells from patients with SLE. Activation of Stat3 induces the expression of a number of cytokines. Since Stat3 activation is enhanced in several autoimmune/inflammatory disorders, including SLE, we concluded that Stat3-mediated effects on gene expression are most likely not limited to just IL-10 expression in SLE. The herewith reported observations suggest high potential for the application of dysregulated transcription factor networks, particularly CREMα and Stat3, as biomarkers and/or molecular targets for future therapeutic interventions in SLE. However, it remains to be investigated whether and to what extent CREMα and/or Stat3 are involved in chromatin remodeling during the physiological generation of DN and CD4+ T helper cell subsets, or whether they contribute exclusively to the generation of effector T cell phenotypes in SLE and other autoimmune/inflammatory disorders. The translational importance of our observations is underscored by the recently initiated application of JAK/Stat inhibitors in the treatment of autoimmune/inflammatory conditions.

Obesity accounts for $168 billion in annual medical expenses and increases the risk of cardiovascular disease, cancer, and type-2 diabetes, three diseases responsible for over 50% of deaths in the United States. It is well established that the pattern of adiposity is an important factor in the relationship with disease risk and that visceral adiposity, which favors hypertrophy (characterized by enlarged cells) is more dangerous than subcutaneous adiposity, which tends to be hyperplastic (characterized by an increase in cell number). Hypertrophy is associated with inflammation and insulin resistance, and hyperplasia (adipogenesis, i.e., the formation of new adipocytes), is associated with improved insulin sensitivity. Tumor necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory cytokine that activates a nuclear factor-kappa B (NFKB) intracellular pathway that is an important mediator of obesity-associated insulin resistance and increased risk of type-2 diabetes. Interestingly, obesity has been positively associated with both low vitamin D status and elevated levels of TNF-alpha. Our studies focused on examining the influence of the active vitamin D hormone, 1,25-dihydroxyvitamin D, and TNF-alpha on adipogenesis and inflammation in human primary adipocytes and determining whether the balance of these two factors influences the extent to which adipocytes accumulate lipid or express pro-inflammatory cytokines. We found no effect of 1,25-dihydroxyvitamin D on adipogenesis or pro-adipogenic gene expression despite a clear upregulation of a vitamin D responsive gene, 24-hydroxylase, in response to treatment with 1,25-dihydroxyvitamin D. TNF-alpha clearly inhibited adipogenesis and expression of PPAR-gamma and C/EBP-alpha and enhanced expression of the pro-inflammatory cytokines IL-6 and MCP-1, but not IL-8. There was a trend towards a dose-dependent downregulation of MCP-1 by 1,25-dihydroxyvitamin D in three individuals; however, this effect was not statistically significant. While we found no interaction between TNF-alpha and 1,25-dihydroxyvitamin D on adipogenesis, there is a potential anti-inflammatory action of 1,25-dihydroxyvitamin D in human primary adipocytes. Future studies into this potential are warranted in light of the growing obesity epidemic and the interest in finding nutritionally modifiable treatment or prevention strategies to mitigate the negative consequences of obesity.

Many human diseases, including cancer, share an inflammatory component but the molecular underpinnings remain incompletely understood. We report that physiological and pathological Dickkopf1 (DKK1) activity fuels inflammatory cytokine responses in cell models, mice and humans. DKK1 maintains the elevated inflammatory tone of cancer cells and is required for mounting cytokine responses following ligation of toll-like and cytokine receptors. DKK1- controlled inflammation derives from cell-autonomous mechanisms, which involve SOCS3- restricted, nuclear RelA (p65) activity. We translate these findings to humans by showing that genetic DKK1 variants are linked to elevated cytokine production across healthy populations. Finally, we find that genetic deletion of DKK1 but not pharmacological neutralization of soluble DKK1 ameliorates inflammation and disease trajectories in a mouse model of endotoxemia. Collectively, our study identifies a cell-autonomous function of DKK1 in the control of the inflammatory response, which is conserved between malignant and nonmalignant cells. Additional studies are required to mechanistically dissect cellular DKK1 trafficking and signaling pathways.

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