(En) Paneth cells are one of the major player in the maintenance of the homeostatic relationship between intestinal microbiota and the immune system. This function is largely achieved by their production of bactericidal enteric α-defensins (ED) and other antimicrobials. Disruption of Paneth cell functions is associated with severe human disorders such as Crohn's disease (CD) and Autoimmune Polyendocrinopathy- Candidiasis-Ectodermal Dystrophy (APECED). However, there is only a very limited information regarding the interactions and regulatory circuits operating between Paneth cells and intestinal immune system in either health or under pathological conditions. The previous study conducted in our laboratory described a new mechanism for the initiation and maintenance of Paneth cells targeted autoimmunity. The suggested model was that ED-specific T cells escape the selection in the thymus, infiltrate the intestine and diminish Paneth cell numbers through autoimmune destruction. This process also lead to the accumulation of inflammation- inducing bacteria, which were implied to exacerbate the inflammatory autoimmunity. Since this model of intestinal autoimmunity is of correlative nature, its intrinsic mechanism and functional relationships between immune system, Paneth cells and microbiota are largely...

La muqueuse intestinale puis le foie sont en contact récurrent avec la flore microbienne issue du tube digestif. L’activation des récepteurs reconnaissant des motifs moléculaires microbiens (PRR :Pattern Recognition Receptors) constitue l’élément initial de la réponse inflammatoire de l’immunité innée et oriente le type d’activation de l’immunité adaptative. La première étape entraînera l’expression de médiateurs inflammatoire (cytokines (IL-1, IL-6, TNFα), activation de la réponse de phase aiguë) et le recrutement des cellules effectrices de l’immunité innée (neutrophiles, puis monocytes). L’équilibre entre la tolérance microbienne et l’exacerbation inflammatoire afin d’éliminer l’agent microbien et menant à la destruction tissulaire permet d’atteindre la situation d’homéostasie.<p>Si l’inflammation se perpétue, par exemple en cas de défaut de clairance de l’agent microbien ou de trouble de l’intégrité de la barrière muqueuse, l’inflammation peut devenir chronique. L’IL-6 exerce alors un rôle central dans la transition de l’immunité innée à l’immunité adaptative, en modulant différentiellement l’expression de chimiokines et l’apoptose des cellules immunes menant au remplacement de l’infiltrat neutrophilique par un infiltrat lympho-monocytaire. Par ailleurs, ce climat cytokinique particulier est propice au développement de lignées spécifiques de lymphocytes tels que les lymphocytes T CD4+ sécrétant de l’IL-17 (Th17). Ceux-ci, surtout étudiés dans les défenses anti-bactériennes et fungiques, et dans les maladies autoimmunes, ont été incriminés dans les phénomènes de cytotoxicité et de renouvellement inflammatoire par l’induction d’expression de chimiokines.<p><p>Une fois la barrière intestinale franchie, le foie est le premier organe en contact avec la flore microbienne issue de l’intestin. Certains TLRs ont été démontrés impliqués dans la physiopathologie de la stéatohépatite et dans le processus de fibrose. Ce climat constant d’exposition antigénique est associé en cas de maladie chronique du foie à une exacerbation d’expression de médiateurs infammatoires (IL-1, IL-6, TNFα).<p>Nous avons étudié la modulation d’expression des différents TLRs au cours d’un modèle de maladie alcoolique du foie chez la souris. Cette étude démontrait qu’il existait une majoration d’expression des TLR1, 2, 4, 6, 7, 8 et 9 dépendante du stress oxydatif suite à l’exposition chronique du foie à l’alcool ;celle-ci entraînant davantage de lésions hépatiques lors de l’injection des ligands respectifs de ces différents TLRs.<p>Dans ce contexte, nous avons également étudié l’expression des différentes sous-unités du récepteur à l’IL-6 au cours de deux maladies chroniques du foie chez l’homme :les maladies alcooliques du foie et l’hépatite C chronique. Nous avons mis en évidence que les taux plasmatiques d’IL-6 et de la forme soluble de gp130 augmentaient au cours des maladies chroniques du foie, de manière corrélée à la sévérité. Nous avons également démontré l’effet inhibiteur de sgp130 sur la réponse de phase aiguë dépendante du trans-signaling de l’IL-6 in vitro. Ces données suggèrent que sgp130 contribue au déficit de réponse de phase aiguë observé chez les patients atteints de cirrhose. <p><p>Par ailleurs, vu le contexte « cytokinique » chronique des maladies alcooliques du foie (IL-1 et IL-6), nous avons étudié l’activation de la voie de l’interleukine-17 et des Th17 au cours des maladies alcooliques du foie chez l’homme. Nous avons mis en évidence qu’il existait une activation de cellules circulantes sécrétant de l’IL-17 (comprenant des Th17) au cours de la cirrhose alcoolique stable. Par contre, au sein du foie, l’activation de cellules sécrétant de l’IL-17 était davantage augmentée lors de l’hépatite alcoolique. Nous avons également mis en évidence que les cellules stellées (cellules responsables de la fibrose hépatique) stimulées à l’IL-17 recrutaient les neutrophiles suite à l’expression d’IL-8 et de GROα. Cette nouvelle voie inflammatoire démontrée lors d’une maladie du foie chez l’homme met en évidence l’activation de la voie de l’IL-17 au cours des maladies alcooliques du foie et sa contribution potentielle au recrutement hépatique de neutrophiles au cours de l’hépatite alcoolique aiguë. Cette voie sera explorée dans l’avenir en termes de fonctionnalité et de potentielle cible thérapeutique. <p> <p>En conclusion, tout comme le suggère la littérature pour les maladies autoimmunes (maladie de Crohn, arthrite, sclérose en plaque), il semble que les maladies alcooliques du foie partagent avec ces dernières diverses caractéristiques inflammatoires. La flore microbienne intestinale participe à la physiopathologie des lésions hépatiques, de même que l’activation de PRR (TLR). Par ailleurs, un climat inflammatoire chronique (IL-6,…), contre-régulé par certains mécanismes (sgp130), est associé à la présence périphérique et hépatique de lymphocytes Th17. Cette dernière découverte ouvre de nouvelles perspectives dans la compréhension de la physiopathologie des maladies alcooliques du foie, et peut-être de nouvelles cibles thérapeutiques concernant l’hépatite alcoolique aiguë.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

T lymphocytes are distinguished by the expression of αβ TCR or γδ TCR on their cell surface. The kinetic differences in the effector functions classifies γδ T cells as innate-like lymphocytes and αβ T cells as adaptive lymphocytes. Although distinct, αβ and γδ T cell lineages produce a common array of cytokines to mount an effective immune response against a pathogen. The production of cytokine IL-17 is a shared characteristic between the γδ T (Tγδ17) cells and the CD4 T (Th17) cells. γδ T cells develop into Tγδ17 cells in the thymus whereas CD4 T cells differentiate into Th17 cells in response to antigens in the peripheral lymphoid tissues. γδ T cells exported from the thymus, as pre-made effectors, are the early IL-17 producers compared with the late IL-17 producing Th17 cells. In this thesis we describe how TGFβ-SMAD2 dependent pathway selectively regulates Th17 cell differentiation but not Tγδ17 cells generation. We further illustrate the requirement of WNT-HMG box transcription factor (TF) signaling for the thymic programming of Tγδ17 cells. Cytokine TGFβ in co-operation with IL-6 induces the differentiation of Th17 cells. Conversely, TGFβ signaling also regulates the differentiation and maintenance of CD4+FOXP3+ regulatory T cells. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. While mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-/- T cells are impaired in their response to TGFβ in vitro and in vivo and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is essential for TGFβ signaling in CD4+ T effector cell differentiation and that it possesses functional capabilities distinct from SMAD3. Although SMAD2 is essential for the differentiation of Th17 cells, TGFβ signaling via SMAD2 is not required for the thymic programming of innate Tγδ17 cells. Among different γδ T cells, Vγ2+ (V2) γδ T cells are the major IL-17 producing subsets. We demonstrate that Sry-high mobility group (HMG) box TFs regulate the development of V2 Tγδ17 cells. We show that the HMG box TF, SOX13 functions in a positive loop for the intrathymic generation of V2 Tγδ17 cells. SOX13 regulates the programming of Tγδ17 cells by controlling the expression of B-lymphoid kinase (BLK) in developing immature V2 γδ T cells. BLK is an Src-family kinase expressed by all Tγδ17 cells. Furthermore, we show another HMG box TF, TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of IL-17 production by all γδ T cells. We propose that the antagonism of SOX13 and TCF1 determines the generation of IL-17 producing γδ T cells. We also show that extrinsic cues from αβ T cells do not affect the generation of IL-17 producing γδ T cells. Using OP9-DL1 culture system, we demonstrate that the progenitors of V2 Tγδ17 cells are the c-Kit+ early thymic precursors.

Dendritische Zellen (DZ) sind essentiell für die Aktivierung von Immunantworten. Drei Flt3-abhängige DZ Populationen aus dem Blut bestehend aus konventionellen (kDZ) BDCA1+ DZs und BDCA3+ DZs und plasmazytoide DZs wurden bereits beschrieben. Hier wurden zum ersten Mal sich aus Monozyten entwickelnde DZ (moDZ), genauer BDCA1+CD14+ pro-inflammatorische DZ (pro-iDZ) aus periphärem Blut unter homöostatischen Bedingungen identifiziert. Isolierte pro-iDZ sekretierten spontan große Mengen an pro-inflammatorischen Zytokinen, die kDZ reifen ließen und T Zell Proliferation unterstützten. Sie waren BDCA1+CD14- DZ und CD14+CD16- Monozyten in der TH17 Zell Induktion überlegen. Pro-iDZ ähnliche BDCA1+CD14+ Zellen konnten durch imaging cycler microscopy in Geweben von Patienten die an Psoriasis, Dermatomyositis oder entzündetem Halonävus erkrankt waren identifiziert werden. Ihr Fehlen in gesunder Haut deutete eine Rekrutierung von pro-iDZs in entzündetes Gewebe an. Eine Verwandtschaftsanalyse von pro-iDZ zwischen Monozyten, kDZ des Blutes und in vitro generierten moDZ auf genomweiter Ebene wies auf einen monozytären Ursprung hin. Anylse mittels funktioneller Annotation auf differentiell exprimierten Genen zwischen pro-iDZ und Monozyten zeigte eine DZ spezifische Gensignatur auf. Diese Gene waren insgesamt in der gleichen Weise wie in kDZ und moDZ reguliert, das eine Entwicklung von Monozyten nach DZ nahelegte. Dieses Entwicklungskonzept wurde insofern unterstützt, indem unter entzündlichen Bedingungen kultivierte CD14+CD16- Monozyten BDCA1 Expression und DZ Funktion erlangten. Da pro-iDZ sehr ähnlich zu BDCA1+CD14+ Zellen aus entzündeter Haut waren und beide große Konvergenz mit zuvor beschriebenen BDCA1+CD14+ inflammatorischen DZ (infDZ) aus entzündeten Geweben aufwiesen, können pro-iDZ als direkte infDZ Vorläufer angesehen werden. Dadurch und wegen ihrer monozytären Herkunft können pro-iDZ als Beweis für die humane Differenzierung von Monozyten nach DZ in vivo betrachtet werden. / Dendritic cells (DCs) are critical for the activation of immune responses. Three Flt3-dependent blood DC populations including conventional BDCA1+ DCs and BDCA3+ DCs (cDCs) and plasmacytoid DCs were described previously. This work identifies for the first time human peripheral blood monocyte derived BDCA1+CD14+ pro-inflammatory DCs (pro-iDCs) during steady state. Isolated pro-iDCs spontaneously secreted high amounts of pro-inflammatory cytokines, which matured cDCs and promoted T cell proliferation. They were superior in priming TH17 cells when compared to BDCA1+CD14- DCs and CD14+CD16- monocytes. BDCA1+CD14+ cells resembling blood pro-iDCs as identified by imaging cycler microscopy were found in samples from patients suffering from psoriasis, dermatomyositosis and inflamed halo nevus. Their absence in healthy donor’s skin indicated a recruitment of pro-iDCs to sites of inflammation. Analysis of the developmental relationship of pro-iDCs between monocytes, blood cDCs and in vitro generated monocyte derived DCs (moDCs) on whole genome level strongly suggested a monocytic origin. Functional annotation analysis of differentially regulated genes between monocytes and pro-iDCs revealed a DC specific gene signature. In addition, these genes were overall regulated in the same way in blood cDCs and moDCs, indicating an ongoing development of pro-iDCs from monocytes towards DCs. This developmental concept was supported as CD14+CD16- monocytes cultured under inflammatory conditions gained BDCA1 expression and DC function. Since pro-iDCs were highly similar to BDCA1+CD14+ cells found in inflamed skin and as both showed a marked convergence with BDCA1+CD14+ inflammatory DCs (infDCs) present in inflamed tissues described previously, pro-iDCs can be regarded as immediate precursors of infDCs. Thus, in respect of a monocytic origin and a presumably inflammatory DC fate, pro-iDCs may constitute a missing link to prove human moDC differentiation in vivo.

Abstract Glucocorticoids are the most important and widely used treatment modality in dermatology. A large variety of topical as well as systemic preparations is available. Most patients treated with glucocorticoids respond quickly to the treatment, but some are considered insensitive or even resistant to glucocorticoid therapy. Currently, there is no known measurable variable, through which the response can be predicted. Glucocorticoids mediate their actions through glucocorticoid receptors (GR). Several isoforms of GR exist, but the α (GRα) and β (GRβ) isoforms are clinically the most important. Based on previous studies, it has been proposed that the abundance of GR isoforms or the GRβ: GRα –ratio could affect individual responsiveness to corticosteroid treatment. In particular, up-regulation of GRβ expression has been shown to be linked to resistance to corticosteroid treatment. This thesis comprises three sub-studies. Firstly, we wanted to determine whether GRα and GRβ are expressed in inflammatory skin diseases. Secondly, we examined if the expression is altered by corticosteroid treatment in eczema atopicum, bullous pemphigoid and psoriasis. Finally, we measured the effects of a topical vitamin D3 analogue (calcipotriol) combined with betamethasone compared with betamethasone monotherapy on inflammatory biomarkers of psoriasis. Our studies provide detailed novel data about the expression of GRα and GRβ. GRα and GRβ were shown to be expressed in the blood lymphocytes and lesional skin of patients with eczema atopicum, bullous pemphigoid and psoriasis, as well as in the skin of patients with eczema nummulare, lichen simplex chronicus and lichen ruber planus. Systemic corticosteroid treatment was shown to affect the expression of GRα and GRβ in eczema atopicum and bullous pemphigoid, but the inconsistent variation in their expression between patients prevented us from drawing firm conclusions. Neither GRα nor GRβ as a single marker were found to be a suitable predictor of corticosteroid responsiveness. Clinical and laboratory analyses showed that topical treatment of psoriasis with calcipotriol/betamethasone combination ointment is more beneficial measured by both than betamethasone monotherapy. / Tiivistelmä Glukokortikoideja (”kortisoni”) käytetään tulehduksellisten ihotautien hoidossa paikallisesti tai systeemisenä lääkkeenä. Suurin osa potilaista reagoi hoitoon nopeasti, mutta osalla hoitovaste on heikompi tai ilmenee hitaasti. Tällä hetkellä ei tunneta keinoja ennustaa luotettavasti kortisonihoidon vastetta. Glukokortikoidit vaikuttavat elimistössä glukokortikoidireseptorien (GR) kautta. Glukokortikoidireseptorista tunnetaan useita alatyyppejä, joista tärkeimmät ovat α (GRα) ja β (GRβ). Aiemman tiedon pohjalta on pidetty mahdollisena, että GR-alatyyppien suhteella tai määrällä on merkitystä kortisonivasteen syntymisessä. Erityisesti on arveltu, että ylimäärä GRβ:aa voisi estää kortisonihoidon vaikutusta. Tässä väitöskirjassa tavoitteena on ollut selvittää, tapahtuuko GR-alatyyppien ilmenemisessä muutoksia tulehduksellisia ihosairauksia sairastavilla potilailla sekä tutkia, miten kortisonihoito vaikuttaa GR-tasoihin atooppista ihottumaa, pemfigoidia ja psoriaasia sairastavilla potilailla. Lisäksi olemme verranneet paikallishoitoa pelkällä kortisonivoiteella D-vitamiinijohdos kalsipotriolin ja kortisonin yhdistelmähoitoon psoriaatikoilla. Tutkimus on antanut uutta yksityiskohtaista tietoa GRα:n ja GRβ:n esiintymisestä ihossa ja tulehdussoluissa ihosairauksia sairastavilla potilailla. Tutkimustulosten perusteella voidaan todeta, että GRα ja GRβ esiintyvät atooppista ihottumaa, pemfigoidia ja psoriaasia sairastavien potilaiden ihossa ja veren tulehdussoluissa sekä nummulaari-ihottumaa, neurodermatiittia ja punajäkälää sairastavien potilaiden ihossa. Suun kautta annettu kortisonihoito vaikuttaa GRα- ja GRβ–lähetti-RNA:n ilmenemiseen, mutta potilaskohtaiset erot ovat suuret, eikä kumpikaan, GRα tai GRβ, sovellu yksinään ennustamaan kortisonihoidon vastetta. Paikallisella kortisonihoidolla D-vitamiinijohdos kalsipotrioliin yhdistettynä on suotuisampi vaikutus psoriaasin tulehduksellisiin välittäjäaineisiin ja tulehdussoluihin kuin pelkällä paikallisella kortisonihoidolla.

Indiana University-Purdue University Indianapolis (IUPUI) / IL-17-producing T cells are critical to the development of pathogen and tumor immunity, but also contribute to the pathology of autoimmune diseases and allergic inflammation. CD8+ (Tc17) and CD4+ (Th17) IL-17-secreting T cells develop in response to a cytokine environment that activates Signal Transducer and Activator of Transcription (STAT) proteins, though the mechanisms underlying Tc17/Th17 development and stability are still unclear. In vivo, Tc17 cells clear vaccinia virus infection and acquire cytotoxic potential, that is independent of IL-17 production and the acquisition of IFN-γ-secreting potential, but partially dependent on Fas ligand, suggesting that Tc17-mediated vaccinia virus clearance is through cell killing independent of an acquired Tc1 phenotype. In contrast, memory Th cells and NKT cells display STAT4-dependent IL-23-induced IL-17 production that correlates with Il23r expression. IL-23 does not activate STAT4 nor do other STAT4-activating cytokines induce Il23r expression in these populations, suggesting a T cell-extrinsic role for STAT4 in mediating IL-23 responsiveness. Although IL-23 is important for the maintenance of IL-17-secreting T cells, it also promotes their instability, often resulting in a pathogenic Th1-like phenotype in vitro and in vivo. In vitro-derived Th17 cells are also flexible when cultured under polarizing conditions that promote Th2 or Th9 differentiation, adopting the respective effector programs, and decreasing IL-17 production. However, in models of allergic airway disease, Th17 cells do not secrete alternative cytokines nor adopt other effector programs, and remain stable IL-17-secretors. In contrast to Th1-biased pro-inflammatory environments that induce Th17 instability in vivo, during allergic inflammatory disease, Th17 cells are comparatively stable, and retain the potential to produce IL-17. Together these data document that the inflammatory environment has distinct effects on the stability of IL-17-secreting T cells in vivo.