Physiologische Konsequenzen eines gestörten Ceramid Stoffwechsels unter Beteiligung von Ceramidsynthase 3 und saurer Ceramidase = Physiological consequences of a disturbed ceramide metabolism due to altered activities of ceramide synthase 3 and acidic ceramidase

Tiefes Wissen über den Ceramid Stoffwechsel ist rudimentär für das Verständnis der Haut-Pathophysiologie (z.B. für atopische Dermatitis oder Psoriasis ) und unabdingbar für gezielte Therapieansätze. Wenn die zwei wichtigen Barriere Funktionen, gegen transepidermalen Wasserverlust und Pathogene Invasionen undicht werden, sind bestimmte Barriere Komponenten wie z.B. Ceramide stark verändert. In Haut und Hoden führt die Deletion der Ceramid-Synthase 3 zu einem Arrest der epidermalen Reifung und der Spermatogenese, welches ihre Bedeutung für eine intakte Barriere heraushebt. Sphingosin (So), ein Abbauprodukt von Cer, wurde als antimikrobielles Mittel identifiziert. So konnte das Wachstum von Candida albicans hemmen und die Invasion von Pathogenen in tiefere Hautschichten verringern, wodurch ihre mögliche Rolle in der Therapie von Hauterkrankungen gezeigt wurde. Auch eine neue Klasse von Ceramiden, die 1-O-acylceramide, wurde entdeckt. 1-O-acylceramide könnten zu einer funktionellen Wasserdurchlässigkeit Barriere beitragen, da sie zu den hydrophobesten der epidermalen Cers gehören. Die neutrale Glucosylceramidase scheint topologisch mit der 1-Oacylceramid Produktion verbunden zu sein, sowie die Enzyme der Diacylglycerol O-Acyltransferase-2 (DGAT2) Familie eine Rolle dabei spielen könnten. Die Identifizierung der für die 1-O-acylceramid Synthese verantwortlichen Enzyme wir Gegenstand weiterer Forschung sein, jedoch zeigten Untersuchungen an Mäusen, defizient für die saure Ceramidase (Farber-Krankheit), dass Makrophagen ein weiterer potenzieller Produktionsort sein könnten.

Review: leucocyte-endothelial cell crosstalk at the blood-brain barrier: a prerequisite for successful immune cell entry to the brain

Leucocyte migration into the central nervous system is a key stage in the development of multiple sclerosis. While much has been learnt regarding the sequential steps of leucocyte capture, adhesion and migration across the vasculature, the molecular basis of leucocyte extravasation is only just being unravelled. It is now recognized that bidirectional crosstalk between the immune cell and endothelium is an essential element in mediating diapedesis during both normal immune surveillance and under inflammatory conditions. The induction of various signalling networks, through engagement of cell surface molecules such as integrins on the leucocyte and immunoglobulin superfamily cell adhesion molecules on the endothelial cell, play a major role in determining the pattern and route of leucocyte emigration. In this review we discuss the extent of our knowledge regarding leucocyte migration across the blood-brain barrier and in particular the endothelial cell signalling pathways contributing to this process.

Immune cell migration across the blood–brain barrier: molecular mechanisms and therapeutic targeting

The endothelial blood–brain barrier (BBB) and the epithelial blood–cerebrospinal fluid barrier protect the CNS from the constantly changing milieu within the bloodstream. The BBB strictly controls immune cell entry into the CNS, which is rare under physiological conditions. During a variety of pathological conditions of the CNS, such as viral or bacterial infections, or during inflammatory diseases, such as multiple sclerosis, immunocompetent cells readily traverse the BBB and subsequently enter the CNS parenchyma. Most of the available information on immune cell entry into the CNS is derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Consequently, our current knowledge on traffic signals mediating immune cell entry across the BBB during immunosurveillance and disease results mainly from experimental data in the EAE model. Therefore, a large part of this review summarizes these findings. Similarly, the potential benefits and risks associated with therapeutic targeting of immune cell trafficking across the BBB will be discussed in the context of multiple sclerosis, since elucidation of the molecular mechanisms relevant to this disease have largely relied on the use of its in vivo model, EAE.

The mucosal immune system at the gastrointestinal barrier

The immune system faces a considerable challenge in its efforts to maintain tissue homeostasis in the intestinal mucosa. It is constantly confronted with a large array of antigens, and has to prevent the dissemination and proliferation of potentially harmful agents while sparing the vital structures of the intestine from immune-mediated destruction. Complex interactions between the highly adapted effector cells and mechanisms of the innate and adaptive immune system generally prevent the luminal microflora from penetrating the intestinal mucosa and from spreading systemically. Non-haematopoietic cells critically contribute to the maintenance of local tissue homeostasis in an antigen-rich environment by producing protective factors (e.g. production of mucus by goblet cells, or secretion of microbicidal defensins by Paneth cells) and also through interactions with the adaptive and innate immune system (such as the production of chemotactic factors that lead to the selective recruitment of immune cell subsets). The complexity of the regulatory mechanisms that control the local immune response to luminal antigens is also reflected in the observation that mutations in immunologically relevant genes often lead to the development of uncontrolled inflammatory reactions in the microbially colonized intestine of experimental animals.

Effect of intramammary administration of prednisolone on the blood-milk barrier during the immune response of the mammary gland to lipopolysaccharide

OBJECTIVE: To investigate effects of intramammary administration of prednisolone on the immune response of mammary glands in cows. ANIMALS: 5 lactating Red Holsteins. PROCEDURES: Cows received a different intramammary infusion in each mammary gland (10 mg of prednisolone, 100 μg of lipopolysaccharide [LPS], 100 μg of LPS and 10 mg of prednisolone, or saline [0.9% NaCl] solution). Milk samples were collected before (time 0) and 3, 6, 9, 12, 24, and 36 hours after treatment. Somatic cell count (SCC), lactate dehydrogenase (LDH) activity, and concentrations of serum albumin (SA) and tumor necrosis factor (TNF)-α in milk and mRNA expression of TNF-α, interleukin (IL)-8, and IL-1β in milk somatic cells were analyzed. RESULTS: Saline solution or prednisolone did not change SCC, LDH activity, and SA and TNF-α concentrations in milk and mRNA expression of TNF-α, IL-1β, and IL-8 in milk somatic cells. The SCC and TNF-α concentration in milk increased similarly in glands infused with LPS, independent of prednisolone administration. However, the increase of LDH activity and SA concentration in milk after LPS infusion was diminished by prednisolone administration. The mRNA expression of TNF-α, IL-8, and IL-1β in milk somatic cells increased after LPS infusion and was unaffected by prednisolone. CONCLUSIONS AND CLINICAL RELEVANCE: Intramammary administration of prednisolone did not induce an immune response and did not change mRNA expression of TNF-α, IL-8, and L-1β during the response to intramammary administration of LPS. However, prednisolone reduced disruption of the blood-milk barrier. This could influence the severity and cure rate of mastitis.

Modeling immune functions of the mouse blood-cerebrospinal fluid barrier in vitro: primary rather than immortalized mouse choroid plexus epithelial cells are suited to study immune cell migration across this brain barrier.

BACKGROUND The blood-cerebrospinal fluid barrier (BCSFB) established by the choroid plexus (CP) epithelium has been recognized as a potential entry site of immune cells into the central nervous system during immunosurveillance and neuroinflammation. The location of the choroid plexus impedes in vivo analysis of immune cell trafficking across the BCSFB. Thus, research on cellular and molecular mechanisms of immune cell migration across the BCSFB is largely limited to in vitro models. In addition to forming contact-inhibited epithelial monolayers that express adhesion molecules, the optimal in vitro model must establish a tight permeability barrier as this influences immune cell diapedesis. METHODS We compared cell line models of the mouse BCSFB derived from the Immortomouse(®) and the ECPC4 line to primary mouse choroid plexus epithelial cell (pmCPEC) cultures for their ability to establish differentiated and tight in vitro models of the BCSFB. RESULTS We found that inducible cell line models established from the Immortomouse(®) or the ECPC4 tumor cell line did not express characteristic epithelial proteins such as cytokeratin and E-cadherin and failed to reproducibly establish contact-inhibited epithelial monolayers that formed a tight permeability barrier. In contrast, cultures of highly-purified pmCPECs expressed cytokeratin and displayed mature BCSFB characteristic junctional complexes as visualized by the junctional localization of E-cadherin, β-catenin and claudins-1, -2, -3 and -11. pmCPECs formed a tight barrier with low permeability and high electrical resistance. When grown in inverted filter cultures, pmCPECs were suitable to study T cell migration from the basolateral to the apical side of the BCSFB, thus correctly modelling in vivo migration of immune cells from the blood to the CSF. CONCLUSIONS Our study excludes inducible and tumor cell line mouse models as suitable to study immune functions of the BCSFB in vitro. Rather, we introduce here an in vitro inverted filter model of the primary mouse BCSFB suited to study the cellular and molecular mechanisms mediating immune cell migration across the BCSFB during immunosurveillance and neuroinflammation.

Innate and adaptive immunity at mucosal surfaces of the female reproductive tract : stratification and integration of immune protection against the transmission of sexually transmitted infections

This review examines the multiple levels of pre-existing immunity in the upper and lower female reproductive tract. In addition, we highlight the need for further research of innate and adaptive immune protection of mucosal surfaces in the female reproductive tract. Innate mechanisms include the mucus lining, a tight epithelial barrier and the secretion of antimicrobial peptides and cytokines by epithelial and innate immune cells. Stimulation of the innate immune system also serves to bridge the adaptive arm resulting in the generation of pathogen-specific humoral and cell-mediated immunity. Less understood are the multiple components that act in a coordinated way to provide a network of ongoing protection. Innate and adaptive immunity in the human female reproductive tract are influenced by the stage of menstrual cycle and are directly regulated by the sex steroid hormones, progesterone and estradiol. Furthermore, the effect of hormones on immunity is mediated both directly on immune and epithelial cells and indirectly by stimulating growth factor secretion from stromal cells. The goal of this review is to focus on the diverse aspects of the innate and adaptive immune systems that contribute to a unique network of protection throughout the female reproductive tract.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

The Innate Immune System in Acute and Chronic Wounds.

Significance: This review article provides an overview of the critical roles of the innate immune system to wound healing. It explores aspects of dysregulation of individual innate immune elements known to compromise wound repair and promote nonhealing wounds. Understanding the key mechanisms whereby wound healing fails will provide seed concepts for the development of new therapeutic approaches. Recent Advances: Our understanding of the complex interactions of the innate immune system in wound healing has significantly improved, particularly in our understanding of the role of antimicrobials and peptides and the nature of the switch from inflammatory to reparative processes. This takes place against an emerging understanding of the relationship between human cells and commensal bacteria in the skin. Critical Issues: It is well established and accepted that early local inflammatory mediators in the wound bed function as an immunological vehicle to facilitate immune cell infiltration and microbial clearance upon injury to the skin barrier. Both impaired and excessive innate immune responses can promote nonhealing wounds. It appears that the switch from the inflammatory to the proliferative phase is tightly regulated and mediated, at least in part, by a change in macrophages. Defining the factors that initiate the switch in such macrophage phenotypes and functions is the subject of multiple investigations. Future Directions: The review highlights processes that may be useful targets for further investigation, particularly the switch from M1 to M2 macrophages that appears to be critical as dysregulation of this switch occurs during defective wound healing.

Langerin expressing cells promote skin immune responses under defined conditions.

There are conflicting data in the literature regarding the role of epidermal Langerhans cells (LC) in promoting skin immune responses. On one hand, LC can be extremely potent APCs in vitro, and are thought to be involved in contact hypersensitivity (CHS). On the other hand, it seems counterintuitive that a cell type continually exposed to pathogens at the organism\'s barrier surfaces should readily trigger potent T cell responses. Indeed, LC depletion in one model led to enhanced contact hypersensitivity, suggesting they play a negative regulatory role. However, apparently similar LC depletion models did not show enhanced CHS, and in one case showed reduced CHS. In this study we found that acute depletion of mouse LC reduced CHS, but the timing of toxin administration was critical: toxin administration 3 days before priming did not impair CHS, whereas toxin administration 1 day before priming did. We also show that LC elimination reduced the T cell response to epicutaneous immunization with OVA protein Ag. However, this reduction was only observed when OVA was applied on the flank skin, and not on the ear. Additionally, peptide immunization was not blocked by depletion, regardless of the site. Finally we show that conditions which eliminate epidermal LC but spare other Langerin(+) DC do not impair the epicutaneous immunization response to OVA. Overall, our results reconcile previous conflicting data in the literature, and suggest that Langerin(+) cells do promote T cell responses to skin Ags, but only under defined conditions.

Bacterial peptidoglycan biosynthesis and recognition by the innate immune system

Dissertation presented to obtain the Ph.D degree in Biology

The importance of the Hedgehog signaling pathway at the level of the blood-brain barrier

La barrière hémato-encéphalique (BHE) protège le système nerveux central (SNC) en contrôlant le passage des substances sanguines et des cellules immunitaires. La BHE est formée de cellules endothéliales liées ensemble par des jonctions serrées et ses fonctions sont maintenues par des astrocytes, celles ci sécrétant un nombre de facteurs essentiels. Une analyse protéomique de radeaux lipidiques de cellules endothéliales de la BHE humaine a identifié la présence de la voie de signalisation Hedgehog (Hh), une voie souvent liées à des processus de développement embryologique ainsi qu’au niveau des tissus adultes. Suite à nos expériences, j’ai déterminé que les astrocytes produisent et secrètent le ligand Sonic Hh (Shh) et que les cellules endothéliales humaines en cultures primaires expriment le récepteur Patched (Ptch)-1, le co-récepteur Smoothened (Smo) et le facteur de transcription Gli-1. De plus, l’activation de la voie Hh augmente l’étanchéité des cellules endothéliales de la BHE in vitro. Le blocage de l’activation de la voie Hh en utilisant l’antagoniste cyclopamine ainsi qu’en utilisant des souris Shh déficientes (-/-) diminue l’expression des protéines de jonctions serrées, claudin-5, occcludin, et ZO-1. La voie de signalisation s’est aussi montrée comme étant immunomodulatoire, puisque l’activation de la voie dans les cellules endothéliales de la BHE diminue l’expression de surface des molécules d’adhésion ICAM-1 et VCAM-1, ainsi que la sécrétion des chimiokines pro-inflammatoires IL-8/CXCL8 et MCP-1/CCL2, créant une diminution de la migration des lymphocytes CD4+ à travers une monocouche de cellules endothéliales de la BHE. Des traitements avec des cytokines pro-inflammatoires TNF-α and IFN-γ in vitro, augmente la production de Shh par les astrocytes ainsi que l’expression de surface de Ptch-1 et de Smo. Dans des lésions actives de la sclérose en plaques (SEP), où la BHE est plus perméable, les astrocytes hypertrophiques augmentent leur expression de Shh. Par contre, les cellules endothéliales de la BHE n’augmentent pas leur expression de Ptch-1 ou Smo, suggérant une dysfonction dans la voie de signalisation Hh. Ces résultats montrent que la voie de signalisation Hh promeut les propriétés de la BHE, et qu’un environnement d’inflammation pourrait potentiellement dérégler la BHE en affectant la voie de signalisation Hh des cellules endothéliales.

Caractérisation neuro-immunitaire d'un modèle d'encéphalomyélite auto-immune expérimentale spontanée

La sclérose en plaques est une maladie neuroinflammatoire idiopathique caractérisée par la formation de lésions focales de démyélinisation, qui apparaissent suite à l’infiltration périvasculaire de cellules immunitaires et à l’augmentation de la perméabilité de la barrière hémato-encéphalique. L’encéphalomyélite auto-immune expérimentale (EAE) est le modèle animal de cette maladie. Cependant, ce modèle présente des différences importantes avec la sclérose en plaques. L’objectif de ce projet de maîtrise était d’approfondir la caractérisation d’un nouveau modèle transgénique d’encéphalomyélite auto-immune expérimentale spontanée, le modèle TCR1640, afin de valider celui-ci pour l’étude des phénomènes physiopathologiques qui surviennent à différents stades de la sclérose en plaques, ainsi que pour le développement de nouveaux traitements de la maladie. La souris TCR1640 porte un récepteur des cellules T (TCR) transgénique autoréactif, qui reconnaît un peptide de la myéline et déclenche une réaction auto-immune contre la myéline endogène au sein du système nerveux central (SNC). Des observations faites in situ et in vitro ont permis d’identifier des changements qui surviennent de façon très précoce dans l’unité neurovasculaire chez les animaux TCR1640 présymptomatiques, et qui sont liés à la présence d’un profil immunitaire périphérique proinflammatoire. Lors des phases actives de l’EAE spontanée, les animaux TCR1640 au stade chronique présentent une inflammation accrue du système nerveux central associée à une infiltration leucocytaire massive, par rapport aux animaux au stade aigu de la maladie. Une étude in vivo a également permis de moduler la maladie développée par des animaux ayant subi une immunisation passive avec des cellules T auxiliaires en provenance de souris TCR1640. Enfin, l’implication de nouvelles molécules d’adhésion cellulaire dans le développement et le maintien de l’EAE spontanée a été suggérée par des observations in vitro. L’ensemble de ces résultats suggère que le modèle TCR1640 présente plusieurs avantages pour l’étude de la physiopathologie de maladies neuroinflammatoires telles que la sclérose en plaques, et servira d’outil afin de valider de nouvelles stratégies thérapeutiques.

Hypothesis about mechanisms through which nicotine might exert its effect on the interdependence of inflammation and gut barrier function in ulcerative colitis

Ulcerative colitis (UC) is characterized by impairment of the epithelial barrier and the formation of ulcer-type lesions, which result in local leaks and generalized alterations of mucosal tight junctions. Ultimately, this results in increased basal permeability. Although disruption of the epithelial barrier in the gut is a hallmark of inflammatory bowel disease and intestinal infections, it remains unclear whether barrier breakdown is an initiating event of UC or rather a consequence of an underlying inflammation, evidenced by increased production of proinflammatory cytokines. UC is less common in smokers, suggesting that the nicotine in cigarettes may ameliorate disease severity. The mechanism behind this therapeutic effect is still not fully understood, and indeed it remains unclear if nicotine is the true protective agent in cigarettes. Nicotine is metabolized in the body into a variety of metabolites and can also be degraded to form various breakdown products. It is possible these metabolites or degradation products may be the true protective or curative agents. A greater understanding of the pharmacodynamics and kinetics of nicotine in relation to the immune system and enhanced knowledge of out permeability defects in UC are required to establish the exact protective nature of nicotine and its metabolites in UC. This review suggests possible hypotheses for the protective mechanism of nicotine in UC, highlighting the relationship between gut permeability and inflammation, and indicates where in the pathogenesis of the disease nicotine may mediate its effect.

Peanut allergens alter intestinal barrier permeability and tight junction localisation in Caco-2 cell cultures

Allergen absorption by epithelia may play an important role in downstream immune responses. Transport mechanisms that can bypass Peyer's patches include transcellular and paracellular transport. The capacity of an allergen to cross via these means can modulate downstream processing of the allergen by the immune system. The aim of this study was to investigate allergen-epithelial interactions of peanut allergens with the human intestinal epithelium.