Thèse de doctorat : Sciences de la vie et de la santé. Physiologie et physiopathologie de l'appareil respiratoire : Paris 12 : 2007. / Titre provenant de l'écran-titre. 81 p. Bibliogr. : 316 réf.

Primary barriers to drug delivery include mucus and biofilms, which can hinder drug and gene delivery by several orders of magnitude, preventing effective therapeutic effects. By understanding the physical and chemical properties of these ubiquitous barriers, one may employ drug delivery approaches, such as design of nanoparticle and microparticle systems, to attempt to overcome the transport barriers. Nanoparticles are a growing interest in drug delivery, specifically as drug carriers, though most will become entrapped within these extracellular barriers further limiting their desired affects. Previous studies have generally manipulated the surface chemistries or size of these nanoparticles to allow for nearly a 2-fold increase in passive diffusion through barriers. To expand the current ideas of overcoming these barriers, studies in presented in this dissertation were performed using a type of active nanoparticle, superparamagnetic iron oxide nanoparticles. It was first investigated whether these particles would disrupt extracellular barriers under an oscillating magnetic field, which resulted in a 2-fold increased diffusion of particles upon biopolymer breakage. Secondly, influences of an external static magnetic field on diffusion of these nanoparticles through model barriers were determined. Both of these methods resulted in higher fold increases, reaching up to 28-fold compared to 2-fold as described in the literature. Next an examination of drug permeation enhancement in models of extracellular barriers by nanoparticle interactions was performed, using a passive mechanism as found in the literature. With a range of different nanoparticles including diesel particulate matter, barrier function was disrupted resulting in a 5-fold increase in drug permeation. To further manipulate drug diffusion an assisted delivery systems was observed, where magnetic nanoparticles could influence un-associated drug diffusion, resulting in 4-fold increase in drug diffusion. Finally formulations of nanosuspensions were created for aerosol delivery and their performance evaluated in vitro. A dry powder formulation containing drug and nanoparticles was formulated using a spray-drying technique. Upon barrier deposition studies using the dry powder formulation, permeation rates were determined resulting in a 2-fold increase for nanoparticle permeation. When drug diffusion was determined up to a 54-fold increase in drug was seen when co-delivered with nanoparticles, compared to controls containing only drug. / text

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 4 uppsatser.

Inflammatory bowel disease (IBD) is associated with an inappropriate immune response to the gut microbiota and disruption of intestinal homeostasis. IBD patients and experimental animal models have consistently shown alterations in the gut microbiota composition. However, these studies have mainly focused on faecal microbiota samples taken after the onset of inflammation and IBD establishment. The colonic microbiota inhabits both the gut lumen and the mucus layer covering the intestinal epithelium. Thus, information about mucus-resident microbiota is not necessarily conveyed in the routine microbiota analyses of faecal samples. To address potential changes in microbial composition and function before the onset of IBD, we compared both mucus and faecal microbiota in the mdr1a-/- spontaneous model of colitis over times that we histologically defined as before onset of colitis, during and after colitis onset. We showed that alterations in microbiota composition preceded the onset of intestinal inflammation and that these changes were evident in the mucus, but not in faeces. This altered microbiota composition was coupled with a reduced inner mucus layer, indicating a compromised mucus barrier prior to colitis development. Upon emergence of inflammation, compositional differences were found in both mucus and faecal microbial communities. Spatial segregation of microbiota with intestinal mucosa was also disrupted on disease onset which we hypothesise contributes to a more severe intestinal pathology. Therefore, our data indicate that microbial changes start locally in the mucus and then proceed to the faecal matter concomitantly with colitis development. Next, we examined whether microbial gene functional potential and endogenous metabolite profiles followed alterations in gut microbiota taxonomic composition. Our findings showed that the microbial gene content was similar between mdr1a-/- mice and wild-type littermate controls, demonstrating stability of the gut microbiome at the face of ensuing gut inflammation. In further support of these findings, urinary metabolite analysis revealed that metabolite profiles were unaffected by intestinal inflammation. Metabolites previously reported to change in IBD were similar between mdr1a-/- and wild-type mice at stages preceding and during inflammation. We also found that changes in metabolite profiles did not correlate with colitis scores. However, metabolite changes could discriminate mdr1a-/- mice from wild-type controls, suggesting they could have value in predicting risk of IBD with a potential clinical use in at least a subset of individuals with MDR1A polymorphisms. To assess whether changes in antimicrobial proteins (AMPs) accounted for observed differences in mucus microbiota composition, we also investigated the expression of regenerating islet-derived protein 3 γ (Reg3γ), angiogenin 4 (Ang4), β-defensin 1 and resistin-like molecule beta (Relm-β) in the colon. We found similar levels of these AMPs as well as IgA-producing plasma cells between mdr1a-/- and wild-type mice, suggesting that other factors contribute to alterations in microbiota composition. Overall, our data indicate that the mdr1a-/- is a good model of colitis, as it enables us to look at pre-clinical changes in the gut microbiota. This work suggests the importance of mucus sampling for sensitive detection of microbiota changes. Furthermore, metabolite profiling may be a helpful way to discriminate genetic susceptibility to disease.

The intestinal barrier represents a complex environment, composed of different physical barriers and immune cells, which act to prevent the entrance of potentially harmful enteric pathogens and to maintain gut tolerance to food antigens and commensal bacteria. Thus, cross-talk between the different components of the intestinal barrier such as the mucus layer, dendritic cells (DC) and intestinal intraepithelial lymphocytes (IELs) may be important in maintenance of gut homeostasis. This thesis investigates how different components of the intestinal barrier regulate immune responses in the gut. Thus, expression of the transmembrane receptor integrin αvβ8 on DCs is shown to be required for the development of a specific IEL subset marked by expression of CD4 and CD8αα, suggesting that intestinal DC play important roles in regulating the IEL compartment. Moreover, considering that intestinal DCs are likely in close contact with intestinal mucus, it was hypothesized that interactions between DCs and mucins, the predominant proteins that form the mucus layer, may modulate DC function. To test this hypothesis, intestinal mucin was purified and used to treat human monocyte-derived DCs. It was found that that expression of the chemokine IL-8 and co-stimulatory DC markers CD86 and CD83 are significantly upregulated on human DCs in the presence of intestinal mucins. Additionally, IL-8 produced by mucin-treated DCs is able to recruit neutrophil-like cells in transmigration assays. These effects were not due to mucin sample contaminants such as LPS, DNA or contaminant proteins. Instead, mucin glycans seem to be important for the induction of these effects on moDCs. Thus, in contrast to recent published results, intestinal mucins appear capable of inducing important pro-inflammatory functions in DC. To investigate whether mucins modulated DCs found in the intestinal environment, intestinal mucins were used to treat murine intestinal DCs, and gene changes explored using microarray analysis. It was found that, amongst several genes modulated in intestinal DC, up-regulation of the mucosal cytokine IL-22 was induced by intestinal mucin. Therefore, interactions between different components of the intestinal barrier might be crucial for maintaining gut homeostasis. Understanding how different components of the intestinal barrier system work together to maintain homoeostasis may identify pathways that can be targeted to restore this balance in inflammatory disorders such as inflammatory bowel disease.

Chez les mammifères, les spermatozoïdes, au cours du transit à travers le tractus génital femelle, rencontrent des environnements qui influencent leur mobilité et leur pouvoir fécondant. L’analyse par spectrométrie de masse de ces milieux (plasma séminal, mucus cervical, fluides de l’utérus et de l’oviducte) a permis une caractérisation approfondie de leur protéome et l’identification de protéines modifiant l’activité des gamètes comme la Zinc Alpha Glycoprotein. L’étude du protéome du tractus génital femelle au cours du cycle oestral a montré une régulation différentielle de la sécrétion des protéines selon l’organe et le stade du cycle. La comparaison du protéome et des propriétés mécaniques du mucus cervical a montré l’impact de la mucine 5AC sur la viscosité du mucus et la mobilité des spermatozoïdes dans ce mucus. Cette interaction entre le mucus cervical et les spermatozoïdes se traduit par la fixation à leur surface de nouvelles protéines, comme la myosine 9, ayant un impact potentiel sur leur pouvoir fécondant. / During their transit through the female genital tract, mammalian spermatozoa encounter several environments that influence their mobility and fertilizing ability. Analysis by mass spectrometry of these environments (seminal plasma, cervical mucus, fluids from the uterus and the oviduct) enabled characterization of their proteomes and the identification of proteins able to modify gamete function such as Zinc Alpha Glycoprotein. The study of the female genital tract proteome during the oestrous cycle showed differential regulation of the protein secretion according to the organ and the stage of the cycle. Comparison of the proteome and the mechanical properties of cervical mucus showed the impact of mucin 5AC on the viscosity of mucus and the motility of spermatozoa in its presence. 52 cervical mucus proteins, such as myosin 9, bind to the surface of spermatozoa which may impact sperm function and fertilizing ability.

Le mucus recouvre et protège les épithéliums des tractus respiratoire, gastro-intestinal et reproducteur. Les mucines sécrétées sont des molécules mosaïques de très grande taille moléculaire, fortement O-glycosylées et responsables des propriétés rhéologiques du gel de mucus. MUC5B est l’une des cinq mucines gélifiantes et est principalement sécrétée dans le tractus respiratoire. Chez l’Homme, MUC5B est impliquée dans le cancer mammaire et la mucoviscidose. MUC5B est exprimée dans plus de 80% des cancers mammaires alors que MUC5B n’est pas exprimée dans le tissu mammaire sain. Par ailleurs, MUC5B pourrait jouer un rôle majeur dans le poumon de patients atteints de mucoviscidose. Pour étudier la fonction de MUC5B, nous avons utilisé des protéines recombinantes composées de différents domaines de MUC5B et des modèles animaux. Nous avons montré que MUC5B favorise la prolifération cellulaire et l’invasion in vitro et favorise la croissance tumorale et la dissémination métastatique chez des souris immunodéprimées. MUC5B apparait donc comme une cible thérapeutique intéressante pour ralentir la prolifération cellulaire et la dissémination métastatique dans le cancer du sein. Nous avons également montré que l’expression de Muc5b et le nombre de cellules Muc5b positives sont plus élevés dans un modèle murin de mucoviscidose (Cftr–/–) que chez les souris frères-sœurs contrôles, ce qui suggère que les souris Cftr–/– sont prédisposées à former des bouchons bronchiques. Les souris Cftr–/– infectées expérimentalement par Pseudomonas aeruginosa développent des bouchons bronchiques AB-PAS positifs principalement composés de la mucine Muc5b et d’ADN. MUC5B semble être une cible thérapeutique intéressante pour diminuer la viscosité du mucus dans la mucoviscidose. Enfin, nous avons montré que la protéine Muc5b est exprimée très précocement (dès E8.5) au cours du développement embryonnaire murin. A E11.5, Muc5b est exprimée dans le bourgeon pulmonaire, dans le bourgeon formant la trachée et l’œsophage et dans l’estomac. De manière inattendue, Muc5b est exprimée au niveau baso-latéral des cellules épithéliales suggérant un rôle clé de MUC5B dans la morphogenèse. / Respiratory, gastrointestinal and reproductive tracts are protected by a mucus layer. Secreted mucins are large, high molecular weight and heavily O-glycosylated mosaic proteins that are responsible for the rheological properties of mucus gel. MUC5B is one of the five secreted gel-forming mucins and is mainly secreted in the respiratory tract. In human pathology, MUC5B has been implicated in breast cancer and cystic fibrosis (CF). This ‘non mammary’ mucin is expressed in more than 80% of breast cancer and it has been suggested that MUC5B may represent the main mucin to be implicated in the lung disease of CF.To investigate the function of MUC5B, we used recombinant proteins of several MUC5B domains and animal models. We show that MUC5B promotes cell proliferation and invasion in vitro and tumor growth and metastasis using SCID mice suggesting that MUC5B may represent a therapeutic target to slow down the tumor growth and dissemination in breast cancer. We show also that Cftr–/– mice harbor more Muc5b positive Clara cells than do control littermates suggesting that CF mice are predisposed to develop mucus plugs. Experimental infection with Pseudomonas aeruginosa increased the number of Muc5b-positive cells and the formation of mucus plugs in bronchi or bronchioles of Cftr–/– mice. These plugs are mainly composed of Muc5b and DNA suggesting that MUC5B may be a valuable target for decreasing mucus viscosity in CF. Finally, Muc5b protein was detected very early in mouse embryogenesis (at day 8.5). At E11.5, Muc5b is expressed in pulmonary bud, in tracheo-esophagal groove and in stomach. Unexpectedly, Muc5b is expressed at the basal and lateral membrane of epithelial cells of the buds suggesting a key role in epithelial morphogenesis.

La pollution particulaire de l'air provient de différentes sources dont le trafic routier et est reconnue pour avoir un impact significatif sur la santé. Grâce à un meilleur contrôle des émissions à l'échappement, la contribution des émissions non liées à l’échappement à la pollution de l’air s’accroit. Dans celles-ci- se trouvent les particules d'usure des freins dont la toxicité est mal caractérisée. Dans cette thèse nous avons travaillé sur des particules d’usure de freins recueillies sur différents véhicules avec pour objectif une caractérisation physico-chimique et toxicologique de ces particules et de leur fraction nanométrique. Ces particules présentent une hétérogénéité de forme et de taille avec une fraction nanoparticulaire dont le fer et le cuivre sont les composants principaux. L’étude toxicologique a été réalisée sur un modèle 3D de cellules épithéliales bronchiques humaines (Calu-3) cultivées en Transwell® permettant d’obtenir une barrière épithéliale respiratoire. Les nanoparticules (NPs) jusqu’à 100 µg/cm² n’affectent pas la viabilité cellulaire à 24h et n’induisent pas de réponse-proinflammatoire. La technique de « single Particle ICP-MS » nous a permis de démontrer qu’elles sont capables de traverser la barrière respiratoire en faible quantité, probablement par transcytose. De plus ces NPs induisent l’expression et la production de la mucine MUC5AC par un mécanisme dépendant de la voie du récepteur à l'EGF. Dans un test de blessure réalisé sur un épithélium pré-exposé aux NPs, la vitesse de réparation de la lésion n’est pas modifiée mais la prolifération est plus importante que dans un épithélium non pré-exposé. En conclusion, la fraction nanoparticulaire métallique présente dans les particules d'usure des freins traverse faiblement la barrière épithéliale bronchique, induit la production de mucus et active la prolifération lors de la réparation ce qui pourrait suggérer une implication dans le remodelage des voies respiratoires. / Particulate air pollution results from a variety of sources including road traffic and is known to have a significant impact on health. With improved exhaust emissions control, the contribution of non-exhaust emissions to air pollution is increasing. In these are brake wear particles whose toxicity is poorly characterized. In this thesis we worked on brake wear particles collected on different vehicles with the aim of achieving a physico-chemical and toxicological characterization of these particles and their nanometric fraction. These particles have a heterogeneity of shape and size with a nanoparticulate fraction of which iron and copper are the main components. The toxicological study was performed on a 3D model of human bronchial epithelial cells (Calu-3) grown in Transwell® to obtain a pertinent respiratory epithelial barrier. Nanoparticles (NPs) do not affect cell viability up to 100 μg/cm² at 24 hours and do not induce a proinflammatory response. The technique of "Single Particle ICP-MS" has allowed us to demonstrate that particles are able to cross the respiratory barrier in small quantities, probably by transcytosis. In addition, these NPs induce expression and production of MUC5AC mucin by a mechanism dependent on the EGFR pathway. In a wound healing test carried out on an epithelium pre-exposed to the NPs, kinetic repair of the lesion is not modified but the proliferation is greater than in a non-pre-exposed epithelium. In conclusion, the metal nanoparticulate fraction present in brake wear particles weakly crosses the bronchial epithelial barrier, induces production of mucus and activates the proliferation during the repair which could suggest an implication in the remodeling of the airways.

Coral reefs are the proverbial rainforests of the ocean, but these spectacular structures are under threat from globally rising sea surface temperatures and ocean acidification. The Red Sea and the Persian/Arabian Gulf (PAG) display unusually high sea surface temperatures, and therefore, provide a model for studying environmental change. Corals are so-called holobionts consisting of the coral host, photosynthetic algae (Symbiodiniaceae), along with other microorganisms, such as bacteria, archaea, fungi, and viruses. While the importance of bacteria to coral holobiont functioning is acknowledged, little is known about changes in the microbial communities under natural environmental stressors in the Red Sea and the PAG. Accordingly, I investigated microbial community and mucus differences in bleached, healthy, and diseased corals. Analysis of the composition of mucus-associated microbial communities of bleached and healthy Porites lobata colonies from the Red Sea and the PAG were stable, although some regional differences were present. In a distinct study investigating coral disease, a broad range of corals in the Red Sea were shown to be infected with black band disease (BBD). Investigating the microbial community associated with BBD revealed the presence of the three main indicators for BBD (cyanobacteria, sulfate-reducing bacteria (SRB), and sulfide-oxidizing bacteria (SOB). Last, I investigated the chemical composition (carbohydrates) of the surface mucus layer of a range of Red Sea corals. Given that coral mucus represents a first line of defense, I was interested to examine whether mucus carbohydrate composition would point to a role of adaptation to the extreme environment of the Red Sea. This analysis showed that mucus consists of conserved sugars that are globally conserved. In summary, this thesis characterizes the microbial communities associated with a range of coral species in different health states (bleached, healthy, and diseased). The microbial community patterns I characterized support the notion that bacteria contribute to coral holobiont health and possibly adaptation to extreme environmental conditions in the Red Sea and PAG.

Une multitude de voies endocytiques existent à la surface de la membrane plasmique, ce qui conduit à l'endocytose de la majeure partie des membranes ainsi que de leurs protéines associées, des molécules de signalisation, des facteurs de croissance et autres cargos (Smith et al. 2017). Pendant des décennies, la voie majeure dite clathrine dépendante a été la plus étudiée, Cette voie d’endocytose se caractérise par la polymérisation de molécules de clathrine et de protéines adaptatrices au niveau de récepteurs liés à leurs ligands, entrainant la courbure de la membrane, avant sa scission et son endocytose (Smith et al. 2017). Récemment, plusieurs mécanismes alternatifs ont été découverts facilitant l'absorption endocytique des protéines cargo et des récepteurs membranaires, même en l'absence de la voie clathrine dépendante (Mayor et al.2014). Un modèle d'endocytose qui ne requiert pas de clathrine mais à la place des galectines et des glycolipides a été proposé par le groupe de Ludger Johannes (Lakshminarayan et al. 2014). Les galectines sont des lectines qui se lient aux bêta-galactosides et qui, à ce jour, regroupent 15 membres (galectine-1 à galectine-15) chez les mammifères et sont retrouvées dans de nombreux types de cellules et tissus (Leffler et al., 2004). Les galectines sont probablement sécrétées extra-cellulairement par une voie inconnue et non-classique (Hughes, 1999). Les glycosphingolipides (GSL) sont des constituants membranaires ubiquitaires, divisés en fractions neutres ou acides. Le terme GSL s'applique aux composés contenant au moins un monosaccharide et une céramide. Il est à noter que l’enzyme UDP-glucose céramide glucosyltransférase (Ugcg) catalyse l’étape initiale de la biosynthèse de GSLs à base de glycosylcéramides.Notre modèle de travail actuel, impliquant les glycolipides et les lectines, a été qualifié d'hypothèse GL-Lect (Johannes et al. 2016), et préliminairement soutenu par des données expérimentales comme décrit par Lakshminarayan et al. en 2014. Il peut être décrit comme suit:i) le monomère Gal3 se lie aux glycoprotéinesii) Gal3 commence alors à oligomériseriii) Gal3 oligomérisé a la capacité de se lier aux glycosphingolipides, ce qui peut induire la formation de clusters de GSLsiv). Ces clusters Gal3-GSL induisent une invagination de la membrane plasmique, une endocytose des protéines cargo liées à Gal3 et la formation subséquente de CLIC (clathrin-independent carriers ), endosomes pré-précoces.Selon ce modèle, l’oligomère Gal3 est capable de se lier aux GSLs et d’induire une déformation de la membrane de la même manière qu’une autre lectine, la sous-unité pathogène shiga toxine-B (STxB). Par conséquent, les deux processus pourraient être résumés sous la même hypothèse GL-Lect, où GL représente les glycosphingolipides (Gb3 pour STxB et des GSLs non identifiés pour Gal3) et Lect corresponds aux lectines (STxB, Gal3 et éventuellement d'autres). Comprendre si les voies d'internalisation indépendantes de la clathrine, mais dépendantes de la galectine 3, sont conservées, non seulement pour les modèles in vitro mais également in vivo, est un défi majeur dans le domaine du trafic cellulaire.Nous avons caractérisé, pour la première fois, un nouveau mécanisme dans l’intestin facilitant le transport endocytique d’un cargo. Ce mécanisme est conduit par la Galectine 3 et agit dans les entérocytes intestinaux dans le processus analogue de transcytose et dépend des glycosphingolipides. En effet, nous avons découvert que la lactotransferrine (LTF), un cargo de Gal3 que nous avons identifié par Mass spec, dépendait fortement de la Galectine3 pour son endocytose efficace, et des GSLs pour son mode de distribution analogue à la transcytose. Sur la base de ces découvertes dans l'épithélium intestinal de souris, nous avons établi un système modèle in vivo fonctionnel dans lequel le mécanisme endocytique récemment proposé, appelé dans notre laboratoire GL-Lect, a été étudié physiologiquement. / A host of endocytic pathways exist at the surface of eukaryotic cells, which lead to the internalization of the bulk of membranes along with membrane proteins, signaling receptors, growth factors, and other cargoes (Smith et al. 2017). For decades, the clathrin-mediated pathway has been the major well characterized endocytic process where clathrin polymerizes along with the associated adaptor proteins to include ligand-bound receptors, leading to membrane bending, membrane scission, and endocytosis (Smith et al. 2017). Recently, multiple alternative mechanisms have been uncovered which facilitate the endocytic uptake of cargo molecules and membrane receptors even in the absence of clathrin machinery (Mayor et al.2014). A model of endocytosis that doesn’t require clathrin but rather sugar-binding galectins and glycolipids has been proposed by my host laboratory (Lakshminarayan et al. 2014). Galectins constitute a family of beta-galactoside–binding lectins, which to date consists of 15 members in mammals. Galectins are broadly distributed in a variety of cells and tissues (Leffler et al. 2004). They are translocated from the cytosol to the extracellular space by a process of non-classical secretion (Hughes 1999). Glycosphingolipids (GSLs) are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions. The term GSLs applies to compounds that contain at least one monosaccharide and a ceramide. Of note, the enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step for the biosynthesis of glycosylceramide-based GSLs.Our current working model, which involves glycolipids and lectins, was termed the GL-Lect hypothesis (Johannes et al. 2016). It is backed up by experimental data as described in Ref. (Lakshminarayan et al. 2014) and can be described as follows:i) Monomeric Gal3 binds to glycoproteinsii) Gal3 then starts to oligomerizeiii) Oligomerized Gal3 has the capacity to bind to glycosphingolipids and this may induce clustering of GSLsiv) Gal3-GSL cluster are inducing the invagination of the plasma membrane to generate tubular endocytic pits from which clathrin-independent carriers (CLICs, which are pre-early endosomes) are generated.Oligomeric Gal3 is indeed able to bind to GSLs and to induce membrane deformation (Lakshminarayan et al. 2014) in a similar way the pathogenic lectin Shiga toxin-B subunit (STxB) does. Therefore, both processes could be summarized under the same hypothesis, the GL-Lect hypothesis, where GL stands for the glycosphingolipids (Gb3 for STxB and gangliosides for Gal3) and Lect summarizes the lectins (STxB, Gal3 and possibly others as well).Understanding if this clathrin-independent but Gal3-dependent internalization mechanism is conserved not only in vitro model systems but in vivo is a main challenge in the field of trafficking.We characterized for the first time that in the gut a new mechanism facilitates endocytic uptake of cargo. This mechanism is driven by Galectin3 and operates in intestinal enterocytes for transcytosis like process and is glycosphingolipid dependent. Indeed, we have found that the lactotransferrin (LTF), a Gal3 cargo that we have identified by Mass spec, strongly required Gal3 and GSLs for its efficient endocytosis and its transcytosis like distribution pattern, respectively. Based on these findings in mouse intestinal epithelium, we established a functional in vivo model system where the newly proposed endocytic mechanism termed in our lab as GL-Lect, was physiologically investigated.