Effet de la stimulation des TLR sur l'infection par le VIH-1 des lymphocytes T CD4+ et des cellules dendritiques primaires

Depuis sa découverte en 1983, le virus du SIDA ne cesse de faire des ravages. Malgré l’apparition de la thérapie HAART, qui permet de prolonger l’espérance de vie des individus atteints, aucun traitement n’est actuellement disponible pour prévenir ou éradiquer complètement l’infection. Le virus mute beaucoup ce qui lui confère rapidement une résistance à l’ensemble des traitements utilisés. L’objectif actuel du monde scientifique est donc de développer d’autres cibles qui pourraient restreindre les effets indésirables des médicaments actuels tout en diminuant l’émergence de souches virales résistantes. Afin de développer ces nouveaux traitements, il est essentiel de mieux comprendre le comportement du virus et ce, sous diverses conditions de l’organisme. Mon projet de doctorat s’inscrit dans cette thématique puisqu’il a permis d’étudier le comportement du virus suite à la stimulation du TLR2, un récepteur essentiel à la reconnaissance, entre autre, de structures spécifiques des bactéries Gram positives. L’objectif global consistait à déterminer si la stimulation de ce TLR, par un ligand synthétique, avait des effets sur l’état d’activation et le taux d’infection des lymphocytes T CD4+ naïfs et mémoires de même que celui des cellules dendritiques. Les résultats obtenus indiquent que la stimulation du TLR2 augmente l’infection de ces deux types cellulaires et favorise également le transfert du virus des cellules dendritiques aux lymphocytes T CD4+. Sachant que l’activation des cellules favorise l’infection par le VIH 1 et que les produits bactériens présents dans la circulation sanguine lors de la translocation microbienne activent les lymphocytes T CD4+ et les cellules dendritiques, toutes les conditions sont alors réunies pour mener à une hyperactivation des cellules immunes. Ces résultats démontrent que la reconnaissance des produits microbiens par les TLR participe à la dissémination de l’infection en favorisant l’activation de cellules permissives à l’infection.Since its discovery in 1983, HIV-1 continues to destroy many people lives and no treatment is actually available to treat or prevent infection. Despite the utilization of HAART therapy, the epidemy is still increasing and the worldwide really need new treatments. Due to his high capacity of mutations, HIV-1 rapidly develops resistance against all available treatments. The major aim of scientific community is to develop new therapeutics using different targets to try to avoid and control the resistance phenomenon. To develop these treatments, it is very important to acquire a better understanding of the HIV-1 biology. My doctorate project is tightly related to this because it permits to study the reaction of HIV-1 following TLR2 stimulation, a receptor very useful to sense pathogens. Since microbial translocation is a phenomenon frequently observed in HIV-1-patients, characterized by the presence of bacterial compounds into bloodstream, we evaluated if TLR2 stimulation, with a synthetic ligand, could modulated infection process. Our global objective was to determine if this stimulation could change the activation state and influence the infection in both CD4+ T lymphocytes and dendritic cells. Our results confirmed that TLR2 stimulation increases infection in both cell subtypes and also increases the transfer of viruses from dendritic cells to CD4+ T lymphocytes. Knowing that cell activation favors HIV-1 infection and that bacterial compounds present into bloodstream during microbial translocation stimulate TLR2 stimulation activates CD4+ T cells and dendritic cells, all conditions are put together to favors hyperactivation of the immune system. Our results demonstrate that TLR2 stimulation participates to HIV-1 dissemination by triggering activation of immune cells

Investigating human natural killer cell subsets and their responses to a Herpes Simplex Virus lipopeptide, a potential vaccine candidate

Herpes Simplex Virus (HSV) type 1 and 2 cause lifelong, latent infection with recurrences. Serious complications can occur and no vaccine is available. Natural killer (NK) cells play a role in the interface between innate and adaptive immunity, and in the antiviral response. Nonetheless, NK cells have been largely overlooked in designing vaccine candidates. Previously, NK cells were activated by a TLR2 agonist, Pam2Cys, conjugated to an HSV gD peptide: Pam2Cys-30-5, and stimulated CD4+T cells in the absence of other antigen-presenting cells. Human NK cells are comprised of functionally diverse subsets. CD56dimCD16+ are the predominant subset in circulation, >95%, and CD56brightCD16-/dim are <5%. They have been characterised as highly cytolytic or as potent cytokine producers, respectively. The selective responses of CD56bright NK cells to the cytokines IL-15, IL-2 and IL-7 were compared to enrich this subset in culture. IL-7 selectively enhanced CD56bright survival and proliferation, and perforin expression was not increased. IL-7 CD56bright NK cells expressed similar immune function genes as CD56bright NK cells. CD56bright and CD56dim NK cell responses to Pam2Cys-30-5 were investigated. Both subsets were activated by Pam2Cys-30-5, indicated by expression of CD69 and cytokines. The CD56dim subset produced higher concentrations of cytokines, whilst significant upregulation of HLA-DR was exclusive to the CD56bright subset. CD16 was significantly downregulated on CD56dim NK cells, and both shedding and internalisation were detected as mechanisms. Soluble factors produced by both NK cell subsets induced increased expression of maturation markers CD83 and CD80 on monocyte-derived dendritic cells (MDDCs). Finally, normal foreskin and an initial penile herpes lesion biopsy were examined for NK cells. CLA was expressed on CD16- NK cells in normal foreskin, indicating a regular turnover of these cells. In infected tissue the majority of NK cells were CD16+. NK cells may play a role in innate control of initial genital HSV infection and should be considered in vaccine development. Furthermore, understanding the mechanism of NK cell responses to TLR2 agonists will provide insight into their use as vaccine adjuvants

Different Journeys, Same Destination: Exploring the Role of a PYHIN Protein and Involvement of Caspase-8 in the Regulation and Activation of Inflammasomes

Interferon-inducible PYHIN protein family includes the DNA-binding proteins, AIM2 and IFI16, which form ASC-caspase 1 dependent inflammasomes, important in immunity against cytosolic bacteria, DNA viruses and HIV. The role of other members of this family in the recognition of DNA and/or regulation of immune responses is unclear. We identified an immune regulatory function of p205, another member of the PYHIN family, in the transcriptional control of immune genes. Knockdown of p205 in macrophages revealed that inflammasome activation due to dsDNA and ligands that engage the NLRP3 inflammasome were severely compromised. Detailed mechanistic analysis showed that loss of p205 was associated with a decrease in Asc mRNA and protein levels. p205 knockdown resulted in reduced RNA Polymerase II-mediated endogenous Asc gene transcription and mRNA processing, suggesting a co-transcriptional control of Asc gene expression. Ectopically expressed p205 induced expression of an Asc gene-luciferase reporter and collaborated with other transcription factors, such as c/EBPβ, p65/RelA, to further enhance expression. p205 knockdown also affected the expression of the immune genes Cd86, Cox2, Cxcl2, Il1α, Il10, Il12α, Il6 and Ifnα in LPS-stimulated macrophages. Together these findings suggest that p205 regulates inflammation through control of Asc gene expression, and other immune genes. Fungal infections activate both caspase 1-dependent and -independent inflammasomes. In an independent study, we show that Paracoccidioides brasiliensis fungal infection also induces caspase 8-dependent non-canonical inflammasome. Caspase 8-dependent IL-1β processing required dectin-1, Syk and Asc. Rip3-/- Casp8-/- mice infected with P. brasiliensis displayed increased fungal load and showed worse disease progression compared to wild type and Rip3-/- mice. These results revealed the importance of caspase 8 in activating and regulating inflammasome responses during fungal infection in vivo

Polymeric nanocapsules as versatile platform for the development of new dendritic cell-directed nanovaccines

Therapeutic vaccination against tumor diseases remains a major challenge in immune therapy. The effective activation of dendritic cells by a combination of distinctly acting adjuvants and antigens is essential for success. While conventional vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines may offer unique properties to tackle that challenge. In this doctoral thesis, we report the use of polymeric nanocapsules as a versatile platform for the development of new dendritic cell-directed nanovaccines. Those nanocarriers are characterized by a high biocompatibility and modifiability, low cytotoxicity as well as by a large loading capacity for active substances. The resulting nanovaccine comprises a shell consisting of protein antigen and allows an efficient loading with superadditively acting combinations of adjuvants, even when their corresponding receptors are located intracellularly. Furthermore, the capsule surface can be modified with stealth components to increase blood circulation time, allows the functional encapsulation of small interfering RNA and can also be equipped with specific antibodies to substantially increase dendritic cell targeting. Initially, we identified the combination of resiquimod and muramyl dipeptide to exert a superadditive stimulatory potential on dendritic cells. This adjuvant combination maintains its superadditive character and stimulates murine dendritic cells more effective when encapsulated in dextran nanoparticles than when applied directly. At the same time, nanocapsules, consisting of the model antigen ovalbumin, were evaluated as a suitable antigen source for the induction of antigen-specific T cell responses. Subsequently, the aforementioned adjuvant combination was encapsulated in these ovalbumin-based nanocapsules to generate a nanocarrier comprising antigen and superadditive adjuvant combination. Its immunostimulatory potential for dendritic cell stimulation was extensively tested by i) measuring the expression of co-stimulatory markers, ii) the secretion of pro-inflammatory cytokines, iii) the upregulation of immunologically relevant genes on RNA level by transcriptome sequencing, and iv) the capability of accordingly pre-treated dendritic cells to mediate antigen-specific T cell responses. The created nanocapsule, including antigen and adjuvants, triggered strong dendritic cell stimulation and potent antigen-specific T cell proliferation. Moreover, numerous relevant genes were massively upregulated upon treatment. The second step was to equip the protein-based nanocapsule with stealth components to increase its blood circulation time and to reduce unspecific cell interaction. Thereby, a special focus was set on the influence of the molecular weight of the used components as well as on the shielding density and the mass density of the modification. It turned out that such a modification can significantly reduce cellular interaction but is highly dependent on molecular weight, shielding and mass density of the used stealth component as well as on the protein environment. To establish small interfering RNA and targeting moieties in our portfolio of available modifications for polymeric nanocapsules, we switched to a similar, antigen-independent polymeric nanocapsule made of hydroxyethyl starch. Regarding small interfering RNA, we showed that the synthesized nanocapsules were capable of transporting them into dendritic cells and to release them resulting in a functional activity. In terms of targeting, a surface modification with targeting antibodies significantly increased the nanocapsule binding by dendritic cells. Since the introduced protein-based type of nanocapsule provides the option to replace ovalbumin, for instance, by a tumor-related antigen, it also allows a further optimization for personalized tumor treatment by employing a patient’s tumor-specific antigen. In combination with the demonstrated advantages and available modifications, polymeric nanocapsules as presented here constitute a promising platform for the design and generation of new, innovative nanovaccines for tumor treatment.Die therapeutische Impfung gegen Tumorerkrankungen stellt bis heute eine große Herausforderung der Immuntherapie dar. Die Aktivierung von dendritischen Zellen durch die kombinierte Gabe von gezielt wirkenden Adjuvantien und Antigenen ist dabei essentiell. Konventionelle Vakzine sind im Falle einer therapeutischen Tumorbehandlung häufig nicht effizient genug um eine ausreichend starke T-Zellantworten zu induzieren. Demgegenüber weisen Nanovakzine spezielle Eigenschaften auf, die eine therapeutische Vakzinverwendung ermöglichen bzw. erleichtern könnten. In der vorliegenden Doktorarbeit wird die Verwendung von polymeren Nanokapseln als vielseitige Plattform für die Entwicklung neuartiger Nanovakzine beschrieben. Diese Kapseln zeichnen sich vor allem durch eine hohe Biokompatibilität und Modifizierbarkeit, eine geringe Zytotoxizität, sowie durch eine große Ladungskapazität für biologisch aktive Substanzen aus. Das resultierende Nanovakzin weist eine Hülle aus Proteinantigen auf und erlaubt die effiziente Beladung mit superadditiv wirkenden Adjuvanzkombinationen, selbst wenn sich deren Rezeptoren intrazellulär befinden. Zudem kann die Kapseloberfläche mit ‚Stealth‘-Molekülen modifiziert werden, wodurch die Blutzirkulationszeit erhöht wird. Ebenso ermöglicht dieses System die funktionale Verkapselung von small interfering RNA zur Immunmodulation und die Anbindung von Antikörpern zur verstärken Adressierung von dendritischen Zellen. Zu Beginn wurde die Kombination der Adjuvantien Resiquimod und Muramyl-Dipeptid als superadditiv für die Stimulation von dendritischen Zellen identifiziert. Es zeigte sich, dass die stimulierenden Eigenschaften dieser Kombination durch Partikulierung in Dextran-basierte Nanopartikel im Vergleich zur solublen Applikation noch gesteigert werden konnten. Gleichzeitig wurde die Verwendung von Nanokapseln, die aus dem Modellantigen Ovalbumin bestanden, als geeignete Antigenquelle zur Einleitung antigenspezifischer T-Zell-Antworten etabliert. Um ein vollständiges Nanovakzin zu generieren, dass sowohl Antigen als auch eine superadditive Adjuvanzkombination beinhaltet, wurde die zuvor genannte Adjuvanzkombination in diese Antigenkapseln integriert. Das resultierende immunstimulatorische Potential dieser Kapseln wurde anhand i) der Expression kostimulatorischer Marker, ii) der Sekretion proinflammatorischer Zytokine, iii) der Aufregulation immunologisch relevanter Gene auf RNA-Ebene durch Transkriptom-Sequenzierung und iv) der Fähigkeit entsprechend vorbehandelter dendritischer Zellen zur Induktion antigenspezifischer T-Zell-Antworten untersucht. Die generierte Nanokapsel, welche sowohl Antigen als auch Adjuvantien enthielt, induzierte eine potente Stimulation dendritischer Zellen und eine starke Proliferation antigenspezifischer T-Zellen. Zudem wurden unter Gabe der Nanokapseln zahlreiche relevante Gene stark aufreguliert. In einem zweiten Schritt wurden diese proteinbasierten Nanokapseln mit ‚Stealth‘-Komponenten ausgestattet um ihre Blutzirkulationsdauert zu erhöhen und um die unspezifische Interaktion mit Zellen zu reduzieren. Dabei wurde ein spezieller Fokus auf den Einfluss des Molekulargewichts der verwendeten Komponenten sowie auf die Beschichtungs- und Massendichte der Modifikation gesetzt. Es zeigte sich, dass solche eine Modifikation die Interaktion der Nanokapseln mit Zellen signifikant reduzieren kann, der resultierende Effekt allerdings stark von den genannten Einflüssen sowie dem vorherrschenden Proteinmilieu abhängt. Um die Verkapselung von small interfering RNA und die Kopplung von Antikörpern für polymere Nanokapseln zu etablieren, wurden Nanokapseln aus Hydroxyethylstärke verwendet. Bezüglich small interfering RNA, konnte gezeigt werden, dass die synthetisierten Nanokapseln geeignet waren diese in dendritische Zellen zu transportieren und dort freizusetzen. Die resultierende funktionale Aktivität konnte gemessen werden. In Bezug auf die Adressierung von dendritischen Zellen, konnte eine Oberflächenmodifikation mit spezifischen Antikörpern die Nanokapselbindung durch dendritische Zellen signifikant erhöhen. Da das präsentierte proteinbasierte Nanokapselsystem die Option bietet, das Ovalbumin durch beispielsweise ein tumorassoziiertes Antigen zu ersetzen, kann es durch die Verwendung von patientenspezifischen Tumorantigenen weiter in Richtung personalisierte Tumortherapie optimiert werden. Zusammen mit den hier aufgezeigten Vorteilen und möglichen Modifikationen, stellen polymere Nanokapseln eine vielversprechende Plattform für die Synthese von neuartigen, innovativen Nanovakzinen für die therapeutische Tumorbehandlung dar

Dissecting the Role of a lncRNA and Involvement of \u3cem\u3ePlasmodium\u3c/em\u3e Infections in the Innate Immune Response: A Dissertation

The innate immune system is a multicomponent response governed by intricate mechanisms of induction, regulation and resolution to elicit antimicrobial defenses. In recent years, the complexity of eukaryotic transcriptomes has become the subject of intense scrutiny and curiosity. It has been established, that RNA polymerase II (RNAPII) transcribes hundreds to thousands of long noncoding RNAs (lncRNAs), often in a stimulus and cell-type specific manner. However, the functional significance of these transcripts has been particularly controversial. While the number of identified lncRNAs is growing, our understanding of how lncRNAs themselves regulate other genes is quite limited. In chapter 2, a novel lncRNA is identified, more specifically, a natural antisense transcript, that mediates the transcription of the pro-inflammatory cytokine IL-1α. Through loss-of-function studies, I report the necessity of this transcript in mediating IL-1α mRNA expression by affecting RNAPII binding to the IL-1α promoter after toll-like receptor signaling. For the first time, I show that IL-1α is regulated at the transcriptional level. As a second independent component of this thesis, we explore the role of the innate immune response after infection by the malaria-causing parasite, Plasmodium berghei ANKA (PbA), and how innate immune components are both beneficial and detrimental to the host depending on when and where inflammation is triggered during infection. We attempt to identify the “malarial toxin” responsible for aberrations in the immune response that is detrimental for disease outcomes and the innate signaling pathways that are involved. Many pathogens induce pathological inflammatory conditions that lead to irreparable homeostatic imbalances and become fatal to the host. Here, type I Interferon signaling is required to dampen parasite load during liver-stage infections, but leads to host mobidity if these pathways are activated in the erythrocytic phase of infection. Together, this thesis provides new insights on how components of the innate immune system are regulated, and how dysregulation of immunity can potentially lead to adverse effects during active infections

Immunology of the genital tract - a review

The objective of this work was to systematically review and discuss recent studies and articles dealing with the subject of the immunology of female genital tract mucosal tissue. The emphasis hereby lies on the evaluation of studies concerning the basics of female reproductive immunology, research on immunology of the most important genital infections and vaccination strategies, immunologic principles at the fetomaternal interface during normal pregnancy and its complications as well as on immunologic data on infertility and immunocontraception. It is now established that the mucosal immune system is a distinct and separate component of the host`s immune apparatus and differs from the lymphoid tissues in peripheral sites. Furthermore, despite some common features, the female genital tract mucosal system displays some distinct characteristics which outlines its special role. Analysis of the female genital tract indicates that the key cells of the innate and adaptive immune systems are present and functionally responsive to antigens; however, there is a certain degree of compartmentalization within the tract. The identification of TLRs in the fallopian tubes, uterus, cervix, and vagina and the presence of ECs, macrophages, DCs, NK cells, and neutrophils throughout the reproductive tract along with their responsiveness to selected PAMPs indicate that the female reproductive tract has evolved to meet the challenges of STDs, while at the same time supporting an immunologically distinct fetal placental unit. To meet these diverse challenges, innate and adaptive immune system in the female genital tract are precisely regulated not only by a network of cytokines and chemokines, but also by the sex hormones estrogen and progesterone. Understanding the specialty of the genital tract immune system is of critical importance, because STDs are and will be a major worldwide health problem. Despite extensive efforts, only limited success has been achieved in dealing with a growing list of STDs. The role of immune factors in the control of genital viral and bacterial infections appears complex and needs further studying, also with respect to creating vaccines. Despite the recognition that innate immunity as the first line of defense and adaptive immunity, especially Th1 immune responses, play a critical role in preventing infection and in limiting viral replication, factors such as antimicrobials and TLRs that contribute to the mucosal response in the female genital tract have only recently begun to receive attention. Further studies are also needed to elucidate the relationship between mucosal immunity, the hormonal environment, and response to pathogen challenge. More data must be collected on the mechanisms of immune evasion by several pathogens such as HSV, N. gonorrheae or Chlamydia. While considerable information can be obtained from animal experiments, important differences in the physiology of reproduction and the immune sytem result in the need for studies in humans. Further knowledge on female tract immunology will also impact on immunological approaches to contraception, immunological infertility and the immunological aspects of pregnancy. This does not only involve new options for diagnostics but also for treatment of pregnancy complications such as preeclampsia, preterm birth and early pregnancy loss as well as for infertility. Pregnancy involves maternal tolerance of the semiallogenic histoincompatible fetus and is characterized by the enhancement of the innate immune system and suppression of the adaptive immune response, probably with progesterone as the important regulator. In opposite to normal pregnancy, improper immune responses and an unbalanced cytokine network may characterize implantation failures, pregnancy loss and obstetric complications. These are the presence of elevated Th1/Th2 cell ratios, high concentrations of Th1 cytokines, elevated NK cell cytotoxicity and levels, and emergence of various autoantibodies. These immunological approaches needs to be investigated and evaluated further with respect to widening of treatment options by modification of immune responses

Interactions of nanoparticles and innate immunity system

Τις τελευταίες δεκαετίες, η νανοτεχνολογία και η αξιοποίηση των νανοσωματιδίων για κλινική χρήση έχουν διερευνηθεί εκτενώς στην ιατρική και σε διάφορους τομείς των βιολογικών επιστημών. Οι τεχνικές που βασίζονται στη νανοτεχνολογία έχουν τη δυνατότητα να βελτιώσουν σημαντικά τη θεραπεία, την πρόληψη και τη διάγνωση ενός ευρέος φάσματος ασθενειών. Τα πλεονεκτήματα των νανοσωματιδιακών συστημάτων, όπως η εξειδικευμένη χορήγηση φαρμάκων, η ανθεκτικότητα in vitro και in vivo και οι λιγότερες παρενέργειες σε σύγκριση με τα παραδοσιακά φάρμακα, τα έχουν καταστήσει τη θεραπεία επόμενης γενιάς για ένα ευρύ φάσμα ασθενειών. Μετά τη χορήγηση, τα νανοσωματίδια αλληλεπιδρούν τόσο με το έμφυτο όσο και με το προσαρμοζόμενο ανοσοποιητικό σύστημα, προκαλώντας ανοσολογικές αποκρίσεις. Οι φυσικοχημικές ιδιότητες είναι οι κινητήριοι παράγοντες που κάνουν κάθε νανοσωματίδιο να δρα διαφορετικά στον οργανισμό. Οι τοξικολογικές μελέτες έχουν αποκαλύψει ότι η πρόσληψη νέων νανοσωματιδίων μπορεί να οδηγήσει σε απρόσμενες επιδράσεις λόγω απρόβλεπτων αλληλεπιδράσεων με βιολογικές διεπιφάνειες, οι οποίες αποτελούν τις βασικές ανησυχίες για τη βιοασφάλεια που προκύπτουν από τη χρήση νανοϋλικών. Η παρούσα ανασκόπηση εξετάζει την τρέχουσα κατάσταση των γνώσεων σχετικά με τον τρόπο αλληλεπίδρασης των νανοσωματιδίων (ΝΡs) με τα συστατικά του έμφυτου ανοσοποιητικού συστήματος, με έμφαση στους κυτταρικούς και διαλυτούς παράγοντες (συμπλήρωμα), και τον τρόπο με τον οποίο οι φυσικοχημικές και επιφανειακές τους ιδιότητες (συνθετική ταυτότητα) επηρεάζουν τη συμπεριφορά τους σε διάφορα φυσιολογικά περιβάλλοντα. Η αποκάλυψη της σχέσης δομής-δραστικότητας των νανοσωματιδίων θα έχει ως αποτέλεσμα την περαιτέρω κατανόηση των ανοσοτροποποιητικών επιδράσεών τους και τη βελτίωση της κλινικής αποτελεσματικότητάς τους, ενώ θα ελαχιστοποιηθούν οι ανεπιθύμητες τοξικολογικές επιδράσεις.In recent decades, nanotechnology and the exploitation of nanoparticles for clinical use have been extensively investigated in medicine and various fields of biological sciences. Nanotechnology-based techniques have the potential to significantly improve the treatment, prevention, and diagnosis of a wide range of diseases. The advantages of nanoparticulate systems, such as site-specific drug delivery, in vitro and in vivo durability, and fewer side effects compared to traditional medications, have made them the next-generation therapy for disease treatment. Following administration, nanoparticles interact with both the innate and adaptive immune systems, eliciting immune responses. The physicochemical properties are the driving factors that make each nanoparticle act differently in the organism. Toxicological studies have revealed that the uptake of novel nanoparticles may result in unexpected effects due to unpredicted interactions with biological interfaces that have been the underlying biosafety concerns raised by the use of nanomaterials. This review examines the current state of knowledge on how nanoparticles (NPs) interact with innate immune system components, with a focus on cell and soluble factors (complement), and how their physicochemical and surface properties (synthetic identity) influence their behavior in various physiological environments. Uncovering the structure–activity relationship of NPs would result in the further understanding of their immunomodulatory effects and improvement of their clinical efficacy, while undesired toxicological effects would be minimized

Innate immune potential of primary lymphatic endothelial cells

Lymphatic endothelial cells (LECs) are loosely overlapping cells that line the lymphatic vasculature. In tissues, LECs are often located directly beneath the mucosal epithelial layer, and therefore, are likely to be among the first cells that come in contact with incoming pathogens and/or vaccine antigens and adjuvants when there is a breakage at the epithelial barrier. We are only starting to appreciate the role of LECs in the development of host innate and adaptive immune responses during infection or vaccine administration. This is largely due to the difficulties in studying LECs in vivo and the challenges in obtaining pure LEC cultures for study ex vivo. My work focused on isolation and establishment of primary LEC cultures derived from different tissue origins of commonly used animal models – ferrets and rhesus macaques – and assessment of the potential of these LECs to respond to pathogen-associated molecular patterns (PAMPs) or a subset of microbes (SIV/HIV-1). In addition, I also partially compared the phenotype and functionality of LECs to dendritic cells (DCs), an immune cell type that acts as a “bridge” between the host innate and adaptive immunity. My findings revealed that LECs were highly heterogeneous in their gene expression profiles. They also endogenously expressed multiple known viral entry and restriction factors for SIV/HIV-1. LECs responded to several PAMPs by producing proinflammatory cytokines/chemokines that are known to recruit immune cells to sites of inflammation. However, LECs were not highly susceptible or permissible to infection using genetically engineered, single-cycle competent SIV/HIV-1 or wild-type SIV. Interestingly, LECs expressed phenotypic markers that have been shown to be expressed by DCs and showed some functional similarities. LECs were able to take up and process model antigens, although it was not determined if these antigens were presented by MHC I and II molecules. These findings are of public health importance because they expand our knowledge of the emerging potential of LECs as key players in innate immunity during pathogen-host interactions or vaccinations. Improving our understanding of LECs will positively impact our knowledge of mucosal infections and will help in development of improved treatment and vaccination strategies. These primary cells will serve as tools to study LEC immunobiology and will also be useful in development of vaccines/therapeutics for human diseases of public health importance that target LEC and/or its crosstalk with other immune cells

Immunology of the genital tract - a review

The objective of this work was to systematically review and discuss recent studies and articles dealing with the subject of the immunology of female genital tract mucosal tissue. The emphasis hereby lies on the evaluation of studies concerning the basics of female reproductive immunology, research on immunology of the most important genital infections and vaccination strategies, immunologic principles at the fetomaternal interface during normal pregnancy and its complications as well as on immunologic data on infertility and immunocontraception. It is now established that the mucosal immune system is a distinct and separate component of the host`s immune apparatus and differs from the lymphoid tissues in peripheral sites. Furthermore, despite some common features, the female genital tract mucosal system displays some distinct characteristics which outlines its special role. Analysis of the female genital tract indicates that the key cells of the innate and adaptive immune systems are present and functionally responsive to antigens; however, there is a certain degree of compartmentalization within the tract. The identification of TLRs in the fallopian tubes, uterus, cervix, and vagina and the presence of ECs, macrophages, DCs, NK cells, and neutrophils throughout the reproductive tract along with their responsiveness to selected PAMPs indicate that the female reproductive tract has evolved to meet the challenges of STDs, while at the same time supporting an immunologically distinct fetal placental unit. To meet these diverse challenges, innate and adaptive immune system in the female genital tract are precisely regulated not only by a network of cytokines and chemokines, but also by the sex hormones estrogen and progesterone. Understanding the specialty of the genital tract immune system is of critical importance, because STDs are and will be a major worldwide health problem. Despite extensive efforts, only limited success has been achieved in dealing with a growing list of STDs. The role of immune factors in the control of genital viral and bacterial infections appears complex and needs further studying, also with respect to creating vaccines. Despite the recognition that innate immunity as the first line of defense and adaptive immunity, especially Th1 immune responses, play a critical role in preventing infection and in limiting viral replication, factors such as antimicrobials and TLRs that contribute to the mucosal response in the female genital tract have only recently begun to receive attention. Further studies are also needed to elucidate the relationship between mucosal immunity, the hormonal environment, and response to pathogen challenge. More data must be collected on the mechanisms of immune evasion by several pathogens such as HSV, N. gonorrheae or Chlamydia. While considerable information can be obtained from animal experiments, important differences in the physiology of reproduction and the immune sytem result in the need for studies in humans. Further knowledge on female tract immunology will also impact on immunological approaches to contraception, immunological infertility and the immunological aspects of pregnancy. This does not only involve new options for diagnostics but also for treatment of pregnancy complications such as preeclampsia, preterm birth and early pregnancy loss as well as for infertility. Pregnancy involves maternal tolerance of the semiallogenic histoincompatible fetus and is characterized by the enhancement of the innate immune system and suppression of the adaptive immune response, probably with progesterone as the important regulator. In opposite to normal pregnancy, improper immune responses and an unbalanced cytokine network may characterize implantation failures, pregnancy loss and obstetric complications. These are the presence of elevated Th1/Th2 cell ratios, high concentrations of Th1 cytokines, elevated NK cell cytotoxicity and levels, and emergence of various autoantibodies. These immunological approaches needs to be investigated and evaluated further with respect to widening of treatment options by modification of immune responses