ArticlePDF Available

Establishment and first characterization of a sublingual epithelial and immune cell co-culture model

Authors:

Abstract and Figures

We describe here the establishment and first characterization of a co-culture model of human epithelial sublingual cells (HO-1-u-1 cell line) and human dendritic cells derived from human peripheral blood monocytes (PBMC). Cell culture conditions for HO-1-u-1 cells were optimized. First characterization of phenotypic features by electron microscopy and fluorescence imaging revealed resemblance to sublingual tissue specimen from healthy donors. Successful co-culturing of epithelial and dendritic cells (DCs) was confirmed by confocal laser scanning microscopy. Stimulation of HO-1-u1 cells alone and the epithelial/DC co-culture by incubation with liposomes, virosomes and influenza virus lead reproducibly to the release of inflammatory cytokine GM-CSF. This co-culture model may be suitable for elucidation of mechanisms involved in the immune response at the sublingual epithelium as well as for the evaluation of novel topical vaccines, potentially replacing cumbersome ex vivo and in vivo methods currently in place. Copyright © 2014 Elsevier B.V. All rights reserved.
Content may be subject to copyright.
A preview of the PDF is not available
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
The sublingual locus has recently received great attention as a delivery site for various immunotherapies, including those that induce allergen-specific tolerance, and for vaccines that generate protective immunity. To further understand the immune functions of the human sublingual mucosa, we characterized the distribution of various immunocytes therein by immunohistochemistry. We identified professional antigen presenting cells (APCs), including Langerhans cells (LCs) and macrophages. CD1a + and langerin + LCs were further found to be distributed in the basal and supra-basal layers of the epithelium, and macrophages were identified in the lamina propria. HLA-DR + cells were observed in both the epithelium and the lamina propria, which mirrors the tissue distribution of LCs and macrophages within these tissues. CD3 + , CD4 + , and CD8 + T cells were found to be distributed along the basal layer of the epithelium and also in the lamina propria. Although B cells, plasma cells, and Foxp3 + regulatory T cells (Tregs) were only occasionally observed in the human sublingual mucosa in the absence of inflammation, they did show enrichment at inflammatory sites. Hence, we have further elucidated the immune cell component distribution in human sublingual mucosa.
Article
Full-text available
Mucosal immunization offers the promises of eliciting a systemic and mucosal immune response, as well as enhanced patient compliance. Mucosal vaccination using defined antigens such as proteins and peptides requires delivery systems that combine good safety profiles with strong immunogenicity, which may be provided by virus-like particles. Virus-like particles are assembled from viral structural proteins, and thus devoid of any genetic material. They excel by mimicking natural pathogens, therefore providing antigen-protecting particulate nature, inherent immune-cell stimulatory mechanisms and tissue-specific targeting depending on their parental virus. Nevertheless, despite the number of promising pre-clinical results, virus-like particles remain rarely investigated in clinical studies. This review is intended to give an overview of obstacles and promises of virus-like particle-based mucosal immunization, as well as identifying the strategies to further improve suitability of virus-like particle while maintaining a good safety and tolerability profile.
Article
Full-text available
Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.
Article
Full-text available
Influenza virosomes have been used for more than 10 years in commercial vaccines. The technology has been further developed as a carrier and adjuvant system for subunit vaccines, in particular for synthetic peptides. The extensive amount of preclinical and clinical data supports the notion that influenza virosomes represent a platform technology that ensures robust and long-lasting immune responses against subunit antigens with an excellent safety profile. Structurally and functionally, virosomes are enveloped virus-like particles, although they are assembled in vitro. This unique feature ensures a tight control of their composition and at the same time provides the flexibility to adapt the particle to various types of antigens. The mode of action of virosomes is complex and includes carrier as well as immune-stimulatory functions.
Article
Full-text available
The sublingual route has been used for many years to deliver drugs and small molecules to the bloodstream. Surprisingly, the potential of this route for delivering vaccines has received very little if any attention until recently. During the past few years, a number of laboratories have documented the efficacy of sublingual immunization for inducing a broad range of immune responses in different experimental animal systems using a variety of antigens, including soluble proteins, inert particulate antigens (killed viruses, virus-like particles, bacterial extracts) as well as live-attenuated viruses. In most cases, systemic and mucosal immune responses, including humoral and cytotoxic T-cell responses were induced in both mucosal and extra-mucosal tissues. Overall, sublingual immunization was comparable to nasal immunization regarding the magnitude, breadth, and anatomic dissemination of the induced immune responses. Importantly, and contrary to nasal administration, sublingual administration did not redirect antigens and/or adjuvants to the brain. Here we review the results of pre-clinical studies using animal models of respiratory, intestinal and genital infections. These promising results provide a foundation for testing the approach in humans.
Article
We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.
Article
Allergic rhinitis and asthma have a very high prevalence and constitute a health problem with a relevant burden of disease, concerning medical and economical issues. Among the treatments of allergy, specific immunotherapy (IT) has the capacity to favourably alter the natural history of the disease both during and after its performance, and thus to reduce the direct and indirect costs of allergic rhinitis and asthma. A number of studies reported a cost reduction for traditional, subcutaneous IT (SCIT). We analyzed the available studies on economic evaluation of sublingual immunotherapy (SLIT) in children and adults. Articles were retrieved from Medline and Embase using the terms economic aspects, pharmacoeconomics, cost effectiveness, and sublingual immunotherapy. 18 articles were found in English language, 7 studies investigated the economic advantage of SLIT over standard drug treatment in subjects with respiratory allergy, and 2 of them included a comparison of costs of SCIT and SLIT. The data obtained provide evidence that SLIT is associated to economic advantages and/or monetary savings, specifically in terms of reduction of economic burden of the disease. Although the number of studies is still limited, the available data support a SLIT effect on sparing costs for respiratory allergy.
Article
Needle-free vaccine delivery has become a global priority, both to eliminate the risk of improper and unsafe needle use and to simplify vaccination procedures. In pursuit of greater ease of vaccination, a number of needle-free delivery routes have been explored, with mucosal routes being perhaps the most prominent. Since the vaccine administration route significantly affects immune responses, numerous researchers are attempting to develop alternative vaccine delivery methods including a mucosal route. My group's recent studies demonstrate the potential of the sublingual (s.l.) route for delivering vaccines capable of inducing mucosal as well as systemic immune responses. Sublingual administration conferred effective protection against a lethal challenge with influenza virus (H1N1) or genital papillomavirus. Moreover, CCR7-CCL19/CCL21-regulated dendritic cells are responsible for activation of T and B cells following s.l. administration. This review highlights current knowledge about the safety and effectiveness of s.l. vaccination and describes how s.l. vaccination can induce both systemic and mucosal immunity.
Article
The development of mucosal vaccines for prevention of infectious diseases caused by pathogens entering through the mucosal surfaces is an important and challenging objective. To this purpose, we evaluated the efficacy and durability of immune response induced by sublingual immunization with tetanus toxoid (TT) as an antigen in the presence of mucosal adjuvants, such as E. coli Heat-Labile enterotoxin (LT) or the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Both serum anti-TT IgG and mucosal anti-TT IgA antibodies reached a peak after four immunizations and decreased over time, maintaining detectable titers up to 4 months after the last immunization. Similarly, antigen-specific antibody secreting cells in bone marrow and TT-specific CD4+ and CD8+ T cells in draining lymph nodes and spleen were present up to 4 months from the last immunization. Overall, LT-treated mice showed significantly higher responses compared to LTK63 immunized mice. The efficacy and persistence of the immune response induced by sublingual immunization with different adjuvants strongly suggest that this route represents an appealing and promising alternative to the other mucosal routes of vaccine delivery.