Cigarette smoking alters intestinal barrier function and Peyer's Patch composition

Smokers have a two-fold increased risk to develop Crohn’s disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Interestingly, the Peyer’s patches in the terminal ileum are the sites where the first CD lesions develop. To investigate whether smoke exposure causes alterations in Peyer’s patches, we studied C57BL/6 mice after exposure to air or cigarette smoke for 24 weeks. First, barrier function of the follicle-associated epithelium overlying Peyer’s patches was evaluated. We demonstrate that chronic smoke exposure is associated with increased apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in the ileal Peyer’s patches. We observed significant increases in total dendritic cells (DC), CD4+ T-cells (including regulatory T-cells) and CD8+ T-cells after smoke exposure compared with air-exposed animals. The CD11b+ DC subset almost doubled. Interestingly, these changes were accompanied by an up-regulated mRNA expression of the chemokines CCL9 and CCL20, which are known to attract CD11b+ DC towards the subepithelial dome of Peyer’s patches. Our results demonstrate that cigarette smoke exposure induces apoptosis in follicle-associated epithelium and is associated with immune cell accumulation in Peyer’s patches, changes which can predispose to the development of CD

Lymphoid follicles in (very) severe COPD: beneficial or harmful?

Abstract: Inflammation is a main pathogenetic factor in the development and progression of chronic obstructive pulmonary disease (COPD) Recently, it has become clear that not only the innate, but also the specific immune response plays a role. A striking finding, in particular in lungs of patients with severe COPD, often with a predominant emphysema phenotype, is the presence of B-cell follicles. As seen in other tissues, these follicles are the result of lymphoid neogenesis. The finding of oligoclonality in B-cell follicles in COPD suggests that they play a role in local antigen specific immune responses. To date, it is not known which antigens may be involved; microbial antigens, cigarette smoke-derived antigens and antigens from extracellular matrix breakdown products have been suggested. Consequently, the pathogenetic role of this follicular B-cell response is not yet clear. It might be protective against microbial colonisation and infection of the lower respiratory tract and, therefore, beneficial, or it could be of a more harmful (autoimmune) nature, directed against lung tissue components. It is necessary to determine the specific antigen(s) and to explore the exact role of the COPD related B-cell response in order to include modulation of this response and develop therapeutic options

CCR7 Modulates Pulmonary and Lymph Node Inflammatory Responses in Cigarette Smoke-Exposed Mice

Peribronchial lymphoid follicles have recently been identified as one of the hallmark features of (severe) chronic obstructive pulmonary disease (COPD). However, little is known about the relative contribution of peribronchial lymphoid follicles vs mediastinal lymph nodes in inflammatory responses in COPD patients and animal models. In a murine model of COPD, we studied inflammatory responses in airways, lungs, and mediastinal lymph nodes of wild-type (WT) vs CCR7 knockout (CCR7(-/-)) mice upon subacute or chronic exposure to cigarette smoke (CS). Although crucial for the organization of the secondary lymphoid organs, CCR7 was not required for the development of chronic CS-induced pulmonary lymphoid follicles. Moreover, T cell numbers were significantly increased in airways and lungs of air-exposed CCR7(-/-) mice, and they continued to increase upon chronic CS exposure. Unexpectedly, subacute CS-induced inflammation in airways and lungs, including airway neutrophilia and the recruitment of inflammatory-type CD11b(+) dendritic cells, depended greatly on CCR7. In the draining lymph nodes, chronic CS exposure induced CCR7-dependent recruitment of airway-derived dendritic cells, accompanied by increases in CD4(+) and CD8(+) T cells. Correspondingly, CS exposure up-regulated mRNA expression of CCR7 ligands CCL19 and CCL21-Ser in lymph nodes of WT mice, but not CCR7(-/-) mice. In the lungs of WT mice, chronic CS exposure significantly increased CCL19 mRNA and protein. Furthermore, double staining for CCL19 and pro-surfactant protein C showed that alveolar type II cells express high levels of CCL19. These data unveil a so far unappreciated role for CCR7 in modulating inflammatory responses in airways and lungs

Evaluation of beta-glucan particles as mucosal delivery systems in the Peyer's patch regions of the murine small intestine

Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microcapsules (3-5 µm) were prepared from accharomyces cerevisiae and loaded with Alexa Fluor 488-conjugated bovine serum albumin. Particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells. Interestingly, flow-cytometric analysis indicated a modest but clear uptake of particles in the B-cell population. Confocal microscopic images showed yeast particles localized in the Follicle Associated Epithelium. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP

Evaluation of β-glucan particles as mucosal delivery system in the Peyer's patch regions of the murine small intestine

Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microparticles (3-5 µm) were prepared from Saccharomyces cerevisiae and loaded with Alexa Fluor 488-conjugated bovine serum albumin. Particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells. Interestingly, flow-cytometric analysis indicated a modest but clear uptake of particles in the B-cell population. Confocal microscopic images showed yeast particles localized in the Follicle Associated Epithelium. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP

Beta-glucan particles as novel antigen delivery systems: towards oral vaccination

Several enteric pathogens infect the body following oral uptake and cause life-threatening diseases such as cholera, dysentery and typhoid fever. Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. Recently, there has been a lot of interest in the use of microparticles as antigen delivery systems in the development of more efficient mucosal vaccines. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microparticles (2-4 µm) were prepared from Saccharomyces cerevisiae and loaded with FITC or Alexa Fluor 488-conjugated bovine serum albumin. Firstly, the appropriateness of β-glucan particles as antigen delivery systems for oral and intestinal applications was assessed in stability tests. Fluorimetric analysis demonstrated that the BSA concentration within particles is stable during the first 12 hours of simulated gastric or simulated intestinal fluid treatment. Secondly, β-glucan particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells, however, a modest particle uptake was repeatedly detected in the B-cell population. Confocal microscopic images showed localization of β-glucan particles in the follicle associated epithelium and B-cell internalization. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. In conclusion, stability tests show that antigen concentration of β-glucan particles remains stable in gastric and intestinal environment. This means that the β-glucan particles can be administered orally without prior enteric coating and are suitable for mucosal delivery of antigen in the murine gastro-intestinal tract. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP and transcytosis of antigen to underlying antigen presenting cells and immune cells

Beta-glucan particles as novel antigen delivery systems : towards oral vaccination

Gastro-intestinal infections are still a main cause of enteric diseases and mortality among humans and animals. Oral vaccination is crucial in generating an adequate local mucosal immune response, however the hostile environment of the intestinal tract and oral tolerance remain huge obstacles that inhibit the ability to successfully develop new mucosal vaccines. A promising strategy for vaccination with safe, biodegradable non-replicating antigen delivery systems has gained increased interest for eliciting cellular and humoral immune responses. The current study evaluates the potential of β-glucan (BGP) and calcium carbonate (CaCO3)- and mannitol-templated polyelectrolyte particles as mucosal antigen delivery systems and their adjuvant characteristics. All microparticle types are efficiently internalized by Caco-2 and HT-29 cell lines and in particular the BGP triggered the expression of pro-inflammatory cytokines IL-23p19, IL-8 and beta-glucan receptors in activated Caco-2 cells and CCL20 in HT-29 cells. In contrast, the expression level of TGF-b, an important mediator of the active component of oral tolerance, was significantly downregulated in HT-29 cells. Oral administration of BGP induced intestinal adaptive immune responses characterized by an increased sIgA and secretory component production. Interestingly, adoptive transfer experiments pointed out the proliferation of naïve OVA-specific CD4+ OT-II cells and increased IL-17 production in spleens of BGP-fed mice upon antigen restimulation. These results demonstrate that BGP enhances MHC-II-presentation and promotes mucosal immune responses preferably skewed towards an Th17 response and represents a promising strategy for oral vaccination

Cigarette smoking alters epithelial apoptosis and immune composition in murine GALT

Smokers have a twofold increased risk to develop Crohn's disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Especially Crohn's ileitis is negatively influenced by smoking. Interestingly, the ileum and, more in particular, the Peyer's patches in the terminal ileum are also the sites where the first CD lesions are found. Several chemokines are implicated in the pathogenesis, among which is the CCL20-CCR6 pathway. Here, we studied the gut-associated lymphoid tissue in C57BL/6 wild-type mice and in CCR6-deficient mice after exposure to air or cigarette smoke for 24 weeks. Apoptotic index of the follicle-associated epithelium overlying the Peyer's patches was evaluated. We found that chronic smoke exposure induced apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in Peyer's patches. Important changes in immune cell composition were observed: total dendritic cells, CD4+ T cells (including regulatory T cells) and CD8+ T cells increased significantly after smoke exposure. The CD11b+ dendritic cell subset almost doubled. Interestingly, these changes were accompanied by an upregulated mRNA expression of the chemokines CCL9 and CCL20. However, no differences in the increase of dendritic cells were observed between wild-type and CCR6-deficient mice. Our results show that cigarette smoke exposure increases apoptosis in the follicle-associated epithelium and is associated with immune cell accumulation in Peyer's patches