Claudio Nicoletti

Oral delivery of Lactobacillus casei Shirota modifies allergen‐induced immune responses in allergic rhinitis

Background Changes in the composition of the gut microbiota have been implicated in the pathogenesis of allergic disorders, suggesting beneficial interactions between the intestinal immune system and specific bacterial strains. Lactobacilli are naturally present within the complex gastrointestinal microbiota of humans and they are currently present in many probiotic supplements.

Unsolved mysteries of intestinal M cells

M cells are highly specialised cells present within the epithelium overlying organised lymphoid follicles of the small and large intestine. They play a central role in the initiation of mucosal immune responses by transporting antigens and microorganisms to the underlying lymphoid tissue. In this way the mucosal immune system encounters the limitless variety of antigens that enter the body through the gut mucosa and reacts by mounting specific mucosal and systemic immune responses. Despite the role of M cells in mucosal defence many basic aspects of their biology, the most controversial being their origin within the follicle associated epithelium (FAE), still remain the subjects of debate. Recently, new information on the complex interactions of luminal microorganisms, mucosal immune system, and epithelial cells, that are instrumental in the induction of this cell phenotype, have become available. Here, the most novel data and hypotheses on M cell genesis and function in the gut are reviewed and discussed. The main task of the epithelium overlying mucosal surfaces of the intestinal tract is to provide an effective barrier to the vast majority of macromolecules and microorganisms present in the intestinal lumen. This is achieved by several means. Firstly, the epithelium is formed by cells joined by tight junctions that allow passage of water and ions but provide an effective mechanical barrier to macromolecules.1 2 Secondly, mucosal surfaces are covered by local secretions of mucus, secretory IgA antibodies, and by a thick glycocalix.3-6 These features and the closely packed carpet of microvilli present on absorptive cells prevent contact and binding of macromolecules and potential pathogens to the epithelium. On the other hand, the intestinal epithelium must also provide portals through which antigens and microorganisms are delivered to the intestinal immune system in order to induce immune responses. In fact, it is now established …

Up-regulation of microsphere transport across the follicle-associated epithelium of Peyer’s patch by exposure to Streptococcus pneumoniae R36a

Transport of antigens through the follicle‐associated epithelium (FAE) of Peyers patch (PP) is the critical first step in the induction of mucosal immune responses. We have previously described that short‐term exposure to Streptococcus pneumoniae R36a induced dramatic morphological alterations of the FAE in rabbit PP. These results prompted us to investigate whether the pneumococci‐induced modifications were accompanied by enhanced ability of the FAE to transport antigens. We addressed this problem by evaluating the ability of the FAE to bind, internalize, and transport fluorescent polystyrene microparticles, highly specific to rabbit M cells, after exposure to S. pneumoniae. Quantitative study revealed a marked increase in the number of microspheres in PP tissues exposed to S. pneumoniae compared to tissues exposed to either phosphate‐buffered saline or Escherichia coli DH5a as controls. No sign of bacterially induced damage to the epithelial barrier was observed. Further confocal microscopy analysis of the FAE surface showed that a significant increase in the number of cells that showed both morphological and functional features of M cells took place within pneumococci‐treated PP tissues. These data provide the first direct evidence that the FAE‐specific antigen sampling function may be manipulated to improve antigen and drug delivery to the intestinal immune system.—Meynell, H. M., Thomas, N. W., James, P. S., Holland, J., Taussig, M. J., Nicoletti, C. Up‐regulation of microspheres transport across the follicle‐associated epithelium of Peyers patch by exposure to Streptococcus pneumoniae R36a. FASEB J. 13, 611–619 (1999)

Salmonella Induces Flagellin- and MyD88-Dependent Migration of Bacteria-Capturing Dendritic Cells Into the Gut Lumen

BACKGROUND & AIMS Intestinal dendritic cells (DCs) sample bacteria, such as Salmonella, by extending cellular processes into the lumen to capture bacteria and shuttle them across the epithelium; however, direct evidence of bacteria-loaded DCs travelling back into the tissue is lacking. We hypothesized that sampling is paralleled by migration of DCs into the lumen prior to or following the internalization of Salmonella. METHODS The small intestine and the colon of BALB/c and C57BL/6 mice were challenged with noninvasive Salmonella enterica serovar Typhimurium SL1344-DeltaSalmonella pathogenicity island (SPI) 1 or Escherichia coli DH5alpha by using isolated loops or oral administration by gavage. Transepithelial migration of DCs was documented by immunohistochemistry, microscopy, and flow cytometry. The role of flagellin was determined by using flagellin (DeltafliC DeltafljB)- and SPI1-SPI2 (DeltaSPI1 DeltassrA)-deficient Salmonella, flagellated E coli K12, and MyD88 mice. RESULTS Salmonella DeltaSPI1 induced migration of CD11c(+)CX(3)CR1(+)MHCII(+)CD11b(-)CD8alpha(-) DCs into the small intestine, whereas flagellin- and SPI1-SPI2-deficient Salmonella, soluble flagellin, and E coli DH5alpha or flagellated K12, failed to do so. DC migration did not occur in the colon; it was not observed in MyD88 mice, and intraluminal DCs internalized Salmonella but did not cross the epithelium to return into tissues. Finally, DC migration was not linked to Salmonella-induced damage of the epithelium. CONCLUSIONS DC-mediated sampling of Salmonella is accompanied by flagellin- and MyD88-dependent migration of Salmonella-capturing DCs into the intestinal lumen. We suggest that the rapid intraluminal migration of Salmonella-capturing DCs may play a role in the protection of the intestinal mucosa against bacterial infection.

Factors influencing the incidence and prevalence of food allergy

Food allergy is an increasing problem in Europe and elsewhere and severe reactions to food are also becoming more common. As food allergy is usually associated with other forms of allergic sensitisation it is likely that many risk factors are common to all forms of allergy. However the potential severity of the disease and the specific public heath measures required for food allergy make it important to identify the specific risk factors for this condition. Food allergy is unusual in that it often manifests itself very early in life and commonly remits with the development of tolerance. Hypotheses that explain the distribution of food allergy include specific genetic polymorphisms, the nature of the allergens involved and the unique exposure to large quantities of allergen through the gut. Progress has been made in developing more specific and testable hypotheses but the evidence for any of these is still only preliminary. Further collaborative research is required to develop an appropriate public health response to this growing problem.

Improving M cell mediated transport across mucosal barriers: do certain bacteria hold the keys?

Specialized microfold (M) cells of the follicle‐associated epithelium (FAE) of the mucosal‐associated lymphoid tissue (MALT) in gut and the respiratory system play an important role in the genesis of both mucosal and systemic immune responses by delivering antigenic substrate to the underlying lymphoid tissue where immune responses start. Although it has been shown that dendritic cells (DC) also have the ability to sample antigens directly from the gut lumen, M cells certainly remain the most important antigen‐sampling cell to be investigated in order to devise novel methods to improve mucosal delivery of biologically active compounds. Recently, novel information on the interactions between bacteria and FAE have come to light that unveil further the complex cross‐talk taking place at mucosal interfaces between bacteria, epithelial cells and the immune system and which are central to the formation and function of M cells. In particular, it has been shown that M cell mediated transport of antigen across the FAE is improved rapidly by exposure to certain bacteria, thus opening the way to identify new means to achieve a more effective mucosal delivery. Here, these novel findings and their potential in mucosal immunity are analysed and discussed, and new approaches to improve antigen delivery to the mucosal immune system are also proposed.

Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium

This study was undertaken in order to study the effects of short‐term exposure of the follicle‐associated epithelium (FAE) of rabbit Peyers patches to a non‐intestinal, Gram‐positive bacterium. Isolated ileal loops, each containing one Peyers patch (PP), were stimulated for short periods of time (30 and 60 min) with Streptococcus pneumoniae R36a, a micro‐organism normally not present in the intestinal area. Samples from antigen‐stimulated and control Peyers patches were analysed by light (LM), transmission electron (TEM), and scanning electron microscopy (SEM). Stimulation with living pneumococci induced dramatic changes in FAE architecture and morphology. A massive passage of cells from lymphoid tissue to the FAE was rapidly detectable, accompanied by alterations of the FAE surface, with a marked increase of M‐cell area. Furthermore, TEM analysis revealed that M cells were able to internalize living pneumococci. S. pneumoniae R36a is a valid experimental model for the further study of the unique antigen sampling function which characterizes the highly specialized FAE in Peyers patches.

Fast: Towards safe and effective subcutaneous immunotherapy of persistent life-threatening food allergies

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen-specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side-effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site-directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre-clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum-absorbed hypoallergens will be evaluated in phase I/IIa and IIb randomized double-blind placebo-controlled (DBPC) clinical trials, with the DBPC food challenge as primary read-out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

The impact of ageing on the intestinal epithelial barrier and immune system

The vast mucosal surface of the intestine is patrolled by a large number of lymphocytes forming the intestinal immune system. Like any other system in the body, this branch of the immune system is affected by ageing. Although our knowledge on the age-associated changes of the systemic immune system has improved over the past few years, our understanding of the mechanisms of senescence of both adaptive and innate immune system of the gastrointestinal (GI) tract is still largely incomplete. However, recent advances in the field have shown that the identification of the events underlying the ageing process in the gut may have important consequences on health and wellbeing far beyond the GI-tract. The aim of this review is to summarise the impact of ageing on the intestinal immune system, including the gut epithelium and other components of the intestinal barrier that maintain intestinal immune homeostasis and shape antigen-specific immune responses.

The repertoire diversity and magnitude of antibody responses to bacterial antigens in aged mice: I. Age-associated changes in antibody responses differ according to the mouse strain

Aging influences the host immune responses in various ways. In aging mice we have studied the antibody responses to two unrelated bacterial antigens. Streptococcus pneumoniae R36a vaccine (Pn) and TNP coupled to Brucella abortus (TNP-BA). Aged animals (20-24 months old) of the C57BL/6 strain had markedly reduced numbers of IgM antibody plaque-forming cells (PFC) to Pn as compared to young/adult mice (2-3 months old). In contrast, the anti-Pn IgM PFC responses of aged BALB/c mice were consistently higher than they were in the young/adult mice. The increased anti-Pn responses were not due to a nonspecific immunostimulation, because the responses of aged BALB/c mice to TNP-BA were lower as compared to the adults. However, the aged BALB/c mice responded relatively poorly to Pn challenge, and their IgG responses (as determined by ELISA plaque assay) demonstrated a very high individual variability. The clonotypic diversity of anti-Pn response in young BALB/c and C57BL/6 is limited, such that the majority of PFC produce antibody that express all idiotopes (Id) of the T15 immunoglobulin encoded in the VH-S107/Vk22 genes. In contrast, the PFC from aged mice are diverse, expressing incomplete T15 Id or none at all, suggesting that the antibodies are encoded by altered T15 genes and by different, non-T15 genes. Our data demonstrate that the age-related changes in the magnitude of antibody response to certain antigens are influenced by the host genetic make-up, and that the changes in magnitude and diversity of antibody response may be unrelated to each other.