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FP5

MUCIMM Résumé de rapport

Project ID: QLK2-CT-1999-00228
Financé au titre de: FP5-LIFE QUALITY
Pays: Italy

Evaluation of the role of dendritic cells in mucosal immunity

We have studied the role of DC in mucosal immunity. In particular, we have established a method for DC and epithelial co-culture in vitro and we have studied the effects of bacterial vectors on DC using both in vitro and in vivo approaches. Penetration of pathogens expressing invasion genes in the gut mucosa is believed to occur mainly through specialized epithelial cells, called M cells, which are located in Peyer’s patches (PP). However, S. typhimurium deficient in invasion genes encoded by Salmonella Pathogenicity Island 1 (SPI1), are still able to reach the spleen following oral administration, suggesting the existence of an alternative route for bacterial invasion, independent of M-cells.

We have been able to show a new mechanism for bacterial uptake in the mucosa tissues, which is mediated by dendritic cells (DC). DC open the tight junctions between epithelial cells, send dendrites outside of the epithelium and directly sample bacteria. Moreover, as DC expresses tight junction proteins, such as occludin, claudin 1 and Zonula occludens 1, the integrity of the epithelial barrier is preserved. With this knowledge we should be able to design bacterial vectors as suitable delivery systems at the mucosal sites.

Since new evidence suggests that dendritic cells (DC) orchestrate the two types of mucosal immune responses, immunity and tolerance, we wanted to evaluate the mechanism involved in this regulation. Thus, our aim was to study mucosal dendritic cells and their interaction with the mucosal adjuvant molecule, cholera toxin (CT), and its derivative forms. By the use of antigen conjugated to CT we managed to induce DC maturation in vitro, with up-regulation of different co-stimulatory surface markers. In contrast, using the enzymatically inactive binding moiety of CT, the CTB adjuvant, and the immature form of the DC was preserved.

These phenotypically distinct DC stages go along with our in vivo findings, showing that only CT conjugated to the OVA antigen triggered an immune response, while CTB-OVA induced tolerance. The different responses were induced despite equal uptake of antigen coupled to each carrier molecule, emphasizing the importance of enzymatic activity comprised by the holotoxin.

Conjugates traced in vivo were found accumulated at the periphery of the B cell follicles, both after CT-OVA as well as CTB-OVA administration, where the main part of OVA carrying cells were CD11c+ i.e. DC. We continued to use these conjugates to target DC at mucosal membranes, herewith controlling the induction of mucosal responses. Hence, by oral feeding of OVA coupled to CT or its B-subunit we were able to target mucosal DC and manipulate their differential stages.

Antigen bound to CT induced strong local recruitment of DC to the GALT, in line with high production of specific IgA in the gut mucosa. The influx of DC was most evident in the Peyer´s patches (PP) with a huge amount of cells entering the subepithelial dome. This infiltration into the PP was also found after administering CTB-OVA, although poor gut IgA immunity was detected. Interestingly, both CT- and CTB-OVA conjugates triggered influx of DC to the draining mesenteric lymph node, suggesting migration of immature- as well as mature DC to the lymph nodes.

To find out which genes are differentially regulated by CT- and CTB stimuli we have now performed a global gene analysis to analyse the mechanisms involved in tolerance and immunity. Preliminary results are that more than 400 genes in CT treated DC are induced, whereas only 25 are induced using the CTB molecule. These genes will now be further analysed to define their role in vivo. Thus, through the use of distinct immunomodulatory adjuvants, CT and CTB, and their interaction with DC, we are now able to monitor the immune responses in favour of active immunity using the enzymatically active CT-OVA conjugate or tolerance by the use of CTB-OVA conjugate.

In addition, studies expanding mucosal DC with flt3L injection have recently been initiated to thoroughly characterize the DC in the gut mucosa and unravel the cellular features that distinguish mucosal IgA response from oral tolerance.

More information on the MUCIMM project can be found at: http://www.altaweb.it/mucimm/.

Informations connexes

Contact

Paola RICCIARDI-CASTAGNOLI, (Head of Molecular and Cellular Immunology Programme)
Tél.: +39-0264483559
Fax: +39-0264483552
E-mail