Dendritic Cells Shuttle Microbes Across Gut Epithelial Monolayers

Immunobiol. (2001) 204, pp. 572 – 581 © 2001 Urban & Fischer Verlag http://www.urbanfischer.de/journals/immunobiol

Department of Biotechnology and Bioscience, University of Milano-Bicocca, Milano, Italy

Dendritic Cells Shuttle Microbes Across Gut Epithelial Monolayers MARIA RESCIGNO, GIANLUCA ROTTA, BARBARA VALZASINA, and PAOLA RICCIARDICASTAGNOLI

Abstract Understanding the mechanisms governing the type of induced immune response after microbial invasion, could be of crucial importance for the rational design of a bacteria-based vaccine. Targeting a vaccine directly to dendritic cells (DCs), which are considered the most powerful antigen presenting cells, could be extremely effective. Here we describe that CD11b+CD8a– dendritic cells are involved in the direct bacterial uptake across mucosal surfaces. DCs are widely spread in the lamina propria of the gut and are recruited at the site of infection. DCs open the tight junctions between epithelial cells, send dendrites outside of the epithelium and sample bacteria. Moreover, the integrity of the epithelial barrier is preserved because DCs express tight junction proteins, such as occludin, claudin 1 and Junctional Adhesion Molecule (JAM) and can establish tight junctions-like structures with neighbouring epithelial cells.

Introduction The rational design of bacteria-based vaccines requires the knowledge of basic mechanisms controlling microbial entry. Mucosal routes of vaccine administration are most appealing for the facility of application, however, the intestinal mucosa has always been considered almost inaccessible to microbes. The presence of a brush border on the lumenal cell surface opposes steric hindrance whereas a belt of tight junctions (TJ) between cells (1, 2) impedes the paracellular route of bacteria and of their metabolites. Thus, entry of pathogens (3–5), has been shown to occur mainly through specialized epithelial cells, called M cells which lack an organized brush border. Penetration of M cells by bacteria requires the expression of invasin proteins; nevertheless, S. typhimurium deficient in invasion genes encoded by Salmonella Pathogenicity Island 1 (SPI1), are still able to reach the spleen following oral administration (6), suggesting an M cell independent pathway. Dendritic cells (DCs) are migratory and phagocytic cells, ideally located for antigen sampling in tissues which interface the external environment, such as skin and mucosae, where they perform a sentinel function for incoming pathogens (7–9). After encountering the microorganisms DCs upregulate their capacity to present antigens and become 0171-2985/01/204/05-572 $ 15.00/0

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professional antigen presenting cells able to prime naive T cells. DCs with typical features of immature cells (10) have been described in PP forming a dense layer of cells in the subepithelial dome, just beneath the follicle epithelium (11, 12), and in the lamina propria of the gut inserting into the epithelium (13), but they have never been observed facing the gut lumen. It is not clear whether DCs can directly take up bacteria across the mucosal epithelium or if they intervene only after the bacterial internalization has been carried out by M cells. As DCs are able to activate naive T cells, understanding how to target bacterial vaccines directly to DC could have enormous impact in vaccine development. Thus, we have investigated the interaction of DC with epithelial cells both in vitro and in vivo, in the mouse, to dissect out the involvement of DCs in bacterial uptake. We find that DCs express TJ proteins, open the TJ between epithelial cells and can directly take up microorganisms preserving the integrity of the epithelial barrier.

Materials and Methods Cells, bacterial strains and reagents

The D1 cells were cultured in DCs medium (complete IMDM supplemented with 30% R1 conditioned medium containing 30 ng/ml GM-CSF) as described (14). The auxotrophic S. typhimurium aroA strain SL7207 (S. typhimurium 233765 derivative hisG46, DEL407 [aroA::Tn10{Tc-s}]), was kindly provided by Dr. B. A. D. Stocker, Stanford, CA. SL7207-GFP was generated by transformation with a plasmid (pkk223.3) containing the gene encoding green fluorescence protein (GFP). Bacteria were grown in Brain Heart Infusion (BHI) to an OD600 = 0.6. GFP-D1 cells were generated as already described (15), whereas labeling of cells with CellTracker™ Orange CMTMR, Molecular probes, (5-(and-6)-(((4-chloromethyl) benzoyl)amino) tetramethylrhodamine) was according to manufacturer’s instructions. Caco-2-D1 Transwell ® coculture system

Caco-2 cells were seeded either on the lower or on the upper face of 6.5-mm filters (3-mm pore Transwell ® filters, Costar, Cambridge, MA, USA) for 10–15 days in a 24 well plate until a TER of ~330 W cm2 was achieved. 4 ×105 D1 cells, GFP-D1 or CMTMR-D1 cells were cultured either from the basolateral side or from the apical side of Caco-2 monolayer for 2 to 18 h. Fluorescent bacteria (108 CFU) were incubated always from the apical side. At different time points after addition of D1 cells or after subsequent addition of bacteria, TER was measured and the composition of medium facing the basolateral side of Caco-2 cells was analyzed by FACScan (Becton Dickinson). Filters were either fixed with 1% paraformaldehyde for 60 min. at 4 °C and processed for fluorescent confocal microscopy . Immunofluorescence staining

Cells or cryo-sections were fixed in 1% paraformaldehyde for 60 min. at 4 °C and processed for immunofluorescence. FITC- or PE- conjugated anti-CD11c, anti-CD8a and antiCD11b antibodies were purchased from Pharmingen. Rabbit anti-occludin was purchased from Zymed laboratories. Gene-chip analysis

Total RNA was prepared from 15 ×106 LPS-treated and untreated DCs by TRIzol ® reagent (Gibco) according to manufacter’s instructions and mRNA was reverse transcribed to cDNA and cRNA (Enzo kit). cRNA was hybridized to Affimetrix 11 K gene-chips.

574 · M. RESCIGNO et al. Ligated loop experiments

Mice were anesthetized with 2,5% avertin for the entire duration of the experiment. Stretches of the small intestine of nearly three centimeters long were ligated at both extremities with surgical thread. 5 ×108 or 109 bacteria were resuspended in 200 ml and injected in the loop. 30, 60 or 120 min. later, intestines were asported, snap frozen in embedding medium (OCT, Sakura) and stored at –20 °C until cutting. 7 mm cryosections were cut, air-dried for 1 h, fixed in 1% PFA and processed for immunofluorescence as above. Specimens were mounted in Pro-Long anti-fade medium (Molecular Probes).

Results DCs creep between epithelial cells when bacteria are seeded from the apical side

The interaction of DCs with epithelial cells, was studied in a transwell co-culture system in vitro (16). We cultured a monolayer of differentiated human enterocyte cell line Caco-2 on the lower face of 6.5-mm filters (Corning) until a transepithelial electric resistance (TER) of ∼330 W cm2 was achieved. Subsequently, growth factor dependent D1 cells expressing the green fluorescent protein, representing mouse immature DCs (8, 14, 17), were seeded for 4 h on the other side of the filter, facing the basolateral membrane of the Caco-2 cells. After the incubation time, the transwells were extensively washed to eliminate DCs that had not attached to the filter, and bacteria were seeded from the apical side. The filters were fixed with paraformahldehyde and stained with antioccludin antibody. As you can see from Figure 1a, GFP-D1 cells could be detected all the way to the apical surface and this was evident only in the presence of bacteria. Interestingly during the entire experiment, the trans-epithelial resistance was unchanged suggesting that the integrity of the epithelial barrier was preserved (Fig. 1b). DCs can take up bacteria from the apical side

We have already shown that in contrast to lymphocytes (18), DCs did not induce the formation of M cell-like cells. The DCs, however, that had crept between epithelial cells were able to open the epithelial tight junctions without disorganizing the brush border. DCs added to the apical compartment failed to migrate into the epithelial monolayer (not shown), suggesting that the interaction with epithelial cells and/or the release of epithelial chemotactic signals is polarized. The ability of DCs to gain access to the lumenal side of the epithelium and their role in mediating bacterial transport across the monolayer was analyzed by confocal fluorescent microscopy using GFP-Salmonella. The capacity of DCs to internalize bacteria, including attenuated Salmonella, is well established (19–21), while uptake from the apical membrane of differentiated Caco-2 is minimal. Thus we have incubated GFP-salmonellae (108 CFU) from the apical side of the co-cultured transwell and have analyzed the position of the bacteria in the filter throughout their transport across the monolayer. We observed that bacteria were found associated only with DCs stained with CellTracker™ Orange CMTMR, (Molecular probes), but not with Caco-2 cells (Fig. 1c).

Figure 1. DCs creep between a monolayer of differentiated human Caco-2 enterocyte cell line. A monolayer of Caco-2 cells was grown on the lower face of 6.5-mm filters (3-mm pore Transwell filters, Costar, Cambridge, MA) for 10-15 days in a 24-well plate until a TER of ~330 W_cm2 was achieved. Subsequently, D1 cells (4 ×105) were seeded overnight facing the basolateral side of the Caco-2 monolayer. Bacteria (108 CFU) were incubated from the apical side of the epithelium first at 4 °C for 60 min and then at 37 °C. a) D1-GFP migrated all the way to the apical side of the monolayer. b) TER was unchanged throughout the whole experiment. c) Fluorescence for GFP or for CMTMR are recorded independently so as to exclude any interference in their respective channels. Colocalization of DCs with bacteria or contact are shown. The scheme depicts the filter organization: CMTMR-D1 cells, GFPbacteria, and filter.

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Figure 2. DCs express proteins involved in the formation of tight junctions. D1 cells were either untreated (NT) or incubated with 10 mg/ml LPS for the indicated times. The mRNA was extracted, retro-transcribed to cRNA and hybridized to Affimetrix mouse 11K gene-chips.

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DCs express TJ proteins

TJs consist of a continuous, circumferential belt that seals the apex of epithelial and endothelial cells and prevents the paracellular diffusion of solutes, macromolecules and microorganisms. The major constituents of the TJ strands include three integral membrane proteins, i.e. occludin, claudin and Junctional Adhesion Molecule (22), associated with cytoplasmic proteins, i.e. ZO-1, E-cadherin and catenins, that play an important role in the formation and localization of TJs (23). We have tested whether the D1 cells (Fig. 2), express the typical integral membrane tight junctions proteins. We quantified the mRNA expression after treatment of D1 cells with bacterial lypopolysaccharide (LPS) by means of DNA microarray gene expression analysis. As shown in Figure 2 mRNAs for occludin, claudin-1 and JAM was detected already in immature cells, and their expression was differentially regulated during the maturation process of DCs. Therefore, occludin and claudin 1 can mediate the interaction between DCs and epithelial cells by opening preexisting TJs and forming new TJ-like structures. The role of the differential regulation of TJ-proteins expression remains to be elucidated. DCs take up bacteria also in vivo across gut mucosae

In order to assess whether also in vivo DC could take up bacteria across epithelia, we have carried out experiments of ligated loop of the small intestine in the mouse, and have infected the loops with Salmonella-GFP or PBS, as a control. Thirty min, one or two h later, the ligated loops were snap-frozen and processed for immunohistochemistry with anti-CD11c, anti-CD11b and anti-CD8a to distinguish among the different DCs subsets. Already 30 min after bacterial infection, DC dendrites were sent outside of the epithelium (Fig. 3) and some of them were found in close contact with GFP-bacteria (Fig. 3a, b, arrows) or cells were found outside but still in contact with the epithelium (Fig. 3c). Interestingly, DC present in the lamina propria were mainly CD11b+CD8acells (Fig. 3d, e).

Discussion How the immune system can generate a mucosal rather than a systemic response to bacterial invasion remains an open question, but one that needs to be answered. Understanding the mechanisms governing the type of induced immune response could be of crucial importance for the rational design of a vaccine directed to a particular type of microorganism or even to a tumor. In this study, we provide a novel mechanism that allows DCs to sample environmental microorganisms without compromising the epithelial barrier function and to deliver them into lymphoid tissues where an efficient immune response can be mounted. Indeed, we have shown that after bacterial infection, DCs are are induced to modulate TJ proteins and to establish TJ-like structures with epithelial cells in order to take up antigens without changing the transepithelial resistance. This type of junctions is not species-restricted since it occurs between human epithelial cells and mouse DCs, which is not surprising considering the high level of conservation of these proteins in the two species (∼90% identity). The mechanism that allows the DCs

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to destabilize preexisting epithelial TJs is not known. The presence of occludin in DCs may be sufficient to loosen the epithelial TJ, a destabilization that is followed by the rapid formation of new junctions between the epithelium and the infiltrating DCs. Occludin and claudin-1 are constitutively expressed in immature DCs. Upon bacterial infection, DCs are recruited from the blood and are activated probably via epithelial cell signals; they upregulate the expression of occludin and JAM and downregulate that of Claudin-1. This allows DCs to compete for epithelial occludin and open up the TJs like a zip. Infiltrating DCs face then the gut lumen and can directly sample bacteria, without changing the TER. It is also known that neutrophils are able to migrate across epithelia without affecting the TER and that occludin is involved in the modulation of this process (24). Thus neutrophils could employ the same mechanism to get through the epithelial cells without perturbing the epithelial barrier. Moreover, a similar mechanism for the opening of TJ between endothelial cells could also be employed by leukocytes for their transmigration in the cerebrospinal fluid during experimental meningitis, as blockage of the interaction with JAM by a specific antibody, inhibits the recruitment of both monocytes and neutrophils in the brain parenchyma (25). Because DCs are migratory cells, they can transport pathogens to the mesenteric lymph node and to the spleen for the induction of systemic responses (26), suggesting important physiological relevance for this alternative route of bacterial internalization. Indeed the number of CD11c+ cells is only transiently increased in the intestinal villi when the loops are infected with pathogens. Accordingly, it has been recently described that caspase 1-deficient mice are not colonized by Salmonella in the PP, but they still contain bacteria in the spleen (27). We propose that the bacteria are carried to the spleen by DCs directly after their uptake. We have previously described that also non-pathogenic bacteria can enter via this route (16). Interestingly, an IgA responses to commensal bacteria can be induced by a primitive T cell independent mechanism, that does not require organized follicular lymphoid tissue (28), suggesting involvement of DCs in IgA induction towards commensal bacteria directly in the lamina propria. In conclusion, the exact role of this new mechanism remains to be elucidated and in particular its contribution towards the induction of systemic responses to pathogenic bacteria and mucosal responses to commensal bacteria. Acknowledgements

This work was supported by grants from the Italian Association against Cancer (AIRC), the National Research Council (CNR Project in Biotechnology), the EC contract MUCIMM.

Figure 3. CD11b+CD8a– DCs are responsible for bacterial uptake in vivo. Mice were anesthetized and stretches of the small intestine corresponding to » 3 cm were ligated at their extremities. PBS or S. typhimurium (109) were injected in the loop and 30 min. later intestines were isolated and snap-frozen. Cryosections (7 mm) were fixed with 1% PFA and immunostained for CD11c, CD11b or CD8a. a–b DC dendrites were found either contacting GFP-salmonella (a, arrow) or stretching outside of the epithelium (b, arrow). In c-f individual staining and the merge of the two fluorescences are shown.

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