Jean-Pierre Kraehenbuhl

Epithelial M Cells: Gateways for Mucosal Infection and Immunization

M cell transport is an important factor in induction of mucosal immune responses and is exploited by pathogenic microorganisms for invasion of the intestinal mucosa. The specific molecular recognition systems and nonspecific adherence mechanisms that determine the efficiency of the M cell transport pathway are largely unknown. Future studies on the interactions of micoorganisms with this highly specialized epithelial cell will enhance our understanding of microbial pathogenesis and will lead to more effective strategies for targeting of vaccines and live microbial vaccine vectors to the mucosal immune system.

Collaboration of epithelial cells with organized mucosal lymphoid tissues

Immune surveillance of mucosal surfaces requires the delivery of intact macromolecules and microorganisms across epithelial barriers to organized mucosal lymphoid tissues. Transport, processing and presentation of foreign antigens, as well as local induction and clonal expansion of antigen-specific effector lymphocytes, involves a close collaboration between organized lymphoid tissues and the specialized follicle-associated epithelium. M cells in the follicle-associated epithelium transport foreign macromolecules and microorganisms to antigen-presenting cells within and under the epithelial barrier. Determination of the earliest cellular interactions that occur in and under the follicle-associated epithelium could greatly facilitate the design of effective mucosal vaccines in the future.

Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

Enteropathogenic bacteria elicit mucosal innate and adaptive immune responses. We investigated whether gut epithelial cells played a role in triggering an adaptive immune response by recruiting dendritic cells (DCs). Immature DCs are selectively attracted by the CCL20 chemokine. The expression of the CCL20 gene in human intestinal epithelial cell lines was up-regulated by pathogenic bacteria, including Salmonella species, but not by indigenous bacteria of the intestinal flora. The Salmonella machinery for epithelial cell invasion was not required for CCL20 gene activation. Flagellin but not the lipopolysaccharide was found to be the Salmonella factor responsible for stimulation of epithelial CCL20 production. CCL20 in turn triggered a specific migration of immature DCs. Our data show that crosstalk between bacterial flagellin and epithelial cells is essential for the recruitment of DCs, a mechanism that could be instrumental to initiate adaptive immune responses in the gut.

An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses

The EuroVacc 02 phase I trial has evaluated the safety and immunogenicity of a prime-boost regimen comprising recombinant DNA and the poxvirus vector NYVAC, both expressing a common immunogen consisting of Env, Gag, Pol, and Nef polypeptide domain from human immunodeficiency virus (HIV)-1 clade C isolate, CN54. 40 volunteers were randomized to receive DNA C or nothing on day 0 and at week 4, followed by NYVAC C at weeks 20 and 24. The primary immunogenicity endpoints were measured at weeks 26 and 28 by the quantification of T cell responses using the interferon γ enzyme-linked immunospot assay. Our results indicate that the DNA C plus NYVAC C vaccine regimen was highly immunogenic, as indicated by the detection of T cell responses in 90% of vaccinees and was superior to responses induced by NYVAC C alone (33% of responders). The vaccine-induced T cell responses were (a) vigorous in the case of the env response (mean 480 spot-forming units/106 mononuclear cells at weeks 26/28), (b) polyfunctional for both CD4 and CD8 T cell responses, (c) broad (the average number of epitopes was 4.2 per responder), and (d) durable (T cell responses were present in 70% of vaccinees at week 72). The vaccine-induced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but smaller responses against Gag-Pol-Nef were also observed in 48% of vaccinees. These results support the development of the poxvirus platform in the HIV vaccine field and the further clinical development of the DNA C plus NYVAC C vaccine regimen.

Flagellin Promotes Myeloid Differentiation Factor 88-Dependent Development of Th2-Type Response

Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4+ T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-κB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.

Immunization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease

BACKGROUND/AIMS Because Helicobacter pylori is a potentially dangerous human pathogen, the protective potential of oral immunization with H. pylori urease and its subunits was evaluated in an animal model. METHODS Mice were orally immunized with H. pylori sonicate, urease, or recombinant enzymatically inactive urease subunits and then challenged with Helicobacter felis. Control mice were sham-immunized. RESULTS H. felis colonization was present 5 days after challenge in 9 of 10 sham-immunized, 6 of 9 sonicate-immunized, and 3 of 10 urease-immunized animals (P = 0.031 vs. sham-immunized). Twelve days after challenge, urease B-immunized mice had a weaker colonization than sham-immunized controls, whereas urease A had no effect. After 70 days, most urease A- and urease B-immunized mice had cleared the colonization (10/17: P = 0.0019; 16/20: P = 0.00002 vs. sham-immunized). In urease B-immunized animals, protection was often associated with corpus gastritis. CONCLUSIONS Oral immunization with H. pylori urease protects mice against H. felis infection. Enzymatically inactive urease A and B subunits contain protective epitopes. It is unclear whether protection depends on the development of a mononuclear inflammatory response in the gastric corpus. Our observations should encourage the development of a human vaccine.

Transepithelial prion transport by M cells

To the editor—The appearance of a new variant of Creutzfeldt–Jakob disease has raised concerns that bovine spongiform encephalopathies might be communicable to humans by dietary exposure. Membranous epithelial cells (M cells) are key sites of antigen sampling for the mucosa-associated lymphoid system and have been recognized as major ports of entry for enteric pathogens via transepithelial transport. Recently, in vitro systems have been developed in which epithelial cells undergo differentiation to cells resembling M cells by morphological and functional-physiological criteria. Here we investigated whether M cells are a plausible site of prion entry in a coculture model. We seeded 3 × 10 Caco-2 cells (clone TC7) on the upper face of 6.5-mm filters (3μm pore Transwell filters, Costar, Cambridge, Massachusetts) and cultured in culture medium (high-glucose DMEM supplemented with 10% FCS) for 72 h until confluency was reached. Next, we placed 1 × 10 Raji B cells diluted in 80 μl low-glucose culture medium onto the lower chamber facing the basolateral side of the Caco-2 cells by inverting the inserts. Lymphoid cells migrated through the pores of the filter and settled within the epithelial monolayer (Fig. 1a–c), inducing differentiation of some Caco-2 cells into M cells. Raji B cells were exclusively found at the basolateral site within Caco-2 monolayers (Fig. 1c), most likely because Raji cells express the chemokine receptor CCR6 that responds to CCL20, a chemokine secreted basolaterally by Caco-2 cells. We ascertained successful conversion by monitoring active, temperature-dependent transport of inert FITC-conjugated latex beads (FluoSpheres, 0.2 μm, Molecular Probes, Eugene, Oregon). Introduction of beads in the chamber facing the apical surface of epithelial cells allowed fluorescence recovery from the opposite compartment in confluent (as routinely analyzed by transepithelial resistance (TER) of ∼ 200 Ω × cm or occasionally by [H]inulin permeability) and M-cell–containing cocultures at 37 °C, as examined by FACS analysis (Fig. 1d). In contrast, there was no transepithelial transport of beads at 4 °C (data not shown) or in Caco-2 monolayers at 37 °C. Cocultures that combined integrity and active transport of beads (∼ 10% of cocultures) were incubated with 5 or 3 logLD50 Rocky Mountain Laboratory scrapie strain prions administered to the apical compartment. After 24 h, infectivity within the basolateral compartment was determined by bioassay with tga20 mice, which overexpress a Prnp transgene and develop scrapie rapidly after infection. Upon challenge with 5 logLD50 scrapie prions, we consistently recovered prions in the basolateral compartment of cocultures containing M cells (n = 3), indicating transepithelial prion transport (Fig. 1e). Even at very low prion doses (3 logLD50), we found infectivity in at least one M-cell–containing coculture (n = 3) Transepithelial prion transport by M cells

CD4+CD3− Cells Induce Peyer's Patch Development: Role of α4β1 Integrin Activation by CXCR5

CD4+CD3- cells are the predominant hematopoietic cells found in mouse fetal intestine. We prove their role as Peyers patch (PP)-inducing cells by transfer into neonatal PP-deficient mice. To test the requirement of chemokines and adhesion molecules in induction of PP, we studied mice deficient in CXCR5 and/or alpha4beta1 integrin-mediated adhesion. CXCR5-/- mice have CD4+CD3- cells, which are inefficient in inducing PP formation. We show here that CXCR5/CXCL13 signaling activates alpha4beta1 integrin on CD4+CD3- cells. Blocking of beta1 integrin or VCAM-1, the ligand of alpha4beta1 integrin, inhibits PP formation. This study demonstrates the link between chemokine receptors and adhesion molecules that regulates stromal/hematopoietic cell interaction leading to PP formation.

Oral immunization with Helicobacter pylori urease B subunit as a treatment against Helicobacter infection in mice

BACKGROUND & AIMS Eradication of Helicobacter pylori infections in humans results in the healing of gastritis and gastric ulcers. This study used a mouse model to test whether oral vaccination can cure Helicobacter infection and gastritis. METHODS Mice were infected with Helicobacter felis. Three weeks after infection, the mice were orally immunized with H. pylori urease B subunit. Control mice were simultaneously infected but sham immunized. RESULTS Three to 8 weeks after oral immunization of H. felis-infected mice with recombinant H. pylori urease B subunit, the infection cleared and there was no evidence of gastritis. Vaccinated mice remained protected against two consecutive H. felis challenges. CONCLUSIONS These results show that the lack of natural immunity against Helicobacter can be overcome by oral immunization and that vaccination offers a novel therapeutic approach to Helicobacter-induced gastritis.

Regulation of the sodium pump: how and why?

Abstract The sodium pump, Na + , K + -ATPase, controls (directly or indirectly) many essential cellular functions, such as cell volume, heat production, intracellular pH, free calcium concentration and membrane potential. In epithelia, due to its asymmetric cell surface distribution, the sodium pump represents the major driving force for the transepithelial transport of ions, solutes and water, thereby controlling the extracellular volume and its ionic composition. By modulating the concentration of cytosolic calcium in excitable cells, Na + , K + -ATPase regulates the efficiency of muscle contraction in cardiac cells, and the release or uptake of neurotransmitters in neurons. How and why Na + ,K + -ATPase is regulated in response to acute or chronic environmental changes are the questions we address in this review. Alterations in regulation may play key roles in pathological processes such as heart failure, hypertension, or neurological disorders.