Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA Vannary Tieng*, Chantal Le Bougue´ nec†, Laurence du Merle†, Philippe Bertheau‡, Pierre Desreumaux§, Anne Janin‡, Dominique Charron*, and Antoine Toubert*¶ *Laboratoire d’Immunologie et d’Histocompatibilite´, Institut National de la Sante´et de la Recherche Me´dicale Unit 396, Institut Universitaire d’He´matologie, Hoˆpital Saint-Louis, AP-HP, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France; †Unite´de Pathoge´nie Bacte´rienne des Muqueuses, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France; ‡Laboratoire de Recherche Universitaire de Pathologie EA 2378, Institut Universitaire d’He´matologie, 1 Avenue Claude Vellefaux, 75475 Paris Cedex 10, France; and §Equipe Propre Institut National de la Sante´et de la Recherche Me´dicale 0114, Centre Hospitalier Universitaire, 59037 Lille Cedex, France Communicated by Jean Dausset, Fondation Jean Dausset-CEPH, Paris, France, December 13, 2001 (received for review November 20, 2001) MICA are distant homologs of MHC class I molecules expressed in Infection of dendritic cells and epithelial cells by Mycobacterium the normal intestinal epithelium. They are ligands of the NKG2D tuberculosis could also increase MICA expression and enhance activating receptor expressed on most ␥␦ T cells, CD8؉ ␣␤ T cells, the V␥2␦2 T cell recognition via MICA–NKG2D interaction and natural killer cells and therefore play a critical role in innate (12). However, there is no study of MICA expression in response immune responses. We investigated MICA cell-surface expression to enteric bacterial infection (Shigella spp., Salmonella spp., on infection of epithelial cell lines by enteric bacteria and show Escherichia coli), an especially important issue regarding the here that MICA expression can be markedly increased by bacteria normal pattern of MICA tissue distribution. of the diffusely adherent Escherichia coli diarrheagenic group. This We provide here direct evidence that strains of E. coli of the effect is mediated by the specific interaction between bacterial diffusely adherent E. coli (DAEC) pathogenic group trigger a rapid adhesin AfaE and its cellular receptor, CD55, or decay-accelerating MICA expression increase at the surface of Caco-2 intestinal factor. It is extremely rapid after AfaE binding, consistent with a epithelial or HeLa cell lines. Adhesion of DAEC to epithelial cells stress-induced signal. MICA induction on epithelial cells triggered is mediated by adhesins belonging to a family that includes the AfaE IFN-␥ release by the NKG2D expressing natural killer cell line NKL. afimbrial adhesins and the fimbrial F1845 one (13, 14). The specific This host–bacteria interaction pathway could play a role in the cellular receptor for these adhesins is CD55, also known as the pathogenesis of inflammatory bowel disease, a condition that decay-accelerating factor (DAF), a glycophosphatidylinositol- implicates a bacterial trigger in genetically susceptible individuals. anchored protein expressed notably on epithelial cells (15). We This was supported by the increased MICA expression at the show that the increase in MICA expression is triggered by the surface of epithelial cells in colonic biopsies from Crohn’s disease- CD55–AfaE interaction and induces the release of IFN-␥ by the IMMUNOLOGY affected patients compared with controls. NKG2D-expressing NK cell line NKL. Therefore, we describe a new function for MICA as a molecule involved in the innate nvironmental pathogens stimulate host immune responses immune antibacterial response as well as a new function for CD55 Ebecause of recognition of antigenic peptides bound to MHC besides its role of complement regulatory protein. These data could molecules by T lymphocytes mainly expressing ␣␤ T cell recep- be relevant to the pathogenesis of autoimmune conditions triggered tors. In addition to the antigen-specific acquired immunity, by a bacterial infection, for instance Crohn’s disease (CD), where innate cellular immune responses may involve ␥␦ TCR- DAEC have been implicated (16, 17). expressing T lymphocytes, natural killer (NK), or NK T cells, and Methods the recognition of ligands distinct from classical MHC class I or class II molecules. Bacterial Infection of Epithelial Cell Lines. Two human epithelial cell MHC class I-related MICA and MICB are polymorphic lines were used, the cervical carcinoma cell line HeLa (homozy- molecules encoded within the MHC locus centromeric to gous for MICA*008; ref. 9) and Caco-2 (MICA*033) derived HLA-B (1). MICA and MICB are closely related with an 84% from a human colorectal adenocarcinoma. HeLa cells were grown in DMEM with 4.5 g of Glc/liter͞2mML-Gln͞100 amino acid identity (2). MICA molecules are heavily glycosy- ͞ ␮ ͞ lated because of eight potential glycosylation sites accounting for units/ml penicillin 100 g/ml streptomycin 10% FCS in 5% CO2 a 65–75-kDa molecular mass range in the absence of N-glycanase at 37°C. Caco-2 cells were grown in the same medium with 20% treatment (3). MICA crystallographic structure revealed crucial FCS in 10% CO2 and used before being differentiated. Wild-type differences with classical MHC class I folding (4) explaining the bacterial strains were Salmonella typhimurium (CNRSS 96-479), lack of MICA peptide binding, ␤2 microglobulin association, and E. coli A30 (18), and M90T (19), an invasive serotype 5 of CD8 binding. NKG2D, a lectin-like receptor widely distributed Shigella flexneri that carries the 220-kb virulence plasmid on ␥␦ T cells, CD8ϩ ␣␤ T cells, and NK cells, is a functional pWR100. Its mutant derivative BS176 is an avirulent strain cured ligand for MICA (5) to which it binds as a homodimer (6, 7). This of pWR100. Transformed S. flexneri strains derived from M90T interaction mediates effector cell activation through the DAP10 and BS176 (named, respectively, SC301 and SC300) contain the signaling adaptor (8). The importance of MICA expression in plasmid pIL22 including the cloned afa-1 operon (20). E. coli human diseases is further illustrated by their recognition on ␥␦ epithelial tumors by infiltrating T cells (8–10). Two other See commentary on page 2584. properties distinguish MICA molecules: first, they have a limited Abbreviations: NK, natural killer; DAEC, diffusely adherent E. coli; Afa, afimbrial adhesin; normal tissue distribution in the gastrointestinal epithelium; DAF, decay-accelerating factor; CD, Crohn’s disease; SCR, short consensus repeat. second, they are stress-inducible (3). It has been recently dem- ¶To whom reprint requests should be addressed. E-mail: [email protected]. onstrated that MICA molecules were induced on cytomegalov- The publication costs of this article were defrayed in part by page charge payment. This irus-infected fibroblast or endothelial cells and could positively article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Ϫ modulate antigen-specific CD28 CD8␣␤ T cell responses (11). §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.032668099 PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 2977–2982 Downloaded by guest on September 27, 2021 A30 and S. typhimurium were grown in Luria broth at 37°C, and culture medium containing 1.25% DMSO; 48 h after transfec- S. flexneri strains in trypticase soy broth were supplemented with tion, MICA molecules transiently expressed in COS-7 cells were 50 ␮g͞ml ampicillin for SC300 and SC301. Overnight cultures of detected by flow cytometry by using the immune serum. bacteria were diluted in Luria broth or in tryptic soy broth (1:100) and grown to exponential phase for2hat37°C. IFN-␥ Release by NK Cells in Presence of MICA-Expressing Caco-2 Epithelial cells were washed with PBS and infected with washed Targets. To evaluate NKL cell line recognition of MICA, Caco-2 bacteria resuspended in FCS-free and antibiotic-free DMEM. cells infected or not with bacteria during 2 h were harvested and Infections were done unless otherwise mentioned with 100 fixed in 1% PBS–paraformaldehyde for 15 min. After two bacteria͞cell for SC301 and SC300, 200 bacteria͞cell for A30, S. washes, Caco-2 (105 cells͞well) were incubated overnight with typhimurium, and up to 500 bacteria͞cell for M90T and BS176. NKL (105 cells͞well) in round-bottom 96-well plates in 200 ␮lof Cells were incubated1hat37°C for S. flexneri and 2 h for E. coli DMEM supplemented with 10% heat-inactivated human serum, and S. typhimurium. After several washes, cells were incubated 2mML-Gln, 100 units͞ml penicillin, 100 ␮g͞ml streptomycin. in FCS-free DMEM with gentamicin (100 ␮g͞ml) for 30 min. In NKL, grown in RPMI 1640 medium with 10% heat-inactivated addition, M90T and BS176 were centrifuged for 10 min at 2,000 human serum and 100 IU͞ml IL-2, were deprived of IL-2 for 18 h rpm to promote adherence of these bacteria to cells. Bacterial before contact with target cells. In CD55 blocking experiments, adherence on cells was visualized by Giemsa staining. Unin- targets were incubated before addition of bacteria for 30 min at fected cells were treated identically to infected ones at every room temperature with the CD55-specific mAb, BRIC 216 (10 step. ␮g͞ml) or an isotypic control. In MICA-blocking experiments, diluted immune or preimmune sera (1:50) were added after AfaE- and AfaD-Coated Beads. AfaE-III and AfaD products en- bacterial infection. To assess whether the phosphatidylinositol coded by the afa-3 gene cluster were obtained by purification of 3-kinase signaling pathway was involved in NKL activation, NKL histidine-tagged recombinant fusion proteins as described (13). cells were pretreated with 25 mM wortmannin 1 h before These proteins were then coated on 1-␮m-diameter carboxylated incubation with fixed Caco-2 cells. Supernatants from each well microspheres as recommended by the supplier (Molecular were assessed for NKL cell reactivity in duplicate, by measuring Probes). BSA-coated beads were used as a control. The con- specific secretion of IFN-␥ using commercially available ELISA centration of beads was Ϸ1.8 ϫ 1010 beads͞ml, and the amount kits (Diaclone, Besanc¸on, France).