Microbial Pattern Recognition Receptors Mediate M-Cell Uptake of a Gram-Negative Bacterium

Abstract

The receptors involved in the sampling of particulate microbial antigens by the gut are largely unknown. Here we demonstrate for the first time in an in vitro M-cell model and in situ in isolated murine intestinal segments that the receptors TLR-4, PAF-R, and ␣5␤1 integrin are all involved in mediating bacterial uptake associated with transcytosis. The pattern of expression of TLR-4 and ␣5␤1 integrin differed between M cells and enterocytes. There was increased apical expression of TLR-4 in M-cell cultures, and it was present on the apical surface of murine M cells but not enterocytes in situ. In contrast, PAF-R was expressed equally by both cell types in vitro and was abundantly expressed throughout the intestinal epithelium. Inhibition of TLR-4 and PAF-R, but not TLR-2, reduced gram-negative bacterial uptake by both cell types, whereas inhibition of the apically expressed ␣5␤1 integrin significantly reduced the ability of M cells to translocate bacteria. Hence, the involvement of each receptor was dependent not only on differences in the level of receptor expression but the cellular localization. Using bacteria that had mutations that affected the bacterial lipooligosaccharide structure indicated that the oligosaccharide moiety was important in bacterial uptake. Taken together, the data suggest that pathogen-associated molecular pattern interactions with pattern recognition receptors are key factors in M-cell recognition of intestinal antigens for mucosal immune priming.The sampling of particulate microbial antigen by epithelial cells of the gastrointestinal tract resulting in stimulation of the mucosal immune system has long been thought of as a specific receptor-driven event. These receptors are considered to be specific to the microfold (M) cells that are found within the follicle-associated epithelia (FAE) that overlie the mucosal immune inductive tissue of the Peyer's patches of the intestine (6,16,17). Researchers have demonstrated that interactions between pathogenic bacteria and the gut epithelia rely on several pathways, such as receptor-mediated uptake as demonstrated by Yersinia enterocolytica invasin-␤1 integrin interactions (8); type III secretion systems, such as the translocated intimin receptor (Tir)-dependent disruption of the intestinal epithelia by enteropathogenic Escherichia coli (13); and the paracellular invasion strategy used by Campylobacter enteritis (10). However, the species-specific variability of potential Mcell receptors (12) and a failure to find receptors that are specific to human M cells that are not present elsewhere on the gut lining has thwarted research efforts to target mucosal antigen uptake for the induction of mucosal immune responses.Prokaryotes contain molecular motifs that are unique to microorganisms. These motifs, known as common microbial pathogen-associated molecular patterns (PAMPs), are recognized by the innate immune system through pattern recognition receptors (PRRs), either through direct receptor-bacterial ligand or endogenous adaptor-bacterial molecule i...

The M Cell-Targeting Ligand Promotes Antigen Delivery and Induces Antigen-Specific Immune Responses in Mucosal Vaccination

“…For example, it was confirmed using an in vitro M cell coculture model that macrophage inhibitory factor regulates M cell-mediated transport (24). Furthermore, specific expression of TLR4, a5b1 integrin, galectin-9, and caveolin-1 and transcytosis of mucosal pathogens (Salmonella and Gram-negative bacteria) by these molecules were identified using the same in vitro M cell culture model (25)(26)(27).…”

“…A gut loop containing PPs was prepared, and an ex vivo Ag uptake assay was performed as described previously (27)(28)(29). Briefly, either EGFP alone or ligand-fused EGFP protein (0.2 mg/ml) was added to 2-to 3-cm-long ligated gut loops prepared from a male BALB/c mice (Charles River Technology, Boston, MA, through Orient Bio., Sungnam, South Korea) that had been fasted overnight.…”

BOARD-INVITED REVIEW: Porcine mucosal immunity of the gastrointestinal tract1

“…The role of M cells in microbial recognition has not been fully elucidated because of difficulties in establishing suitable in vitro models, but recent work has provided some insight into M-cell function. For example, Tyrer et al (2006) has provided evidence that pattern recognition receptors (e.g., Tolllike receptors) are important for M-cell recognition of Gram-negative bacteria and to induce an appropriate mucosal immune response. Because the in vitro model established by this group consisted of human epithelial cells cocultured with murine Peyer's patch cells, these observations may not be applicable to swine.…”

“…However, antigens and pathogenic microorganisms routinely circumvent the physical barrier provided by IEC. For example, antigen may be taken up by microfold (M) cells found within the follicle-associated epithelium of Peyer's patches (Tyrer et al, 2006). In addition, antigen may be sampled directly by dendritic cells, which open tight junctions between IEC to extend dendrites into the intestinal lumen (Rescigno et al, 2001), and certain species of bacteria overcome the epithelial barrier by using specialized invasion strategies such as the Type III secretion system (Hapfelmeier et al, 2005).…”

Examining the Role of Nasopharyngeal-associated Lymphoreticular Tissue (NALT) in Mouse Responses to Vaccines

“…Located within the NALT are discrete compartments of B and T lymphocytes interspersed with antigen-presenting dendritic cells 4,7,8 . These cells are surrounded by an epithelial cell layer intercalated with M-cells that are responsible for antigen retrieval from the mucosal surfaces of the air passages 9,10 . Naive lymphocytes circulating through the NALT are poised to respond to first encounters with respiratory pathogens 7 .…”