The mucosal surfaces of our body are a primary component of our innate immune system and serve as a barrier against endogenous microflora as well as against external pathogens. This barrier is made of polarized epithelial cells, specialized immune cells, and secreted mucus. Many pathogens have evolved strategies to circumvent this barrier, including entering into cells or traveling through them by transcytosis, crossing through intercellular junctions, or directly disrupting the barrier by killing cells in the epithelium (Kazmierczak et al., 2001a
The mucosal barrier epithelium is comprised of one or more layers of epithelial cells that have specialized and distinct apical and basolateral surfaces, separated by tight junctions (TJs), that form selective permeability barriers between biological compartments (Wang and Margolis, 2007
; Martin-Belmonte and Mostov, 2008
). The apical surface faces the lumen of the cavity, while the basolateral surface faces adjoining cells and the underlying basement membrane. The apical and basolateral membrane domains are distinguished by unique assemblies of proteins and lipids, creating specific membrane domains with distinct roles in formation and maintenance of barrier function, as well as the myriad of physiological barrier functions, such as nutrient exchange.
The apical surface contains transporters and enzymes that are specialized to interact with the external environment. The outer leaflet of the apical surface is highly enriched in glycosphingolipids and cholesterol. The basolateral plasma membrane of the epithelial cell contains many transporters and receptors that are involved in uptake of nutrients and hormones from the circulation. The basolateral surface can be divided into lateral domains, which contact other cells, and basal domains, which contact the basement membrane and blood vessels. The lateral surface contains specialized cell–cell contact domains, including TJs and adherens junctions (AJs).
The TJ is located at the apical-most region of the lateral surface and defines the boundary between the apical and basolateral surfaces (Ebnet, 2008
). The TJ serves two functions. First, it acts as a “gate” or “barrier” to prevent paracellular diffusion between the cells. This function enables the epithelial monolayer to restrict permeability to solutes or larger particles, including pathogens. Second, the TJ acts as a fence to prevent diffusion or intermixing of plasma membrane components between the apical and basolateral domains. The TJ contains three classes of integral membrane proteins: occludins, claudins, and JAMs, each of which forms homophilic interactions that are responsible for gate function of the TJ. The TJ is attached to the cytoskeleton by a set of adaptor proteins including zonula occludens protein 1 (ZO-1).
The AJ lies underneath the TJ. The AJ consists mainly of classical cadherin family members and nectins, which are integral membrane proteins whose large extracellular domains interact in a homophilic or heterophilic manner to connect adjacent cells. Cadherins are linked to the cytoskeleton through β-catenin, α-catenin, and p120-catenin. In addition to providing the structural “linking” of neighboring cells, cadherins function as organizing nodes for multiprotein complexes that regulate cell–cell contacts, an essential function for morphogenesis and remodeling of tissues and organs (Meng and Takeichi, 2009
A wide network of proteins and lipids regulates the formation and maintenance of epithelial cell polarity. The first step in the formation of apical–basolateral polarity is the formation of cell–cell junctions. E-cadherins from adjacent cells interact to create homophilic intercellular adhesions. Activation of small Rho GTPase family members, leads to cytoskeleton rearrangement and recruitment of structural and regulatory proteins, resulting in the formation of mature TJs and AJs (Iden and Collard, 2008
). Junction maturation is coupled to the development of apical–basolateral asymmetry in the cell, where the newly formed AJ serves as a site for basolateral protein sorting (Yeaman et al., 2004
). Maintenance of cell polarity and junction integrity involves continuous sensing of external cues such as extracellular matrix content and cell–cell contacts. These cues are translated into cellular signals that are received by a regulatory core of three protein complexes: Par3/Par6/aPKC, the Crumbs complex (Crumbs-3, PALS1, and PATJ), and the Scribble complex (Scribble, LGL1/2, and DLG1). The mutually exclusive localization of these three complexes helps to stabilize apical–basolateral polarity (Bryant and Mostov, 2008
; Iden and Collard, 2008
In addition to the asymmetric distribution of key polarity proteins described above, phosphoinositides have emerged as important determinants of membrane identity. These lipids bind to specific protein domains, particularly to those involved in the regulation of the cytoskeleton. In mammalian cells, phosphatidylinositol 4,5-bisphosphate (PIP2
) is found primarily on the apical surface whereas phosphatidylinositol 3,4,5-trisphosphate (PIP3
) localizes to the basolateral surface (Martin-Belmonte and Mostov, 2008
The apical–basolateral polarity regulation system is increasingly recognized as an important target for pathogens. Our understanding of the interactions between pathogens with the mucosal barrier has been greatly aided by the use of epithelial cell lines, such as dog kidney (MDCK) cells, Calu-3 (a cell line derived from a human adenocarcinoma), and 16HBE (derived from human bronchial epithelial cells) cells that grow as a single confluent monolayer and recapitulate the development of polarized epithelium when grown on porous filter supports (transwells; Mostov et al., 2005
). When grown at high densities under these conditions, the cells can obtain nutrients from the basolateral medium and will form polarized epithelium with distinct apical and basolateral surfaces and functional TJs and AJs within 24
h. With continued culture, cell polarity develops further. One advantage of this system is that pathogen–epithelial interactions can be studied without confounding effects contributed by immune cells. Furthermore, by using confluent monolayers, it is possible to compare microbe interactions between the apical and basolateral surfaces without having to take into account the effect of increased access to the basolateral surface that occurs in subconfluent cells or in the setting of epithelial injury (Kazmierczak et al., 2001a
). Finally, some of the epithelial cells, including MDCK cells and primary mouse alveolar type II cells, can be grown as three-dimensional (3D) cysts when cultured on extracellular matrix in which the basolateral surface faces outward (Bryant and Mostov, 2008
). These models may more closely mimic organs; in addition, they facilitate the examination of interactions of pathogens with the basolateral surface in the absence of the porous filter support (Barrila et al., 2010
; Bucior et al., 2010
). These reductionist systems provide a platform to analyze host–pathogen interactions, which can then be further validated in animal studies.