text:
The Epithelium–Molecular Landscaping for an Interactive BarrierView this Special Issue
Molecular Modulation of Intestinal Epithelial Barrier: Contribution of Microbiota
The daunting task required of the gut-barrier to prevent luminal pathogens and harmful substances from entering into the internal milieu and yet promoting digestion and absorption of nutrients requires an exquisite degree of coordination between the different architectural units of this barrier. The complex integration and execution of these functions are superbly carried out by the intestinal mucosal (IM) surface. Exposed to trillions of luminal microbes, the IM averts threats by signaling to the innate immune system, through pattern recognition receptors (PRR), to respond to the commensal bacteria by developing tolerance (hyporesponsiveness) towards them. This system also acts by protecting against pathogens by elaborating and releasing protective peptides, cytokines, chemokines, and phagocytic cells. The IM is constantly sampling luminal contents and making molecular adjustments at its frontier. This article describes the topography of the IM and the mechanisms of molecular adjustments that protect the internal milieu, and also describes the role of the microbiota in achieving this goal.
1. Introduction
The single-cell epithelial layer of the intestinal mucosa (IM) confronts the largest antigenic microbial challenge of any other mucosal surface in the human body [1]. The presence of large microbial communities in the lumen is a huge problem for the intestinal immune system [2, 3]. In humans, extensive in utero development of T and B cells takes place, and after birth there is a rapid and massive intestinal colonization of antigenic microbes, ultimately establishing stable microbial communities lasting life-long for the individual host [1, 4, 5].
The molecular mechanisms that are involved in shaping and selecting a stable microbiota for different individuals are areas of considerable research interest [6, 7]. There is evidence emerging that the microbiota modulates a series of processes that result in maturation, differentiation, and proliferation of the IM at both cellular and molecular levels [3, 5, 8]. Through a molecular chain of events, the microbiota provides a major drive for the maturation of the innate and adaptive immune systems. It has a profound effect on the intestinal barrier and on distant organs. In this review, we present molecular microbial-mucosal interactions by providing a summary of the different strata of the IM, beginning with the microbiota as the outermost layer, and describe how different layers of the IM form a physical and immune barrier influenced by the microbiota [2, 4, 5, 8]. In this overview, we also describe how microbiota modulates innate and adaptive immunities.
2. Microbial-Mucosal Interactions
Through a process of "cross talk'' with the mucosal immune system, the microbiota negotiates mutual growth, survival, and inflammatory control of the intestinal ecosystem [9, 10]. The IM is equipped with trans-membrane or intra-cytoplasmic receptors, referred to as pattern recognition receptors (PRRs) that are defined by their ability to specifically recognize and bind distinctive microbial macromolecular ligands (Figure 1). These ligands referred to as microbial-associated molecular patterns (MAMPs) include lipopolysaccharide (LPS-a component of gram-negative bacteria), flagellin, peptidoglycans, and formylated peptides [3, 11–14] (Table 1). The transmembrane PRRs include the family of Toll-like receptors (TLRs), that sample the extracellular and endosomal compartments, and the intracellular NOD (Nucleotide-binding oligomerization domain)-like receptors (NLRs) that confront and protect the cytoplasmic compartment [11–14]. This microbial-mucosal intersignaling cultivates immune tolerance (hyporesponsiveness) resulting in the development of a stable core microbiota. Establishing a core microbiota of diverse and native commensal species is critical and advantageous to the host as it provides competition to the pathogenic microbes [9, 15]. This process prevents pathogens from forming a niche for their persistence and proliferation [16]. In response to viral components, signaling by TLRs results in expression of Interferon 1 (IFN1). For fungal components, C-type lectin receptors serve as PRRs [13]. On the surface of neutrophils, trans-membrane receptors belonging to the family of formylated peptide receptors (FPRs) are present as high affinity PRRs for MAMPs [17]. Upon exposure to MAMPs, signals from FPRs activate neutrophil transduction pathways to generate reduced NADPH (nicotinamide adenine dinucleotide phosphate) oxide (NOX)-dependent reactive oxygen species and promote phagocytic motility [17]. Recently, several neutrophil FPRs have been characterized in intestinal epithelial cells (IECs), suggesting that these epithelial receptors may mediate microbial monitoring in the gut in a manner analogous to their traditional functions in phagocytes [18]. Disruption in the ligand process of PRRs with MAMPs has been linked with inflammatory intestinal diseases [3]. One such example is Crohn's disease, in which mutant forms of NLR NOD2 have been identified [3, 19, 20].
2.1. Regulatory Pathways
In the IM, activation of PRRs initiates regulatory pathways such as nuclear factor B (NFB)/Rel pathways, mitogen-activated protein kinase (MAPK), and caspase-dependent signaling cascades [14, 19–21] (Figure 1). The transcriptional response of the IM to microorganisms can be different. Many PRR ligands are expressed by commensal bacteria yet the IM does not activate an inflammatory response to these bacteria. Conversely, some commensal bacteria exert protective effects by attenuating proinflammatory responses induced by pathogenic bacteria. Inflammatory or apoptotic responses to pathogenic bacteria, or stress signals, are controlled by NFB and caspase-dependent signaling [3, 21–23]. Most commensal organisms limit the signaling of NFB by inhibiting epithelial proteosome function, inhibiting degradation of the IB (the counter regulatory factor to NFB), or by exporting the NFB subunit p65 out of the nucleus through a peroxisome proliferator-activated receptor (PPAR)-dependent pathway. Induction of transforming growth factor- (TGF-) and MMP3K pathways has also been implicated in anti-inflammatory and antiapoptotic effects mediated by commensals [3, 24–28]. The TLRs can also initiate proapoptotic signaling. The bacterial lipoprotein-TLR2 interactions activate the caspase-8 pathway of apoptosis via the myeloid differentiation primary-response gene 88 (Myd88) and by subsequent recruitment of fast activated death domain (FADD) pathways. Recent findings utilizing purified flagellin and flagellate/aflagellate bacteria in both in vitro and in vivo systems have shown that important roles are played by flagellin in the modulation of apoptotic responses [21]. In response to cellular (IEC) attack by flagellate pathogens, signal transduction through TLR5 initiates proinflammatory transcriptional responses. Macrophages utilize intracellular IL-1-converting enzyme protease-activator factor (IPAF)/neuronal apoptosis inhibitory protein (NAIP) 5 to detect intracytoplasmic flagellin and respond with IL-1 release and/or apoptosis. Thus, this interaction between TLR5 and flagellin initiates pro-inflammatory and pro-apoptotic pathways. If pro-inflammatory signaling is unimpeded, transcriptional activation of a battery of anti-apoptotic/cytoprotective genes arrests the apoptotic pathways and allows inflammation without cell death [21]. Analogous to these events, it has been reported that during infection with wild type Salmonella, flagellin-induced pro-inflammatory signaling reduces cellular pro-apoptotic responses to bacterial effectors, whereas the absence of this potent pro-inflammatory determinant in mutants allows unhindered apoptosis. In this case, activation of pro-inflammatory signals protects apoptosis. Thus, the complex process of inflammation is intertwined with the process of apoptosis [21].
When activated, the interactive mucosal-microbial information system relays posttranslational events to the transducer PRR which in turn transmits the message, through transcriptional or post-transcriptional processes, to the effector cell [1, 5, 22–25].
3. Barrier Defense and Innate Immune System
3.1. Microbiota
By adulthood, a vast consortium of microbiota are supported in the intestinal lumen, but with current microbiologic methods, less than 30% of those microbes are culturable [15, 29]. Despite our lack of understanding of the true diversity of luminal microbes, new molecular approaches are allowing census-based culture-independent inventories of the entire microbiota and defining the microbial taxonomy utilizing genomic technology [11, 30]. The most common approach to generate DNA sequence data is to amplify the genes encoding RNA in small ribosomal subunits (16S rRNA) using primers targeting generally conserved regions of the gene. Rapid and cost-effective methods that provide a "fingerprint" of the microbial diversity in the individual components of the microbota are then characterized by cloning and DNA sequencing [15, 30–32].
Animal experiments and comparisons between conventionally raised mice (or rats) with germ-free counterparts have revealed that a series of anatomic, biochemical, and physiologic functions are performed by the microbiota that are crucial for development, maintaining the integrity of the barrier function, and for the repair of the IM [5]. The fact that so many morphological intestinal tissue defects appear in germ-free animals indicates that the development of the IM is inherently connected to microbial luminal colonization. For example, in contrast with conventionally raised mice, the villus capillaries in germ-free mice develop poorly during weaning and remain poorly developed until adulthood, indicating a microbial contribution to angiogenesis of the villus-core. This was further confirmed when it was shown that bacterial colonization of germ-free mice resulted in rapid and dramatic reinduction of angiogenesis [5, 33]. Electrophysiologic studies indicate that microbial colonization improves intestinal motility and modulation of enzyme activity [34]. Several important effects of the microbiota on the development of the immune system have been ascertained by selectively colonizing germ-free animals and then evaluating immune responses that have not been influenced by any other microbial molecules [11, 28, 35]. Germ-free animals show extensive defects in the development of gut-associated lymphoid tissue (GALT) and antibody production, fewer and less cellular lymphoid follicles (Peyer's patches), a thinner and less cellular lamina propria, and fewer plasma cells in germinal centers of the mesenteric lymph nodes compared with animals housed under conventional conditions [28, 36, 37]. These findings indicate that the development of the ultrastructure of the IM is dependent on luminal bacteria. The microbiota also influences the morphology of the IEC. Germ-free mice have been shown to develop an altered morphology of their microvilli and a reduced rate of turnover of their IECs (intestinal restitution) compared to conventionally raised animals [38]. Furthermore, the mircobiota has been shown to direct the glycosylation of surface proteins of the IEC [39].
The microbiota also contributes to the development of intra-epithelial lymphocytes (IEL) as evident by the fact that numbers of T-cell receptor (TCR)-bearing IELs (IELs) are reduced in germ-free mice compared to conventionally raised mice and that TCR-bearing IELs (IELs) isolated from conventionally raised mice are cytolytic but not those isolated from germ-free mice [5, 40–45]. These findings indicate that the microbiota influences maturation and execution of several immune functions that benefit the host IM. In return, after birth, changes occur in the host IM that promote colonization of commensal organisms. Conventionally raised mice begin life by expressing intestinal epithelial glycans that predominantly have sialic acid as their terminal moiety. During weaning there is a shift towards terminal fucose that does not occur in germ-free mice [40]. Fucosylation of glycans facilitates colonization by commensal species, such as B. thetaiotaomicron, that use terminal fucose as an energy source [46]. Thus, the host's IM encourages colonization of commensal organisms by shifting its energy source in their favor so that the commensals can gain control over the pathogenic species in the competitive intestinal ecosystem. Once established, the microbiota then shape their niche in a way that promotes morphological modulation of the host's IM in favor of that host [5, 8]. Another example is that, compared to conventionally raised mice, germ-free mice never achieve high activity of Angiogenin-4 (Ang4), a Paneth cell protein with potent bactericidal activity that plays important role in epithelial cell defense during the postweaning-period in mice, indicating that induction of Ang4 in Paneth cells of the IM is regulated by the microbiota [43].
The microbiota regulates the intestinal innate immune system by modulating expression of TLRs and NOD/CARD (caspase recruitment domain) mediated activation of immunosensory cells through MAMPs [27]. Decreased cytoprotective factors and decreased enterocyte proliferation have been observed in TLR-defective mice [47, 48]. Individual members of the microbiota can dampen TLR-mediated inflammatory signals and exert protective effects by attenuating pro-inflammatory responses [27, 49] (Figure 1). Several studies have shown how commensals, and those bacteria with probiotic function, can suppress inflammatory signals [50, 51]. One of the components of regulation of inflammatory signals is by activating IB, the inhibitory component of NFB activation. Manipulation of the NFB pathways has revealed both anti-inflammatory and pro-inflammatory roles for this transcriptional control pathway, suggesting temporal patterns in which TLR and NFB pathways are activated in response to distinct microbial signals. Mice with IEC-specific knockout genes encoding for I or I, the 2 components of the inhibitory B kinase (I) complex that activates NFB, were found to be susceptible to chemically induced colitis or to spontaneous development of intestinal inflammation, respectively [50–53]. Upon binding to their respective MAMPs, the TLRs act together with the Myd88 adapter protein to induce intracellular signaling events that converge upon the NFB and MAPK pathways to regulate the expression of genes that are involved in commensal-induced fortification of the IM and pathogen-induced inflammation (Figures 1 and 2). TLR ligation triggers recruitment of Myd88 to the receptor complex through TIR-TIR domain interactions. The death domain of Myd88 recruits a death-domain containing protein known as IL-1-R associated protein kinase (IRAK). Activation of IRAK leads to activation of NFB, p38 MAPK, and other regulators of gene expression. These events and expression of pro-inflammatory genes form the basis of innate immune response. Studies demonstrating loss of Myd88 function in dendritic cells established that TLR/Myd88-mediated MAMP recognition activates dendritic cells to produce pro-inflammatory cytokines and promotes T-helper responses [41, 49] (Figure 2).
In another study, IEC-specific deletion of ReIA/P65, which encodes the primary subunit of the NFB transcription factor, increased epithelial cell proliferation and caused apoptosis and increased susceptibility to colitis. These findings indicate that NFB activation through TLRs on the IM promoted antinflammatory responses to microbial signals and promote innate immunity [10–14, 16, 20, 24, 27].
Uncontrolled signaling through TLRs can potentially cause excessive inflammation. Intestinal epithelial cells constitutively, or inducibly, express high levels of the TLR inhibitor Toll-interacting protein (TOLLIP) [11, 21, 26]. Expression of TOLLIP has been shown to correlate with the in vivo luminal bacterial load and is highest in healthy colonic mucosa [26]. There are other regulators, including single immunoglobulin IL-1R-related protein (SIGIRR), IRAK-M, A20, PPAR, and NOD2, which exert inhibitory influence over inflammation. Evidence suggests that similar to TOLLIP, NOD2 might suppress the inflammatory cascade and mutations of NOD2 that are associated with Crohn's disease. These inhibitory molecules are important in maintaining microbial homeostasis [26, 28, 35, 40].
The microbiota also functions as a regulator of nutrient metabolism, allowing the host to digest many nutrients that would otherwise be inaccessible to the IM [3, 25–27, 36]. The complex carbohydrates that would otherwise be indigestible are degraded into short-chain fatty acids (SCFA) and monosaccharides by the mircobiota. These SCFA serve as a source of energy and regulate growth and differentiation of IECs, especially in the colon. The microbiota also degrades mucus glycoproteins and maintains the specific landscape of the IM. Germ-free animals exhibit a dramatic enlargement of the cecum, largely due to accumulation of undegraded mucus. In addition, the microbiota modifies the differentiation programs of intestinal epithelial lineages at key points during their morphogenesis [5].
3.2. Mucin
A coating of mucus on the IM forms the front line of defense segregating virulent organisms and protecting against acidic gastric and duodenal secretions [54, 55] (Figure 3). This mucus gel consists predominantly of high molecular weight glycoproteins (mucins) imparting characteristic polymeric, viscoelastic, and protective properties. Mucins have a high negative surface charge and a large hydration capacity. This mesh-like mucin gel impedes diffusion of offending macromolecules and yet is able to perform functions such as lubrication for the passage of particulate nutrients, maintenance of epithelial hydration, and facilitation of the exchange of gases and nutrients across the IM. So far 21 different mucin genes have been identified. In the intestine, MUC2 is the major mucin produced by goblet cells of the IM. Recent investigations in mice colon have demonstrated that there are two mucus layers of MUC2, an inner mucus layer that is adherent to epithelial layer and an overlying loose mucus layer. The inner layer is densely packed. It is devoid of bacteria suggesting small pore size that physically prevents bacterial penetration [53]. Conversely, the outer loose layer contains high number of bacteria. Because the mucin and protein composition of both the firm and loose layers are identical, it appears that firm layer separates to form loose layers. In small intestine, the same MUC2 is not adherent to epithelial layer but forms the mucus gel that is synthesized in goblet cells. Following synthesis in goblet cells, mucins are packaged in granules, transported to the cell surface, and secreted into the intestinal lumen. Mucins can be secretory or membrane-bound [53–55].
3.2.1. Mucin and Innate Host Defense
Under normal conditions, microbes localize in the mucus, sharing either "self'' signature molecules or pathogen-associated molecular patterns. These patterns are in turn recognized by TLRs or other PRRs. Recognition of LPS is achieved through the combined action of membrane-bound or soluble mucin, LPS-binding protein (LBP), CD14, and TLR4 [55] (Figure 3).
In gram-negative bacteria (e.g., Pseudomonas aeruginosa), both LPS and flagellin play a role in altering mucin production (Figure 3). The Binding of LPS to LBP and then to CD14 leads to activation of Ras-MEK1/2-Erk1/2 (reticulo-activating system-pathway—Meiosis-specific serine/threonine protein kinase-extra-cellular signal-regulated kinase) using TLR-4 as a coreceptor. Flagellin, on the other hand, binds to the surface glycolipid receptor Asialo-GM1. The activation of mucin transcription through Asialo-GM1 is calcium dependent as seen by an increase in calcium levels following administration of flagellin. Binding to Asialo-GM1 leads to ATP release and its subsequent binding to the cell surface G protein-coupled receptor (GPCR) [55]. This activates phospholipase C and creates a subsequent increase in intracellular calcium levels. These events finally lead to downstream activation of the Src (rous sarcoma virus-cytoplasmic protein)-dependent Ras pathway, leading to the activation of NFB and mucin transcription (Figure 3).
Through a different pathway, the gram-positive bacterial product lipoteichoic acid (LTA) binds and activates the platelet-activating factor (PAF) receptor, which is a cell surface G-protein coupled receptor (Figure 3). This leads to activation of ADAM10, which then cleaves the trans-membrane heparin-binding EGF (epidermal growth factor), which in turn activates the EGF receptor. This leads to the engagement of the Ras/Raf/MEK/ERK/pp90rsk/NF-B pathway and MUC2 (mucin) transcription [54–56].
The defensive ability of mucin lies in its capacity to entrap microbes. Adhesion to specific mucin epitopes is thought to facilitate mucus colonization by commensal bacteria, thereby providing a number of growth advantages. Accordingly, intestinal mucin is thought to dictate the composition of the bacterial community [55] (Figure 3).
3.2.2. Other Goblet Cell Secretions in Innate Host Defense
In addition to mucin production, goblet cells also produce two other innate defense molecules: the intestinal trefoil factor and the resistin-like molecule- (RELM-) proteins [55, 56]. There is evidence that these two innate defense molecules may stabilize the mucin polymer and/or regulate mucin secretion [55]. Importantly, these molecules may need a mucin medium to exert their biological functions.
3.2.3. Intestinal Trefoil Factor
This is a small cystein-rich peptide belonging to the family of trefoil factors (TFF) [57]. In humans 3 TFFs have been identified; TFF1, TFF2, and TFF3. Secreted TFFs act on the IM either extracellularly to augment barrier function or intracellularly in transcriptional and signaling events. Trefoils seem to protect the epithelium and heal injured mucosa. With mucosal injury, TFFs are up-regulated and stimulate epithelial restitution [57]. They may also play a role in mucus stabilization by interacting or cross- linking with mucin to aid in the formation of gel. Since TFFs may be coexpressed with secreted mucins, there is possibly a synergistic action in mucosal protection and repair between the two since they are coexpressed in both normal and injured mucosae [55, 57].
3.2.4. Resistin-Like Molecule- (RELM-)
This is a member of the resistin-like molecule (RELM) family. RELM- is expressed in both the small and large intestine within IECs and particularly in goblet cells. RELM- regulates barrier integrity and susceptibility to inflammation. It appears that although mucin and RELM- are secreted by goblet cells, RELM- can also act as an effective luminal secretagogue [55, 58–60].
3.3. Tight Junction (TJ) Assembly
The TJ assembly is a cluster of proteins between intestinal epithelial cells forming an effective barrier between the lumen and lamina propria [61]. In general, there are three types of junctional complexes, the tight junctions (TJs), adherens junctions (AJs) and desmosomes, and gap junctions [61–64]. Of these three, TJs represent the major barrier within paracellular pathways between intestinal epithelial cells [61, 64] (Figure 4). The intestinal TJ-complex-associated proteins consist of intracellular and surface membrane proteins. The intracellular proteins are: zona occludens (ZO)-1, ZO-2, and ZO-3, cingulin, 7H6, symplekin, and ZA-1 [61, 64, 65]. The membrane proteins localized to the TJ are the following: occludin, claudin, junctional adhesion molecules (JAMs), and the coxsackie virus and adenovirus receptor (CAR) proteins [61, 62, 64–68]. Studies suggest that claudin-1 may directly associate with occludin laterally in the membrane within the same cell and the combination of these two proteins functioning together performs the major "gatekeeper'' or barrier function of the tight junction [61, 64–69].
Several signaling pathways of the TJ assembly are being investigated. Studies indicate that regulation of TJ assembly occurs through phosphatidylinositol 3-kinase (PI3K) [63]. Prostaglandins have been found to stimulate the recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of TJA. Other pathways implicate two Rho family GTPases, Rho and Rac, and light chain-associated myosin protein kinase 20 for regulation of tight junction proteins. The latter has been implicated as a mechanism for stress and cytokine-induced increases in TJ permeability [63].
A number of microorganisms have been shown to attack the intercellular TJ proteins [61]. Enteric pathogens can disrupt the TJ of IECs through a number of different virulence factors. Disruption of the TJ by enteropathogenic Escherichia coli (EPEC) has been attributed, in part, to altering occludin distribution from the TJ into the cytosol. C. difficile toxins A and B have been shown to disorganize apical and basal F-actin and cause dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane [65–68]. Rotavirus infection of IECs has been shown to increase paracellular permeability and cause alteration of F-actin. Furthermore, it has been determined that the NSP4 (nonstructural protein) of rotavirus reduces transepithelial electrical resistance, redistributes filamentous actin, and prevents lateral targeting of the TJ-associated ZO-1 protein [65–68].
An endogenous protein called "zonulin,'' which is functionally and immunologically related to zonula-occludin toxin from Vibrio cholera, has been found to disassemble intercellular TJs via interaction with cell membrane receptors [65–68]. It is speculated that dysregulation of zonulin in many diseases may involve loss of cell junction integrity. Serum zonulin is not only up-regulated in celiac disease, but also in type 1 diabetes and multiple sclerosis, suggesting the role for a "leaky gut'' in the development of autoimmunity [61].
Despite their complex organization, neither AJs nor TJs are static structures and they can be rapidly disassembled and reorganized in response to various extracellular stimuli. Internalization of AJs and TJs appears to be a common mechanism that rapidly down regulates cell to cell adhesion and allows remodeling of intercellular junctions. Internalization is also induced by various pathophysiologic stimuli including microbial virulence factors, pro-inflammatory cytokines, and oxidative stress [61, 62, 64, 66–69]. Breakdown in the TJA and in the interepithelial barrier associated with intestinal bacteria has been implicated in several diseases including inflammatory bowel disease (IBD), type I diabetes, and obesity [61, 66]. Recent studies indicate that the modulation of microbiota by selectively increasing Bifidobacterium-spp improves barrier function and function of TJA-proteins [66]. Endotoxemia and low-grade inflammation seen in patients with obesity and type I diabetes is believed to be associated with dysbiosis, the presence of a pathogenic microbiota [61, 66, 69]. Developing specific therapeutic strategies to stabilize the microbiota, such as the use of probiotics and prebiotics as an adjunct to management of some of these diseases holds some promise [70].
3.4. Intestinal Epithelial Layer
Composed of a single layer of cells that is only 20 m thick, the intestinal epithelial layer has evolved strategies to derive maximum nutritional benefit from luminal contents and maintain a beneficial host-microbial relationship while keeping the pro-inflammatory potential of the microbiota under control [36]. As an interface between the host and the environment, the mucosal epithelium consists of well organized crypts and villi supported by microvasculature, as well as lymphatic and connective tissues of the lamina propria and submucosa [1, 25, 27] (Figure 5). Other cellular components include lymphocytes, macrophages, and fibroblasts that perform special functions to maintain the integrity of the IM [1–3, 11, 40, 50, 51, 71].
Studies have shown that several immune functions of IECs are influenced by the microbiota, including secretions of cytokines and chemokines and expression of major histocompatibility complex (MHC) molecules that directly interact with lymphocytes [1, 2]. The expression and localization of PRRs (such as TLRs) is also influenced by the microbiota [3, 24, 26, 28, 41, 47, 48]. Expression of several peptides, such as defensins, is defective in germ-free animals [28]. Microbial colonization of germ-free mice induces the production of regenerating islet-derived 3 (RegIII), a secreted C-type lectin. RegIII is shown to have antimicrobial activity by binding to peptidoglycans, suggesting that microbial species actively shape the intestinal ecosystem [70–72]. In another study, using cocolonization of germ-free mice with B. thetaiotaomicron (a symbiont) and B. longum (a probiotic), Sonnenburg et al. showed that B. longum can increase the diversity of polysaccharides that can be degraded by B. thetaiotaomicron, thus, demonstrating that distinct intestinal bacterial species can affect each other's function [73].
3.4.1. Enteroendocrine Cells (EEC)
More than ten distinct types of EEC have been identified in the adult human [1]. Entero-endocrine cells secrete serotonin, somatostatin, motilin, cholecystokinin (CCK), gastric inhibitory peptide (GIP), neurotensin, vasoactive intestinal peptide (VIP), and enteroglucagon. As EECs migrate to the villus tip the number of cytoplasmic granules increases but the ability to divide decreases. Attempts to map the distribution of EECs using immune-cytochemical techniques in transgenic adult mice have been made [74]. The microbiota has been shown to regulate host energy balance and have an impact on obesity. This effect is mediated through Gpr41, a G protein-coupled receptor expressed by a subset of EECs in the IM [74].
3.4.2. Paneth Cells
These cells are the only cell lineage derived from the crypt stem cells that migrate downward towards the crypt base. Paneth cells produce lysozyme, phospholipase A2, TNF-, cryptdins, and guanylin [75]. The guanylin family of peptides consists of three endogenous peptides, one of which has a similar primary structure and biological activity as that of Escherichia coli heat-stable enterotoxin (STa). The guanylin and STa both activate intestinal guanylate cyclase-C (GC-C) and elicit cyclic guanosine monophosphate (cGMP) accumulation in the intestinal mucosa. The activation of cGMP drives secretion of fluids into the intestinal lumen [40, 42, 43, 76].
The cryptdins are defensin-like peptides in intestinal crypts (Figure 5). Cryptdins kill microbes by forming pores in their limiting membranes [40, 75]. At least 17 isoforms have been characterized from a cDNA library. Cryptdin 3 (Cr3) has been shown to induce IL-8 secretion in a dose-dependant manner when applied to a human intestinal cell line (T84) by activating NF-B and p38 MAPK in a calcium-dependent manner, without influx of extracellular calcium (). Unlike other known inflammatory agonists, signal transduction by Cr3 occurs slowly, suggesting that selective cryptdins may amplify their roles in innate immunity by acting as paracrine agonists to coordinate an inflammatory response with the antimicrobial secretions of Paneth cells [40, 75]. Because of their ability to kill gram-positive and gram-negative bacteria, fungi, spirochetes, and some enveloped viruses, cryptdins are classified as broad-spectrum antimicrobial peptides [40, 75, 77–79]. Although it is the least expressed of the six isoforms, cryptdin-4 is the most bactericidal. Procryptdins, however, are not bactericidal and thus require degradation of the pro-region by matrix metalloproteinase-7 (MMP-7) for activation. In response to bacterial antigens, Paneth cells release their secretory granules into the lumen of intestinal crypts [40, 79–81].
Defensins are small cationic polypeptides and are the predominant antimicrobial proteins present in a large number of expressed genes [1, 80, 81]. Mammalian defensins are divided into two main structural groups, and . -defensins are particularly plentiful in neutrophils and intestinal paneth cells. The antibacterial activity of defensins is generally ascribed to their ability to disrupt membrane integrity and function, which ultimately leads to the lysis of the microorganism [40]. In addition to exerting direct antimicrobial effects, defensins facilitate and amplify innate and adaptive immune responses, such as activation and de-granulation of mast cells, cytokine production and secretion, maturation of dendritic cells, and chemotaxis of immune cells [81–83].
Integrins are receptors that mediate attachment between IECs and the tissues surrounding them, which may be other cells or the extracellular matrix [40, 82, 84, 85]. Integrins also play a role in cell signaling and thereby define cellular shape and mobility, and regulate the cell cycle. Integrins regulate the assembly of adhesive junctions as well as the activation of various signaling pathways involved with complex organization of the epithelial-cell matrix in maintaining the crypt-villus axis [1, 82–84].
3.4.3. Microfold (M) Cells
These cells are restricted to the dome epithelium overlying the lymphoid follicles of Peyer's Patches in the lamina propria and other lymphoid follicles in the gut [1, 11, 83]. The surface of M cells is characterized by microfolds and the numerous vesicles contained within. These cells perform a significant role in sampling the luminal milieu. They also appear to be the avenue of infection for viral agents (e.g., rotaviruses of the family, reoviridae) [82, 83]. The numerous vesicles appear to facilitate transport of luminal antigens, macromolecules, and microorganisms to the underlying lymphoid tissue [83].
4. Barrier Defense and Adaptive Immune System
Exposed to a vast number of antigens, the IM is protected by a large immune system [1, 2]. Both lymphoid cells (T and B cells) and myeloid cells (macrophages, neutrophils, eosinophils, and mast cells) have a copious presence in the gut [14, 16]. The GALT has evolved several modifications to generate a unique local specific immunity. Such modifications can be seen in the mechanisms of antigen processing, innate or acquired immune functions, the presence of M cells, and the segregation of the GALT from other mucosa associated lymphoid tissue (MALT) [11, 25, 36, 86, 87].
The mucosal innate immunity has two components, nonimmune and immune. The non-immune components include physico-chemical barriers including digestive enzymes, mucin, peristalsis, indigenous microbial flora, and the epithelial barrier with TJs [61] (Figure 6). The immune components include cellular and soluble elements. Nearly all classes of cells participate in natural immunity, including phagocytes, mast cells, IECs, and natural killer T cells (NK-T) [1, 2, 35]. Based upon the type of signals received from surface receptors of the IM, these cells phagocytize a microbe or antigen, secrete substances facilitating removal of the offending antigen (e.g., cryptidins), and recruit other cell types to produce pro-inflammatory substances (e.g., IL-8 from IECs and interferon- [IFN-] from NK-T cells) [36, 40]. These cells also remove altered (by injury or infection) host cells (e.g., granzymes and perforins from NK-cells) or modify specific immune responses (e.g., IL-4 from NK-T cells). Several of these responses are initiated by interactions between MAMPs and PRRs [40].
The receptors of the innate immune system are expressed on IECs and antigen presenting cells including monocytes, macrophages, B-cells, and dendritic-cells (DCs), [1, 85] (Figure 6). DCs in the lamina propria are specialized to regulate T cell immunity. Normally traversing through non-lymphoid tissue in immature form, DCs switch to immune-stimulatory mode upon encountering inflammatory stimuli. This process, referred to as maturation, changes the phenotype and function of DCs, including up-regulation of costimulatory and adhesion molecules and expression of distinct chemokine receptors [85, 86, 88, 89].
With respect to adaptive (acquired) immunity, helper T (Th) cells can be divided into four distinct cell types. Th1 cells secrete IL-2 and IFN-, a profile that supports the early events of T-cell and B-cell development (sIgG1 production) as well as cell mediated immunity (delayed-type hypersensitivity) [14, 34, 87, 88, 90–93]. Th2 cells, which predominantly secrete IL-4, IL-5, IL-6, IL-10, and IL-13, have a profile associated with humoral immunity (IgG4 and IgE production). Th3 cells, which predominantly secrete TGF, display a profile associated with suppression [88]. And finally, T-regulatory (T-reg1) cells, which predominantly secrete IL-10, also have a profile associated with suppression. Based on these phenotypes, T cells are either considered effector (Th1, Th2) or regulator (Th3, Treg1) T cells [40, 90]. The cell designated antigen (CD4+) cells which express CD25, the IL-2 receptor chain (CD4 + CD25 + cells), may be particularly important T-regulatory cells capable of secreting IL-10 and TGF-. These cytokines are responsible for diminishing the pro-inflammatory process and seem to prevent autoimmune gastritis and inflammatory bowel disease [86–90]. Through this regulatory mechanism, commensal organisms invoke tolerance (hyporesponsiveness) to self. Th0 cells exhibit a secretory pattern of cytokines that includes those associated with Th1 and Th2 patterns, and also represent newly stimulated T cells. Th0 cells are driven to become either Th1 cells by IL-12, which stimulates the transcription factor T- or Th2 cells by IL-4, which stimulates the transcription factor GATA-3 [90–92].
Regulatory Treg and Th17 cells are two recently described subsets with opposing actions [90]. At a steady state, the lamina propria maintains a balanced population of cells of CD4 lineage, TH17 cells, and Treg cells. Th17 cells induce production of IL-17, a potent inflammatory regulator [88–93]. Production of IL-17 requires the presence of IL-6 and TGF activation, as well as a microbial trigger [93]. It has been suggested that a balance between Th17 and Treg is regulated through microbiota that can influence tolerance (hypo-responsiveness) or inflammatory response [92, 93]. Evidence is accumulating that targeting IL-17 signaling might prove useful in treating a variety of inflammatory diseases [88–93].
Chemokines present another important family of mediators involved in the immune-inflammatory response, activating chemo-attraction and leucocytes [2]. The receptors for chemokines (C, CC, CXC, CX3C) are seven membrane spanning G-protein-linked receptors that are coupled to cell activation through calcium mobilization and respond to cytokines of the CXC chemokine family. Interleukin-8 (IL-8 or CXL-8), induced by interferon gamma (Mig or CXCL9), interferon-inducible protein-10 (IP-10 or CXL10), and Interferon-inducible T cell chemo-attractant (I-TAC or CXCL11), belong to the CXC family that are expressed by enterocytes, presenting another mechanism of immune-inflammatory response by enterocytes [1, 11, 87, 94].
5. Conclusions
External to the IM, the microbiota, mucin, and antibacterial products (such as defensins and immunoglobulins) protect the host against pathogens. The layer of epithelial cells with several components of innate immunity including PRRs on the surface, NOD2/CARD15 intracellularly, and the paracelluar space sealed with TJA [40] present a relatively impermeable brush border. Then, important interactions take place under the control of the adaptive immune system in the lamina propria. The dynamic landscape of the IM in this manner is constantly adjusting to changing conditions while providing nutrition and also maintaining efficient immunity. In this respect, the symbiotic relationship of the IM with the microbiota is critical [3, 26, 40].
Manipulations of the microbiota to enhance its beneficial components that complement the human immune system with barrier-fortification are areas of active research [4, 23, 26, 34]. Disruption in the establishment of a stable microbiota (dysbiosis) is associated with diseases including inflammatory bowel diseases (IBD), obesity, atopy, and neonatal necrotizing enterocolitis (NEC) [44, 47, 70, 73]. The immature immune system and frequent exposure to antibiotics in neonatal intensive care units render premature infants, who are particularly susceptible to dysbiosis, more likely to develop NEC. Available conventional therapies for inflammatory intestinal diseases including NEC primarily target the infectious and inflammatory components of the diseases [3, 61, 73, 84]. The contribution of microbial communities in maintaining the integrity of the IM and in facilitating repair emphasizes the rationale for therapeutic exploitation of commensal organisms. There is evidence supporting a therapeutic role for probiotic strategies in treating certain diseases (e.g., IBD, NEC, diarrhea), but scientific validations and further research investigating the efficacy of such strategies without long-term immunological complications are warranted, particularly when such strategies apply to immune-incompetent hosts such as premature infants or cancer patients [5, 73, 76, 82, 86, 93, 94]. Understanding microbial-mucosal signaling components of inflammatory pathways and the mechanisms of how commensal organisms regulate these pathways should also provide new directions in treating and preventing these diseases.
|intestinal mucosa
|pattern recognition receptors
|microbial-associated molecular patterns
|Toll-like receptor
|nucleotide-binding oligomerization domain
|NOD-like receptors
|interferon 1
|formylated peptide receptors
|nicotinamide adenine dinucleotide phosphate
|NADPH oxide
|intestinal epithelial cells
|nuclear factor kappa B
|proteins coded by rel oncogenes
|mitogen-activated protein kinase
|Inhibitory kappa B
|peroxisome proliferator-activated receptor
|transforming growth factor-
|myeloid differentiation primary-response gene 88
|fast activated death domain
|intra-cellular IL-1-converting enzyme protease-activator factor
|neuronal apoptosis inhibitory protein
|gut-associated lymphoid tissue
|intra-epithelial lymphocytes
|T-cell receptor
|caspase recruitment domain
|inhibitory B kinase
|Toll-interleukin 1 receptor
|IL-1-R associated protein kinase
|Toll-interacting protein
|single immunoglobulin IL-1R-related protein
|short chain fatty acids
|cell designated protein 14
|lipopolysaccharide binding protein
|reticulo endothelial-activating system
|Meiosis specific serine/threonine protein kinase
|extracellular signal-regulated kinase
|adhesion and protease domain metallopeptidase 10
|G protein-coupled receptor
|rous sarcoma virus-cytoplasmic protein
|lipoteichoic acid
|platelet-activating factor
|epidermal growth factor
|resistin-like molecule
|trefoil factors
|tight junction
|adherens junction
|zona occludens
|junctional adhesion molecules
|coxcackie virus and adenovirus receptor protein
|phosphatidylinositol 3-kinase
|nonstructural protein 4
|inflammatory bowel disease
|major histocompatibility complex
|regenerating islet-derived 3 gamma
|tumor necrosis factor
|heat stable enterotoxin
|guanylate cyclase
|cyclic guanosine monophosphate
|matrix metalloproteinase-7
|mucosa associated lymphoid tissue
|natural killer T cells
|dendritic cells
|T helper 2 cells
|T regulatory
|transforming growth factor
|chemokine family
|interferon inducible protein
|T cell chemo-attractant.
This work was supported by the National Institutes of Health Grant RO1 HD05914301.
V. J. McCracken and R. G. Lorenz, "The gastrointestinal ecosystem: a precarious alliance among epithelium, immunity and microbiota," Cellular Microbiology, vol. 3, no. 1, pp. 1–11, 2001.View at: Publisher Site | Google Scholar
T. T. MacDonald and J. N. Gordon, "Bacterial regulation of intestinal immune responses," Gastroenterology Clinics of North America, vol. 34, no. 3, pp. 401–412, 2005.View at: Publisher Site | Google Scholar
A. S. Neish, "Microbes in gastrointestinal health and disease," Gastroenterology, vol. 136, no. 1, pp. 65–80, 2009.View at: Publisher Site | Google Scholar
L. V. Hooper, "Bacterial contributions to mammalian gut development," Trends in Microbiology, vol. 12, no. 3, pp. 129–134, 2004.View at: Publisher Site | Google Scholar
J. Neu, "Perinatal and neonatal manipulation of the intestinal microbiome: a note of caution," Nutrition Reviews, vol. 65, no. 6, pp. 282–285, 2007.View at: Publisher Site | Google Scholar
P. B. Eckburg, E. M. Bik, C. N. Bernstein et al., "Diversity of the human intestinal microbial flora," Science, vol. 308, no. 5728, pp. 1635–1638, 2005.View at: Publisher Site | Google Scholar
P. J. Turnbaugh, R. E. Ley, M. Hamady, C. M. Fraser-Liggett, R. Knight, and J. I. Gordon, "The human microbiome project," Nature, vol. 449, no. 7164, pp. 804–810, 2007.View at: Publisher Site | Google Scholar
I. Adlerberth, "Factors influencing the establishment of the intestinal microbiota in infancy," Nestle Nutrition Workshop Series, vol. 62, pp. 13–29, 2008.View at: Publisher Site | Google Scholar
J. G. Camp, M. Kanther, I. Semova, and J. F. Rawls, "Patterns and scales in gastrointestinal microbial ecology," Gastroenterology, vol. 136, no. 6, pp. 1989–2002, 2009.View at: Publisher Site | Google Scholar
M. J. Wick, J. L. Madara, B. N. Fields, and S. J. Normark, "Molecular cross talk between epithelial cells and pathogenic microorganisms," Cell, vol. 67, no. 4, pp. 651–659, 1991.View at: Google Scholar
S. Akira, "Pathogen recognition by innate immunity and its signaling," Proceedings of the Japan Academy, Series B, vol. 85, no. 4, pp. 143–156, 2009.View at: Publisher Site | Google Scholar
C. G. Figdor, Y. van Kooyk, and G. J. Adema, "C-type lectin receptors on dendritic cells and langerhans cells," Nature Reviews Immunology, vol. 2, no. 2, pp. 77–84, 2002.View at: Google Scholar
L. V. Hooper, M. H. Wong, A. Thelin, L. Hansson, P. G. Falk, and J. I. Gordon, "Molecular analysis of commensal host-microbial relationships in the intestine," Science, vol. 291, no. 5505, pp. 881–884, 2001.View at: Publisher Site | Google Scholar
A. J. V. Thompson and S. A. Locarnini, "Toll-like receptors, RIG-I-like RNA helicases and the antiviral innate immune response," Immunology and Cell Biology, vol. 85, no. 6, pp. 435–445, 2007.View at: Publisher Site | Google Scholar
V. Mai and J. G. Morris Jr., "Colonic bacterial flora: changing understandings in the molecular age," Journal of Nutrition, vol. 134, no. 2, pp. 459–464, 2004.View at: Google Scholar
J. C. Alverdy and E. B. Chang, "The re-emerging role of the intestinal microflora in critical illness and inflammation: why the gut hypothesis of sepsis syndrome will not go away," Journal of Leukocyte Biology, vol. 83, no. 3, pp. 461–466, 2008.View at: Publisher Site | Google Scholar
I. Migeotte, D. Communi, and M. Parmentier, "Formyl peptide receptors: a promiscuous subfamily of G protein-coupled receptors controlling immune responses," Cytokine and Growth Factor Reviews, vol. 17, no. 6, pp. 501–519, 2006.View at: Publisher Site | Google Scholar
B. A. Babbin, A. J. Jesaitis, A. I. Ivanov et al., "Formyl peptide receptor-1 activation enhances intestinal epithelial cell restitution through phosphatidylinositol 3-kinase-dependent activation of Rac1 and Cdc42," Journal of Immunology, vol. 179, no. 12, pp. 8112–8121, 2007.View at: Google Scholar
D. Bouskra, C. Brezillon, M. Berard et al., "Lymphoid tissue genesis induced by commensals through NOD1 regulates intestinal homeostasis," Nature, vol. 456, no. 7221, pp. 507–510, 2008.View at: Publisher Site | Google Scholar
M. T. Abreu, M. Fukata, and M. Arditi, "TLR signaling in the gut in health and disease," Journal of Immunology, vol. 174, no. 8, pp. 4453–4460, 2005.View at: Google Scholar
A. S. Neish, "TLRS in the gut. II. Flagellin-induced inflammation and antiapoptosis," American Journal of Physiology, vol. 292, no. 2, pp. G462–G466, 2007.View at: Publisher Site | Google Scholar
R. A. Caicedo, R. J. Schanler, N. Li, and J. Neu, "The developing intestinal ecosystem: implications for the neonate," Pediatric Research, vol. 58, no. 4, pp. 625–628, 2005.View at: Publisher Site | Google Scholar
A. M. O'Hara and F. Shanahan, "Gut microbiota: mining for therapeutic potential," Clinical Gastroenterology and Hepatology, vol. 5, no. 3, pp. 274–284, 2007.View at: Publisher Site | Google Scholar
C. L. Leaphart and J. J. Tepas III, "The gut is a motor of organ system dysfunction," Surgery, vol. 141, no. 5, pp. 563–569, 2007.View at: Publisher Site | Google Scholar
J. Marchesi and F. Shanahan, "The normal intestinal microbiota," Current Opinion in Infectious Diseases, vol. 20, no. 5, pp. 508–513, 2007.View at: Publisher Site | Google Scholar
A. M. O'Hara and F. Shanahan, "The gut flora as a forgotten organ," EMBO Reports, vol. 7, no. 7, pp. 688–693, 2006.View at: Publisher Site | Google Scholar
J. L. Round and S. K. Mazmanian, "The gut microbiota shapes intestinal immune responses during health and disease," Nature Reviews Immunology, vol. 9, no. 5, pp. 313–323, 2009.View at: Publisher Site | Google Scholar
S. Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov, "Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis," Cell, vol. 118, no. 2, pp. 229–241, 2004.View at: Publisher Site | Google Scholar
M. Mshvildadze, J. Neu, and V. Mai, "Intestinal microbiota development in the premature neonate: establishment of a lasting commensal relationship?" Nutrition Reviews, vol. 66, no. 11, pp. 658–663, 2008.View at: Publisher Site | Google Scholar
S. M. Huse, L. Dethlefsen, J. A. Huber, D. M. Welch, D. A. Relman, and M. L. Sogin, "Exploring microbial diversity and taxonomy using SSU rRNA hypervariable tag sequencing," PLoS Genetics, vol. 4, no. 11, article e1000255, 2008.View at: Publisher Site | Google Scholar
T. Z. DeSantis, E. L. Brodie, J. P. Moberg, I. X. Zubieta, Y. M. Piceno, and G. L. Andersen, "High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone library when sampling the environment," Microbial Ecology, vol. 53, no. 3, pp. 371–383, 2007.View at: Publisher Site | Google Scholar
A. Loy, A. Lehner, N. Lee et al., "Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment," Applied and Environmental Microbiology, vol. 68, no. 10, pp. 5064–5081, 2002.View at: Publisher Site | Google Scholar
T. S. Stappenbeck, L. V. Hooper, and J. I. Gordon, "Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells," Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 24, pp. 15451–15455, 2002.View at: Publisher Site | Google Scholar
M. Conroy and W. Walker, "Intestinal immune health," Nestle Nutrition Workshop Series, vol. 62, pp. 111–121, 2008.View at: Publisher Site | Google Scholar
A. S. Ismail and L. V. Hooper, "Epithelial cells and their neighbors. IV. Bacterial contributions to intestinal epithelial barrier integrity," American Journal of Physiology, vol. 289, no. 5, pp. G779–G784, 2005.View at: Publisher Site | Google Scholar
A. J. Macpherson and N. L. Harris, "Interactions between commensal intestinal bacteria and the immune system," Nature Reviews Immunology, vol. 4, no. 6, pp. 478–485, 2004.View at: Google Scholar
P. G. Falk, L. V. Hooper, T. Midtvedt, and J. I. Gordon, "Creating and maintaining the gastrointestinal ecosystem: what we know and need to know from gnotobiology," Microbiology and Molecular Biology Reviews, vol. 62, no. 4, pp. 1157–1170, 1998.View at: Google Scholar
G. D. Abrams, H. Bauer, and H. Sprinz, "Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of germ-free and conventional mice," Lab Investigation, vol. 12, pp. 355–364, 1963.View at: Google Scholar
L. Bry, P. G. Falk, T. Midtvedt, and J. I. Gordon, "A model of host-microbial interactions in an open mammalian ecosystem," Science, vol. 273, no. 5280, pp. 1380–1383, 1996.View at: Google Scholar
S. Mukherjee, S. Vaishnava, and L. V. Hooper, "Multi-layered regulation of intestinal antimicrobial defense," Cellular and Molecular Life Sciences, vol. 65, no. 19, pp. 3019–3027, 2008.View at: Publisher Site | Google Scholar
Y. Umesaki, H. Setoyama, S. Matsumoto, and Y. Okada, "Expansion of alpha beta T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germ-free mice and its independence from thymus," Immunology, vol. 79, no. 1, pp. 32–37, 1993.View at: Google Scholar
L. Lefrancois and T. Goodman, "In vivo modulation of cytolytic activity and Thy-1 expression in TCR-gamma delta+ intraepithelial lymphocytes," Science, vol. 243, no. 4899, pp. 1716–1718, 1989.View at: Google Scholar
Y. Umesaki, H. Setoyama, S. Matsumoto, A. Imaoka, and K. Itoh, "Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system," Infection and Immunity, vol. 67, no. 7, pp. 3504–3511, 1999.View at: Google Scholar
M. C. Biol, "Nutritional and developmental variations of intestinal glycosylation," Nutrition, vol. 8, no. 5, pp. 368–369, 1992.View at: Google Scholar
L. V. Hooper, T. S. Stappenbeck, C. V. Hong, and J. I. Gordon, "Angiogenins: a new class of microbicidal proteins involved in innate immunity," Nature Immunology, vol. 4, no. 3, pp. 269–273, 2003.View at: Publisher Site | Google Scholar
Y. Umesaki, Y. Okada, S. Matsumoto, A. Imaoka, and H. Setoyama, "Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse," Microbiology and Immunology, vol. 39, no. 8, pp. 555–562, 1995.View at: Google Scholar
D. Kelly, S. Conway, and R. Aminov, "Commensal gut bacteria: mechanisms of immune modulation," Trends in Immunology, vol. 26, no. 6, pp. 326–333, 2005.View at: Publisher Site | Google Scholar
N. Li, W. M. Russell, M. Douglas-Escobar, N. Hauser, M. Lopez, and J. Neu, "Live and heat-killed Lactobacillus rhamnosus GG: effects on proinflammatory and anti-inflammatory cytokines/chemokines in gastrostomy-fed infant rats," Pediatric Research, vol. 66, no. 2, pp. 203–207, 2009.View at: Publisher Site | Google Scholar
F. Yan, H. Cao, T. L. Cover, R. Whitehead, M. K. Washington, and D. B. Polk, "Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth," Gastroenterology, vol. 132, no. 2, pp. 562–575, 2007.View at: Publisher Site | Google Scholar
M. Karin and Y. Ben-Neriah, "Phosphorylation meets ubiquitination: the control of NF-B activity," Annual Review of Immunology, vol. 18, pp. 621–663, 2000.View at: Publisher Site | Google Scholar
A. S. Neish, A. T. Gewirtz, H. Zeng et al., "Prokaryotic regulation of epithelial responses by inhibition of IB- ubiquitination," Science, vol. 289, no. 5484, pp. 1560–1563, 2000.View at: Publisher Site | Google Scholar
P. Dharmani, V. Srivastava, V. Kissoon-Singh, and K. Chadee, "Role of intestinal mucins in innate host defense mechanisms against pathogens," Journal of Innate Immunity, vol. 1, no. 2, pp. 123–135, 2009.View at: Publisher Site | Google Scholar
M. E. Johansson, M. Phillipson, J. Petersson, A. Velcich, L. Holm, and G. C. Hansson, "The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria," Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 39, pp. 15064–15069, 2008.View at: Publisher Site | Google Scholar
Y. Yan, K. Shirakabe, and Z. Werb, "The metalloprotease Kuzbanian (ADAM10) mediates the transactivation of EGF receptor by G protein-coupled receptors," Journal of Cell Biology, vol. 158, no. 2, pp. 221–226, 2002.View at: Google Scholar
B. Deplancke and H. R. Gaskins, "Microbial modulation of innate defense: goblet cells and the intestinal mucus layer," American Journal of Clinical Nutrition, vol. 73, no. 6, pp. 1131S–1141S, 2001.View at: Google Scholar
S. K. Linden, T. H. J. Florin, and M. A. McGuckin, "Mucin dynamics in intestinal bacterial infection," PLoS ONE, vol. 3, no. 12, article e3952, 2008.View at: Publisher Site | Google Scholar
S. Suemori, K. Lynch-Devaney, and D. K. Podolsky, "Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family," Proceedings of the National Academy of Sciences of the United States of America, vol. 88, no. 24, pp. 11017–11021, 1991.View at: Google Scholar
D. K. Podolsky, "Mucosal immunity and inflammation V. Innate mechanisms of mucosal defense and repair: the best offense is a good defense," American Journal of Physiology, vol. 277, no. 3, pp. G495–G499, 1999.View at: Google Scholar
D. Artis, M. L. Wang, S. A. Keilbaugh et al., "RELM/FIZZ2 is a goblet cell-specific immune-effector molecule in the gastrointestinal tract," Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 37, pp. 13596–13600, 2004.View at: Publisher Site | Google Scholar
R. B. Krimi, L. Kotelevets, L. Dubuquoy et al., "Resistin-like molecule regulates intestinal mucous secretion and curtails TNBS-induced colitis in mice," Inflammatory Bowel Diseases, vol. 14, no. 7, pp. 931–941, 2008.View at: Publisher Site | Google Scholar
Z. Liu, N. Li, and J. Neu, "Tight junctions, leaky intestines, and pediatric diseases," Acta Paediatrica, vol. 94, no. 4, pp. 386–393, 2005.View at: Publisher Site | Google Scholar
A. I. Ivanov, A. Nusrat, and C. A. Parkos, "Endocytosis of epithelial apical junctional proteins by a clathrin-mediated pathway into a unique storage compartment," Molecular Biology of the Cell, vol. 15, no. 1, pp. 176–188, 2004.View at: Publisher Site | Google Scholar
N. Li and J. Neu, "Glutamine deprivation alters intestinal tight junctions via a PI3-K/Akt mediated pathway in caco-2 cells," Journal of Nutrition, vol. 139, no. 4, pp. 710–714, 2009.View at: Publisher Site | Google Scholar
Z. Hossain and T. Hirata, "Molecular mechanism of intestinal permeability: interaction at tight junctions," Molecular BioSystems, vol. 4, no. 12, pp. 1181–1185, 2008.View at: Publisher Site | Google Scholar
E. S. Wittchen, J. Haskins, and B. R. Stevenson, "Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3," Journal of Biological Chemistry, vol. 274, no. 49, pp. 35179–35185, 1999.View at: Publisher Site | Google Scholar
P. D. Cani, S. Possemiers, T. Van de Wiele et al., "Changes in gut microbiota control inflammation in obese mice through a mechanism involving GLP-2-driven improvement of gut permeability," Gut, vol. 58, no. 8, pp. 1091–1103, 2009.View at: Publisher Site | Google Scholar
S. Prasad, R. Mingrino, K. Kaukinen et al., "Inflammatory processes have differential effects on claudins 2, 3 and 4 in colonic epithelial cells," Laboratory Investigation, vol. 85, no. 9, pp. 1139–1162, 2005.View at: Publisher Site | Google Scholar
F. Tafazoli, C. Q. Zeng, M. K. Estes, K.-E. Magnusson, and L. Svensson, "NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized epithelial cells," Journal of Virology, vol. 75, no. 3, pp. 1540–1546, 2001.View at: Publisher Site | Google Scholar
J. Meddings, "The significance of the gut barrier in disease," Gut, vol. 57, no. 4, pp. 438–440, 2008.View at: Publisher Site | Google Scholar
H. L. Cash, C. V. Whitham, C. L. Behrendt, and L. V. Hooper, "Symbiotic bacteria direct expression of an intestinal bactericidal lectin," Science, vol. 313, no. 5790, pp. 1126–1130, 2006.View at: Publisher Site | Google Scholar
N. H. Salzman, M. A. Underwood, and C. L. Bevins, "Paneth cells, defensins, and the commensal microbiota: a hypothesis on intimate interplay at the intestinal mucosa," Seminars in Immunology, vol. 19, no. 2, pp. 70–83, 2007.View at: Publisher Site | Google Scholar
S. Vaishnava, C. L. Behrendt, A. S. Ismail, L. Eckmann, and L. V. Hooper, "Paneth cells directly sense gut commensals and maintain homeostasis at the intestinal host-microbial interface," Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 52, pp. 20858–20863, 2008.View at: Publisher Site | Google Scholar
J. L. Sonnenburg, C. T. Chen, and J. I. Gordon, "Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host," PLoS Biology, vol. 4, no. 12, article e413, 2006.View at: Publisher Site | Google Scholar
K. A. Roth, J. M. Hertz, and J. I. Gordon, "Mapping enteroendocrine cell populations in transgenic mice reveals an unexpected degree of complexity in cellular differentiation within the gastrointestinal tract," Journal of Cell Biology, vol. 110, no. 5, pp. 1791–1801, 1990.View at: Publisher Site | Google Scholar
P. W. Lin, P. O. Simon Jr., A. T. Gewirtz et al., "Paneth cell cryptdins act in vitro as apical paracrine regulators of the innate inflammatory response," Journal of Biological Chemistry, vol. 279, no. 19, pp. 19902–19907, 2004.View at: Publisher Site | Google Scholar
B. S. Samuel, A. Shaito, T. Motoike et al., "Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41," Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 43, pp. 16767–16772, 2008.View at: Publisher Site | Google Scholar
M. W. Hornef, K. Putsep, J. Karlsson, E. Refai, and M. Andersson, "Increased diversity of intestinal antimicrobial peptides by covalent dimer formation," Nature Immunology, vol. 5, no. 8, pp. 836–843, 2004.View at: Publisher Site | Google Scholar
N. H. Salzman, D. Ghosh, K. M. Huttner, Y. Paterson, and C. L. Bevins, "Protection against enteric salmonellosis in transgenic mice expressing a human intestinal defensin," Nature, vol. 422, no. 6931, pp. 522–526, 2003.View at: Publisher Site | Google Scholar
O. Froy, N. Chapnik, and R. Miskin, "Mouse intestinal cryptdins exhibit circadian oscillation," FASEB Journal, vol. 19, no. 13, pp. 1920–1922, 2005.View at: Publisher Site | Google Scholar
C. Lussier, N. Basora, Y. Bouatrouss, and J.-F. Beaulieu, "Integrins as mediators of epithelial cell-matrix interactions in the human small intestinal mucosa," Microscopy Research and Technique, vol. 51, no. 2, pp. 169–178, 2000.View at: Google Scholar
S. A. Saenz, B. C. Taylor, and D. Artis, "Welcome to the neighborhood: epithelial cell-derived cytokines license innate and adaptive immune responses at mucosal sites," Immunological Reviews, vol. 226, no. 1, pp. 172–190, 2008.View at: Publisher Site | Google Scholar
G. O. Canny and B. A. McCormick, "Bacteria in the intestine, helpful residents or enemies from within?" Infection and Immunity, vol. 76, no. 8, pp. 3360–3373, 2008.View at: Publisher Site | Google Scholar
T. W. Spahn, M. K. Muller, W. Domschke, and T. Kucharzik, "Role of lymphotoxins in the development of Peyer's patches and mesenteric lymph nodes: relevance to intestinal inflammation and treatment," Annals of the New York Academy of Sciences, vol. 1072, pp. 187–193, 2006.View at: Publisher Site | Google Scholar
J. Neu, M. Chen, and E. Beierle, "Intestinal innate immunity: how does it relate to the pathogenesis of necrotizing enterocolitis," Seminars in Pediatric Surgery, vol. 14, no. 3, pp. 137–144, 2005.View at: Publisher Site | Google Scholar
J. H. Niess and H.-C. Reinecker, "Dendritic cells: the commanders-in-chief of mucosal immune defenses," Current Opinion in Gastroenterology, vol. 22, no. 4, pp. 354–360, 2006.View at: Publisher Site | Google Scholar
T. T. MacDonald and G. Monteleone, "Immunity, inflammation, and allergy in the gut," Science, vol. 307, no. 5717, pp. 1920–1925, 2005.View at: Publisher Site | Google Scholar
M. L. Forchielli and W. A. Walker, "The role of gut-associated lymphoid tissues and mucosal defence," British Journal of Nutrition, vol. 93, supplement 1, pp. S41–S48, 2005.View at: Publisher Site | Google Scholar
E. M. Eisenstein and C. B. Williams, "The Treg/Th17 cell balance: a new paradigm for autoimmunity," Pediatric Research, vol. 65, supplement 5, pp. 26R–31R, 2009.View at: Publisher Site | Google Scholar
M. Niessner and B. A. Volk, "Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR)," Clinical and Experimental Immunology, vol. 101, no. 3, pp. 428–435, 1995.View at: Google Scholar
I. I. Ivanov, R. L. Frutos, N. Manel et al., "Specific microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine," Cell Host and Microbe, vol. 4, no. 4, pp. 337–349, 2008.View at: Publisher Site | Google Scholar
A. Andoh, Y. Yagi, M. Shioya, A. Nishida, T. Tsujikawa, and Y. Fujiyama, "Mucosal cytokine network in inflammatory bowel disease," World Journal of Gastroenterology, vol. 14, no. 33, pp. 5154–5161, 2008.View at: Publisher Site | Google Scholar
K. Honda and K. Takeda, "Regulatory mechanisms of immune responses to intestinal bacteria," Mucosal Immunology, vol. 2, no. 3, pp. 187–196, 2009.View at: Publisher Site | Google Scholar
S. Ivanov and A. Linden, "Interleukin-17 as a drug target in human disease," Trends in Pharmacological Sciences, vol. 30, no. 2, pp. 95–103, 2009.View at: Publisher Site | Google Scholar
A. M. Mowat, "Anatomical basis of tolerance and immunity to intestinal antigens," Nature Reviews Immunology, vol. 3, no. 4, pp. 331–341, 2003.View at: Google Scholar


text:
You know, I know that red dresses are kind of frowned upon on this site from time to time. I know that the prevailing wisdom is that it's impossible to do one well.
To this I say you know what? We're throwing out the rule book. Because Sally Field is fantastic, and she looks spectacular, and she's doing that thing where the dress reminds you of the part without actually being a costume from the show, and I was delighted. It's Valentino Couture. It was note perfect for her, and all the people who are like "oh, Hilary Swank wore it too", need to know that I am not listening.
Because Sally Field probably gets to choose what she does with great precision, and we're not necessarily going to see her at everything all the time, and when we do, we should take it as the gift that it is. I think she would have given a kick-ass speech, too, but we were denied that. You know who to blame on that one.

subset: commoncrawl


text:
Authors work to clear Air Corps pilot's name
At the wake for United States Army Air Corps Lt. William Flynn, Jack Flynn saw a short man pin silver wings to his brother's uniform.
Jack Flynn learned that this man, retired United States Army Air Corps Sgt. Alvin E. Kotler, was a friend of his brother who served with him in World War II.
In addition to being close to William, the two men had another thing in common: They wanted to clear his name.
Kotler and Jack Flynn became friends, and Flynn learned the details of the incident that ended his brother's military career with a dishonorable discharge and sent him to federal prison. Those details became the subject of Kotler and Jack Flynn's book, "We Came to Fight a War."
Kotler, a Canton resident, met William Flynn at an operational training unit in Tennessee. They flew as a crew for three months.
"We trained together," Kotler said. "We were like a family. We lived as a crew, fought as a crew and depended on each other."
Kotler became the radio gunner on William Flynn's B-17 during the course of multiple bombing missions out of Foggia, Italy, in 1944 and 1945.
On May 28, 1945, William Flynn, a Wakefield resident, was awarded The Distinguished Flying Cross for "extraordinary achievement" in combat and Kotler was awarded the Purple Heart for a mission in which they were also cited for destruction of government property.
The crew was flying to Vienna when they came under fire and were shot off course, descendeding through the Italian Alps. The shrapnel struck two engines, which failed and caused the oxygen line to leak. Kotler received a shrapnel wound to his leg. Despite the chaos, William Flynn knew what had to be done. In order to clear the mountains, he told the crew to strip the plane and throw anything they didn't need outside to keep the plane from falling too quickly.
In what Kotler described as incredible flying, William Flynn managed to avoid crashing on a beach and found an emergency landing field and safely put the plane down.
"We were the important part, not the plane," Kotler said.
"[William] was built to fly, to do heroic things," Jack Flynn said. "He had a way of calming people down when horrible things were happening."
On April 15, 1945, Kotler and William Flynn completed their 25th and final bombing mission out of Foggia, Italy. To earn some extra money before they were shipped back home, Kotler said the pair took on training flights. William Flynn was going to get married when he got back, and wanted to save for the wedding. One flight was for the tuxedo, Kotler recalled. Another was for the catering. Each flight brought William Flynn closer to marrying the woman he loved.
There were concerns at the time by the Army Air Corps about pilots "buzzing" – flying below 1,000 feet over heavily populated areas. When William Flynn lost control of his B-17 during a June 22, 1945, flight, Kotler and Jack Flynn said he became the Army's scapegoat.
According to Kotler, William Flynn lost control of his plane over a "tent city" where pilots and co-pilots were staying. The area was known for its rough winds and while William Flynn struggled to keep the plane as high as possible, it jerked to the left and dropped as low as 40 feet above the ground. One of the plane's wings struck a telephone pole cable, which wrapped around a driver who was killed in the accident.
A General Court Martial was convened, and William Flynn was convicted of manslaughter, relieved of all honors and benefits, dishonorably discharged from the service and sentenced to a year of hard labor in a federal prison.
Jack Flynn and Kotler said William Flynn didn't stand a chance during the trial. After his attorney died of a heart attack, he was provided with an inexperienced Judge Advocate General lawyer to defend him.
Many of the witnesses who could have helped William Flynn's case had been shipped out before the trial. One witness who did stay to testify was ordered to sign what Kotler and Jack Flynn call a false statement indicating that William Flynn was "buzzing" at the time of the accident.
After the war and prison time, William Flynn married Dorothy Dunnan of Stoneham, and they raised seven children together. William Flynn ran into Kotler some years later, and Kotler offered him a job at his liquor store in Lakeville after learning he was having money problems. William Flynn worked for Kotler long enough to get back on his feet and later worked for UPS as an area manager until he retired in 1985.
For 15 years, every Memorial Day, William Flynn would fly a small plane over Wakefield and drop flowers on Moulton Field where there was a memorial for fallen veterans.
"They chose Bill to do it because he was the only pilot in the area who could be relied on to hit the target from a thousand feet," Jack Flynn said.
William Flynn died in March 2008 at the age of 83.
"To the day he died, he never spoke bad of his country," Kotler said.
"He never blamed the government," Jack Flynn added. "He was a patriot from the get-go."
After William Flynn died, Kotler and Jack Flynn made it their mission to clear his name and see his military honors restored. Their crusade led to the publication of "We Came to Fight a War," a book that Jack Flynn has sent to former President George H.W. Bush and to Sen. John Kerry, seeking their help.
Bush replied with a personal letter expressing his condolences but apologizing because he couldn't get involved. Kerry sent back a more generic letter saying the past conviction could not be overturned.
But Jack Flynn and Kotler aren't giving up.
"Our goal is to right this wrong," Jack Flynn said.
"We Came to Fight a War" can be found online at www.amazon.com and www.barnesandnoble.com.
Canton Journal


text:
Share this item
Sprinkle Sparkle Earrings Gold
Products marked Considered meet one or more of the following standards.
Ethical Production - The factory manufacturers this item has passed SMETA (Sedex Members Ethical Trade Audit). This audit considers Labor Standards, Health & Safety, Business Ethics and Environment. You can be confident that a SMETA-approved factory adheres to the highest levels of ethical production.
Sustainable Materials - This item is made from materials with a lower impact on our environment. Whether it is recycled nylon, recycled polyester or organically grown cotton, you can shop knowing your new garment is soft on you and gentle on our planet.
We aim to continually assess and improve the standard by which we rate our Considered edit.
- Gold hoop earrings.
- Comes as a set.
- Polished metal construction.
- True to size.
- Crystal star pendant.
- Faux crystal insets.
- Hoop: 2cm
- Earring drop: 1.5cm.
- Lever-back closures for pierced ears.
- Please Note: Avoid contact with water, lotions and perfumes.
It's all glitz and glam with the Sprinkle Sparkle Earrings. These sparkling hoops feature a glamorous star crystal pendant which means bling bling from every angle! We are obsessed!
Hello Molly offers FREE standard shipping to the USA on all orders over $100. Expect delivery within 4-9 business days. Express delivery is also available and takes 2-4 business days. We accept returns for store credit within 30 days of order delivery for full priced items only.


text:
A 4-year-old autistic boy was reported missing Thursday and four people were found slain near his home in Hudson, Fla., authorities say.
The Pasco County Sheriff's Office says Ismael Tristan Santisteban was last seen at his home Aug. 28. During a welfare check Thursday, the child was discovered missing, and there were signs at the home of foul play.
A search of the area revealed four bodies were discovered outside less than a mile from the home. Pasco Sheriff Chris Nocco says the victims appear to be of two adult males and two adult females. They had not been identified Thursday evening.
Authorities say they are looking for 28-year-old Adam Matos, who could be traveling in a 1991 blue Ford F150 pickup truck. Detectives describe Matos as a person of interest.
Nocco said Matos is believed to be Tristan's father. Matos is from Pennsylvania, but investigators say he has family in Florida.
SOURCE: WTSP-TV, Tampa-St. Petersburg


text:
John Christner Trucking
Call Today to Learn More About Our NO CASH DOWN Lease Purchase Program for Solos!
John Christner Trucking is moving critical fresh food loads to restock shelves and need hard working professionals like you. We are all in this together.
That's right – JCT is working harder than ever to make sure our Contractors are earning what they deserve.
We Offer:
* Solos earn $1.00 per mile base pay
* Market Premium Pay – currently $0.08/mile on all miles!!
* $1,000 Orientation Pay (paid upon first dispatch & delivery)
* $40/hour Detention Pay
* Fuel Surcharge paid on all miles – loaded and empty!
* Pilot/Flying J Fuel Rebate of $.15/gallon
* True Lease Purchase Program that saves drivers $10 – $12K of out of pocket expenses
* $.02 cents per mile safety bonus, paid quarterly
* No cash down
* $1 buyout with no balloon payments
* Paid base plates and permits
* Road use and fuel taxes paid
* EZ Pass, Oklahoma Pike Pass and Drivewyze
* Toll and scale reimbursement
* Large fleet buying power
* Pay off your truck quicker with our Variable Lease Program
At the end of the lease term, the truck is yours for $1…That's right, just $1 and the title is yours!
Lease To Own Requirements:
* Must possess a valid Class-A CDL and have 48 state tractor-trailer driving experience
* No more than 3 moving violations in the past 3 years
* No more than 3 preventable accidents in the past 3 years
JCT is big enough to offer all you need to be successful but we're small enough to know you by name. We have the right trucks, the right freight, the right people. If you've ever thought of being a truck driver and owning your own truck, our Lease to Own program is the key to making those dreams come true!
For more information about our Lease to Own program including rate information or to use our expense and compensation calculator, please click here.


text:
Jessica Simpson tweeted the announcement of the her second pregnancy on Christmas. {People}
Lady Gaga will star in a documentary about her life and her new album, made by fashion photographer Terry Richardson. {The Hollywood Reporter}
More From ELLE
Kate Moss, Daria Werbowy and Joan Smalls star in Versace's spring ad campaign, which will debut this January. {WWD}
Kristen Stewart may actually star in the Snow White and the Huntsman sequel. {Vulture}
Spoiler alert: Dan Stevens, aka Downton Abbey's Matthew Crawley, will not return for the period drama's fourth season. {Deadline}
Valentino mixed things up in its spring campaign with white, hazy images that "evoke an expressive urgency," according to the Italian house's co-creative director, Pierpaolo Piccioli. {WWD}
Watch this behind-the-scenes video of Kanye West and his stylist discussing the kilt's fashion merits. {Racked}
Kate Middleton and Prince William made an appearance at St. Mark's Church in Englefield, Berkshire, England with Middleton's family for Christmas mass. {PopSugar}


text:
Language and region
Take a trip into the world of plastic extrusion
Roll film!
BWF Group
As a family business in its fifth generation with 1,800 employees and 16 production sites worldwide, we set international standards through innovation, professionalism and dedication. Our product range is divided into four independent divisions that are each leaders in their respective markets.
You can reach us on weekdays from 8:00 am - 5:00 pm.
Send us an e-mail and we will get back to you as soon as possible.
Or you can use our contact form.
Callback service
A short message is enough – we will call you back,
weekdays from 8:00 am - 5:00 pm.
* Required Fields
Share this page with colleagues and friends.


text:
Watching the political debates this season always puts this writer, perhaps irresponsibly, in mind of seventies movie comedies: Romney seems like the smug country clubber in a hundred National Lampoonish movies, the one Chevy Chase takes the girl away from, while Paul Ryan last night seemed exactly like the authority-pleasing, solemn student-body president who either gets pantsed midway by the stars of "Porkys" or else blissfully turned on to grass in the final reel by Bill Murray. Joe Biden watching Ryan, meanwhile, put me in mind of nothing so much as the great, grouchy, aged Eddie Albert in Elaine May's matchless original, "The Heartbreak Kid," narrowing his eyes in disbelief as he listens to the slick, oleaginous (and already married!) Charles Grodin courting his beautiful blond daughter: " I heard everything you said… and I will tell you, quite honestly, I was very impressed. Very impressed. And I think I can also say, quite honestly… I have never heard such a crock of horseshit in my life."
But beyond the horseshit something genuinely disturbing and scary got said last night by Paul Ryan that is, I think, easily missed and still worth brooding over. It came in response to a solemn and, it seemed to some of us, inappropriately phrased question about the influence of the Catholic Church on both men's positions on abortion. Inappropriately phrased because legislation is made for everyone, not specially for those of "faith." (And one would have thought that, at this moment in its history, the Catholic Church would not have much standing when it comes to defining the relationship between sexual behavior and doctrinal morality. However few in number the sinners might be, the failure to deal with them openly casts doubt on the integrity of the institution.)
Paul Ryan did not say, as John Kennedy had said before him, that faith was faith and public service, public service, each to be honored and kept separate from the other. No, he said instead "I don't see how a person can separate their public life from their private life or from their faith. Our faith informs us in everything we do." That's a shocking answer—a mullah's answer, what those scary Iranian "Ayatollahs" he kept referring to when talking about Iran would say as well. Ryan was rejecting secularism itself, casually insisting, as the Roman Catholic Andrew Sullivan put it, that "the usual necessary distinction between politics and religion, between state and church, cannot and should not exist." And he went on to make it quietly plain that his principles are uncompromising on this, even if his boss's policy may not seem so:
Our system, unlike the Iranians', is not meant to be so total: it depends on making many distinctions between private life, where we follow our conscience into our chapel, and our public life, where we seek to merge many different kinds of conscience in a common space. Our faith should not inform us in everything we do, or there would be no end to the religious warfare that our tolerant founders feared.
Ryan then went on to say something oddly disarming in its inherent lack of self-awareness. He talked about how, looking at a first sonogram of his daughter, he was thrilled by the beating heart in the tiny "bean" on the image, so much that he and his wife still call that child "Bean." As someone who is not often accused of being indifferent to the joys of fatherhood, I recognize the moment—and in fact still have that same early ultrasound picture, two of them. But Ryan's moral intuition that something was indeed wonderful here was undercut, tellingly, by a failure to recognize accurately what that wonderful thing was, even as he named it: a bean is exactly what the photograph shows—a seed, a potential, a thing that might yet grow into something greater, just as a seed has the potential to become a tree. A bean is not a baby.
The fundamental condition of life is that it develops, making it tricky sometimes to say when it's fully grown and when it isn't, but always easy to say that there is a difference and that that difference is, well, human life itself. It is this double knowledge that impacts any grownup thinking about abortion: that it isn't life that's sacred—the world is full of life, much of which Paul Ryan wants to cut down and exploit and eat done medium rare. It is conscious, thinking life that counts, and where and exactly how it begins (and ends) is so complex a judgment that wise men and women, including some on the Supreme Court, have decided that it is best left, at least at its moments of maximum ambiguity, to the individual conscience (and the individual conscience's doctor). The cost of simplifying this truth is immense cruelty—cruelty to the bean when, truly developed, it becomes a frightened teen-ager who is to be compelled by law to carry her unwished-for pregnancy through with all the trauma that involves. This kind of cruelty—cruelty to real persons, killing the infidel in order to hasten him into heaven, stoning the fourteen-year-old girl in pursuit of some prophet's view of virtue, forcing the teenager to complete her pregnancy to fulfill a middle-aged man's moral hunches—is the kind of cruelty that our liberal founders saw with terror.
Ryan talked facilely of what "science" says in this case. But what real science has to tell us, of course, very different; it says that life has no neat on and off, that while life may in some sense begin at conception, the moment when the formed consciousness that distinguishes human life from bean life arises is a very different question, not reducible to a dogma or a simple claim. A bean isn't a baby; a baby was once a bean, and between those two truths it is, or ought to be, every woman for herself.
What is unquestionable is that the kind of fully conscious life that everyone claims to prize already belongs to the woman who happens to be pregnant, and it should be her individual moral conscience that, in a society devoted to the individual, ought to rule. One reason we prize life is because it makes minds. And women, who have them, should be free to make up their own.
See our full coverage of the debates.
Photograph by Eric Gay/AP.


text:
What a difference four years makes. In 2008, the thrill of the nation electing its first African-American president was accompanied for many of us by the bitter disappointment of seeing voters in four states, including California, approving measures that prohibited same-sex marriage or adoption by gay couples. A few days after the election, I wrote that perhaps it was time for those who cared about LGBT rights to abandon the cause of marriage and to seek protections for same-sex relationship by other means or at least by other names. I was tired of watching my community waste its limited energies and precious resources on what felt, after dozens of defeats at the ballot box, like political insanity — doing the same losing thing over and over again and expecting different results. Here is what I proposed as an alternative:
[I]f the issue is gaining legal protections and economic benefits for queer households, does it matter if we call it "marriage"? If the straight majority is dumb enough to grant us all those things – and I do mean all, including the full panoply of federal benefits extended to married couples — as long as we call it anything other than "marriage," then maybe we should take the deal and throw ourselves a victory party. I know, I know, separate is still unequal, but, but, but – Aren't we tired of going through these motions over and over again and never really getting anywhere? Aren't we sick to death of seeing the collective energies and resources of our small community sapped by the ridiculous effort to prove we deserve access to an institution that has been oppressing women and stymieing the relational imagination for centuries?
Yeah, I was tired. And more than a little frustrated. You have to admit, though, that plenty of folks seem willing to extend those benefits to the gayz as long as we call the institution, say, poop on toast rather than marriage, so, hey, why not? Po-tay-toh, po-tah-toh, right?
So, anyhoo, fast forward to Tuesday, November 6, 2012. There are four marriage measures on ballots in four cobalt-blue states, including my home state of Turtle Country. Incredibly, they all win (though we are still waiting on full, final results from Washington State). Just like that, the tide of history is turned! Suddenly, the impossible has become the achievable — indeed, the achieved. Insanity has become like, hey, no big deal, are you guys registered at Pottery Barn?
How did we get here in just four years? Heck if I know. I'm an English prof, not a pundit or a data nerd. My hunch is that generational change and President Obama's endorsement have done a lot to change the climate of opinion on marriage equality. The losers in Tuesday's battles claim they were outspent three to one in the ballot campaigns, so perhaps spending money can help win elections, though that obviously wasn't the case in a lot of the races on Tuesday. On the ground in Turtle Country, I think we can credit smart organizing and good messaging for increasing support, particularly in the African-American and Latino communities. Equality Maryland, the state's largest LGBT civil rights organization (on whose board I happen to serve) worked in coalition with Latino groups to help pass a state Dream Act. About a quarter of registered voters in Maryland are African American, so a lot of pro-6 ads featured civil rights leaders such as Julian Bond and NAACP president Ben Jealous, as well as faith leaders such as the Rev. Delman Coates of Mount Ennon Baptist Church in Clinton, MD. This time, supporters of marriage equality didn't hesitate to frame the cause as a civil rights struggle, and the message seems to have helped drive up support in the African-American community. Question 6 lost by just 4000 votes in Prince George's County, which is 64.5% black, and African-American support throughout the state was close to 50%.
A couple of weeks ago, I mused here on how deeply estranging and uncomfortable it felt to know that my friends, neighbors, coworkers, and countless unknown fellow citizens would be voting on what felt like a referendum on my humanity. I told you that I was feeling reluctant to talk to my neighbors about Question 6 because I wasn't sure I could bear to know that they might vote against me. I never could bring myself to knock on their doors and initiate those conversations, but because the universe is small and endowed with a perverse sense of humor, I ended up spending much of election day handing out pro-Question 6 materials at my own polling place. Which meant, of course, that I saw all of the neighbors I had been avoiding talking to. Minutes before my shift at the poll was scheduled to end, a group of women drove up together in a large, spotlessly clean SUV that I instantly recognized. In the bright sunshine of a beautiful November day, in the parking lot of an elementary school, we smiled and greeted one another warmly, with that giddy kind of excitement those who participate tend to feel on election day. I asked if they would like a Question 6 flier, and they all accepted one. Then I hesitated for just a moment before deciding to make a little leap of faith. "Now," I said, "we haven't talked about this, but I'm hoping we can count on your support." To a woman, they nodded affirmatively or said yes, I could. Mine was the last face they saw before they stepped inside the school to vote.
Look, I'm no fool. I know people lie in social situations in which the truth would be awkward. I've been smiled at a million times by those who believe they can love me while condemning the sin of my lifestyle. I know it's possible my neighbors were merely being polite, but as I walked home on Tuesday afternoon I promised myself that if equality prevailed in the election I was going to believe that my experience at the poll was the universe's way of telling me I should never have doubted the hardworking people who share my lifeworld.
And I do believe that. Because this week, things that felt impossible four years and even six months ago have suddenly become real. This week, it is no exaggeration to claim, as the coalition who helped to pass Question 6 declared, that We made history. We did, and it feels good. The weirdness referred to in my post title is the weirdness of winning after so many losses. After 32 defeats, you begin to feel like Charlie Brown's baseball team or, you know, the Washington Mystics. You can't imagine winning and you try not to as a way to protect yourself from the disappointment of yet another defeat. When victory comes — or four victories in one astonishing night! — you hardly know how to react. You laugh, you cry, you hold tightly to those who are close to you, you shake your head in a mixture of joy and disbelief. Two days later, it still feels slightly unreal. Two days later, I think a message I put up on my Facebook wall after a few hours of not very restful post-election sleep best captures my feelings about this delightfully strange moment:
Friends, my heart is so full and my body so weary that I can't speak yet about what last night meant, here in Maryland and all across the country, for the nation I love and the values I hold dear. For now, all I can say is thank you, to everyone who worked so hard and so faithfully to bring about so many large and small victories for fairness, equality, and inclusion. We are not a perfect nation this morning, but we are better than we were yesterday. And if we keep working the way we have these past few months, then by golly we'll keep getting better, bit by bit, together.
Yes, we can — because we have. Thank you for your hard, good work.
The nation I love and the values I hold dear? Yeah, sometimes a girl just wants to wave a flag. Deal with it, darlings. And don't ask me when the wedding is. This queer against marriage but for marriage equality ain't budging on that point. Yet.