Expression and regulation of the human beta-defensins hBD-1 and hBD-2 in intestinal epithelium

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1alpha stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-kappaB target gene in the intestinal epithelium as blocking NF-kappaB activation inhibits the up-regulated expression of hBD-2 in response to IL-1alpha stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.     

Autophagy in the intestinal epithelium regulates Citrobacter rodentium infection

Autophagy, a ubiquitous degradation pathway, is important for the survival and homeostasis of cells. Previous studies have demonstrated the role of autophagy in host defense against bacterial infection, but the importance of autophagy in the intestinal epithelium for the regulation of bacterial infection has not been fully elucidated. In this study, we showed that the essential autophagy protein Atg7 is required for resistance to Citrobacter rodentium infection in the intestinal epithelium. Infected mice in which Atg7 had been conditionally deleted from the intestinal epithelium exhibited greater clinical evidence of disease and higher expression levels of pro-inflammatory cytokine mRNA in the large intestine. Moreover, C. rodentium clearance was reduced in the Atg7 conditional knockout mice. These results demonstrate that autophagy in intestinal epithelial cells plays an important role in host defense against C. rodentium infection and the regulation of C. rodentium infectious colitis.     

Diversity of tight junctions (TJs) between gastrointestinal epithelial cells and their function in maintaining the mucosal barrier

The gastrointestinal epithelium, which is covered by a single layer of epithelial cells, including enterocytes, intraepithelial lymphocytes, goblet cells, microfold cells, and dendritic cells, serves as a protective barrier separating luminal contents from the underlying tissue compartments. The epithelium plays an important role in the first line of host defense against a variety of pathogens, as well as maintaining the homeostasis in gastrointestinal tract. All these epithelial cells express junction complex proteins and form cell junctions such as adherens and TJs, although the TJs have small differences among different epithelial cells. The TJs, located most apically on the lateral membrane, are required for the proper formation of epithelial cell polarity as well as sustaining of the mucosal barrier. Furthermore, TJs are the key cell junctions modulating the paracellular pathway. Understanding the diversity of the TJs between intestinal epithelial cells and their different roles in defending pathogens' invasion and modifying the paracellular pathway are attractive to exploration.     

Nitric oxide production by human intestinal epithelial cells and competition for arginine as potential determinants of host defense against the lumen-dwelling pathogen Giardia lamblia

Giardia lamblia infection of the human small intestine is a common protozoan cause of diarrheal disease worldwide. Although infection is luminal and generally self-limiting, and secretory Abs are thought to be important in host defense, other defense mechanisms probably affect the duration of infection and the severity of symptoms. Because intestinal epithelial cells produce NO, and its stable end products, nitrite and nitrate, are detectable mainly on the apical side, we tested the hypothesis that NO production may constitute a host defense against G. lamblia. Several NO donors, but not their control compounds, inhibited giardial growth without affecting viability, suggesting that NO is cytostatic rather than cytotoxic for G. lamblia. NO donors also inhibited giardial differentiation induced by modeling crucial environmental factors, i. e., encystation induced by bile and alkaline pH, and excystation in response to gastric pH followed by alkaline pH and protease. Despite the potent antigiardial activity of NO, G. lamblia is not simply a passive target for host-produced NO, but has strategies to evade this potential host defense. Thus, in models of human intestinal epithelium, G. lamblia inhibited epithelial NO production by consuming arginine, the crucial substrate used by epithelial NO synthase to form NO. These studies define NO and arginine as central components in a novel cross-talk between a luminal pathogen and host intestinal epithelium.     

Lipopolysaccharide binding protein and serum amyloid A secretion by human intestinal epithelial cells during the acute phase response.

The acute phase proteins LPS binding protein (LBP) and serum amyloid A (SAA) are produced by the liver and are present in the circulation. Both proteins have been shown to participate in the immune response to endotoxins. The intestinal mucosa forms a large surface that is continuously exposed to these microbial products. By secretion of antimicrobial and immunomodulating agents, the intestinal epithelium contributes to the defense against bacteria and their products. The aim of this study was to explore the influence of the inflammatory mediators TNF-alpha, IL-6, and IL-1beta on the release of LBP and SAA by intestinal epithelial cells (IEC). In addition, the induction of LBP and SAA release by cell lines of intestinal epithelial cells and hepatic cells was compared. The data obtained show that in addition to liver cells, IEC also expressed LBP mRNA and released bioactive LBP and SAA upon stimulation. Regulation of LBP and SAA release by IEC and hepatocytes was typical for class 1 acute phase proteins, although differences in regulation between the cell types were observed. Endotoxin did not induce LBP and SAA release. Glucocorticoids were demonstrated to strongly enhance the cytokine-induced release of LBP and SAA by IEC, corresponding to hepatocytes. The data from this study, which imply that human IEC can produce LBP and SAA, suggest a role for these proteins in the local defense mechanism of the gut to endotoxin. Furthermore, the results demonstrate that tissues other than the liver are involved in the acute phase response.     

The role of autophagy in maintaining intestinal mucosal barrier

The intestinal mucosal barrier is the first line to defense against luminal content penetration and performs numerous biological functions. The intestinal epithelium contains a huge surface that is lined by a monolayer of intestinal epithelial cells (IECs). IECs are dominant mediators in maintaining intestinal homeostasis that drive diverse functions including nutrient absorption, physical segregation, secretion of antibacterial peptides, and modulation of immune responses. Autophagy is a cellular self-protection mechanism in response to various stresses, and accumulating studies have revealed its importance in participating physiological processes of IECs. The regulatory effects of autophagy depend on the specific IEC types. This review aims to elucidate the myriad roles of autophagy in regulating the functions of different IECs (stem cells, enterocytes, goblet cells, and Paneth cells), and present the progress of autophagy-targeting therapy in intestinal diseases. Understanding the involved mechanisms can provide new preventive and therapeutic strategies for gastrointestinal dysfunction and diseases.     

BMP signaling in homeostasis,transformation and inflammatory response of intestinal epithelium

Intestine is the organ for food digestion, nutrient absorption and pathogen defense, in which processes intestinal epithelium plays a central role. Intestinal epithelium undergoes fast turnover, and its homeostasis is regulated by multiple signaling pathways, including Wnt, Notch, Hippo and BMP pathways. BMP signaling has been shown to negatively regulate self-renewal of Lgr5⁺ intestinal stem cells, constrains the expansion of intestinal epithelium, therefore attenuating colorectal cancer formation. BMPs and their receptors are expressed in both epithelial and mesenchymal cells, suggesting a two-way interaction between the mesenchyme and epithelium. In this review, we summarize the current understanding of the function of BMP signaling in homeostasis, cancerous transformation and inflammatory response of intestinal epithelium.     

Specific-sized Hyaluronan Fragments Promote Expression of Human β-Defensin 2 in Intestinal Epithelium

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of virtually all mammalian tissues. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses in immune cells, endothelium, and epidermis through interaction with innate molecular pattern recognition receptors, such as TLR4. Despite these advances, little is known regarding the effect of fragmented HA at the intestinal epithelium, where numerous pattern recognition receptors act as sentinels of an innate defense response that maintains epithelial barrier integrity in the presence of abundant and diverse microbial challenges. Here we report that HA fragments promote expression of the innate antimicrobial peptide human β-defensin 2 (HβD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent up-regulated expression of HβD2 protein in a fragment size-specific manner, with 35-kDa HA fragment preparations emerging as the most potent inducers of intracellular HβD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HβD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice but not in TLR4-deficient mice. Together, our observations suggest that a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium barrier defense through the induction of intracellular HβD2 protein.     

肠道干细胞和潘氏细胞在肠道稳态和疾病中的作用

肠道是最复杂的器官之一,负责营养的吸收和消化。肠道具有多层结构保护整个肠道免受病原体的侵害。肠道上皮是由单层柱状上皮细胞组成,是抵抗病原体的第一道屏障。因此,肠上皮必须保持完整性以保护肠免受感染和毒性剂的侵害。上皮细胞分为两个谱系(吸收型与分泌型),并且每隔3~4天脱落至肠腔中。细胞的快速更替是由于肠道干细胞的存在,肠道干细胞排列在隐窝底部终极分化的潘氏细胞之间并沿隐窝绒毛轴分化成不同的上皮细胞。一旦肠道干细胞受到损伤,潘氏细胞将通过提供WNT配体和Notch刺激来补充肠道干细胞。因此,潘氏细胞充当辅助细胞以维持干细胞微环境,即生态位。该综述探讨了干细胞和潘氏细胞之间的相互作用,进一步探讨了维持肠道稳态的信号通路。     

Beta-defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of beta-defensin expression by IEC. The effect of LPS and peptidoglycan on beta-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of beta-defensin-2 was assessed using promoter-reporter constructs of the human beta-defensin-2 gene. LPS and peptidoglycan stimulated beta-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-kappaB or AP-1 site within the beta-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of beta-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide beta-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.     

Identification of genes involved in mucosal defense and inflammation associated with normal enteric bacteria

Normal luminal bacteria and their products play a role in experimental colitis and inflammatory bowel disease. However, what molecules from what cells are responsible for mounting and maintaining the mucosal defense against luminal flora is still uncertain. The aim of this study was to identify epithelial gene products involved in mucosal defense and inflammation associated with ubiquitous enteric bacteria. Germ-free ICR mice were given an oral bacterial suspension prepared from conventional components (bacterial reconstitution). Small intestinal and colonic epithelial cells were isolated from bacteria-reconstituted, germ-free, and specific pathogen-free mice. Differential gene expression was investigated by differential display, Northern blot, and sequence analysis. Bacterial reconstitution resulted in acute but self-limited colitis. In epithelial cells, we observed the induction of small intestine-specific genes of the cryptdin family and colon-specific expression of serum amyloid A1 gene. This novel approach allows the identification of known and novel gene products involved in mucosal defense against luminal microorganisms and the associated inflammatory response.     

Different epithelia in the distal human male urethra

Summary The distal segment of the human male urethra, in particular the fossa navicularis, was studied with light- and electron microscopy as well as by means of histochemical and immunocytochemical methods. The fossa navicularis of the urethra contains a circumscribed zone of extremely thick, non-keratinized stratified squamous epithelium composed of cells containing a large amount of glycogen. These cells lack acid phosphatase activity and lysozyme-like immunoreactivity, both of which can be demonstrated to varying extents in the other zones of the distal male urethra. These glycogen-rich cells are considered to be the substrate for an endogenous flora of lactobacteria, whereas the acid-phosphatase activity and the lysozyme-like immunoreactivity indicate the presence of macrophages and the secretion of bactericidal agents at the epithelial surface. These observations suggest that the different zones with heterogeneous properties in the distal male urethra probably represent a defense system against the invasion of pathogenic microorganisms. Moreover, the glycogen-rich zone, which resembles the glycogen-rich epithelium of the vagina, is estrogen-dependent. This is demonstrated in cases of sex reversal in which after long-lasting estrogen treatment the glycogen-rich zone becomes extremely extended by displacement of the neighbouring epithelium.     

Innate immune signalling at the intestinal epithelium in homeostasis and disease

The intestinal epithelium-which constitutes the interface between the enteric microbiota and host tissues-actively contributes to the maintenance of mucosal homeostasis and defends against pathogenic microbes. The recognition of conserved microbial products by cytosolic or transmembrane pattern recognition receptors in epithelial cells initiates signal transduction and influences effector cell function. However, the signalling pathways, effector molecules and regulatory mechanisms involved are not yet fully understood, and the functional outcome is poorly defined. This review analyses the complex and dynamic role of intestinal epithelial innate immune recognition and signalling, on the basis of results in intestinal epithelial cell-specific transgene or gene-deficient animals. This approach identifies specific epithelial cell functions within the diverse cellular composition of the mucosal tissue, in the presence of the complex and dynamic gut microbiota. These insights have thus provided a more comprehensive understanding of the role of the intestinal epithelium in innate immunity during homeostasis and disease.     

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using "intestinal sacs" are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (P_app)of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.     

Structure and function of intercellular junctions in human cervical and vaginal mucosal epithelia

The mucosal epithelium is a major portal for microbial invasion. Mucosal barrier integrity is maintained by the physical interactions of intercellular junctional molecules on opposing epithelial cells. The epithelial mucosa in the female reproductive tract provides the first line of defense against sexually transmitted pathogenic bacteria and viruses, but little is known concerning the structure and molecular composition of epithelial junctions at this site. In the present study, the distribution of tight, adherens, and desmosomal junctions were imaged in the human endocervix (columnar epithelium) and ectocervix (stratified squamous epithelium) by electron microscopy, and permeability was assessed by tracking the penetration of fluorescent immunoglobulin G (IgG). To further define the molecular structure of the intercellular junctions, select junctional molecules were localized in the endocervical, ectocervical, and vaginal epithelium by fluorescent immunohistology. The columnar epithelial cells of the endocervix were joined by tight junctions that excluded apically applied fluorescent IgG. In contrast, the most apical layers of the ectocervical stratified squamous epithelium did not contain classical cell-cell adhesions and were permeable to IgG. The suprabasal and basal epithelial layers in ectocervical and vaginal tissue contained the most robust adhesions; molecules characteristic of exclusionary junctions were detected three to four cellular layers below the luminal surface and extended to the basement membrane. These data indicate that the uppermost epithelial layers of the ectocervix and vagina constitute a unique microenvironment; their lack of tight junctions and permeability to large-molecular-weight immunological mediators suggest that this region is an important battlefront in host defense against microbial pathogens.     

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

The cell types of the gut expressing Toll-like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll-like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor-kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin-8 (IL-8). We can conclude that Toll-like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll-like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll-like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen.     

SOS regulatory elements are essential for UPEC pathogenesis

Epithelial cells are highly regarded as the first line of defense against microorganisms, but the mechanisms used to control bacterial diseases are poorly understood. A component of the DNA damage repair regulon, SulA, is essential for UPEC virulence in a mouse model for human urinary tract infection, suggesting that DNA damage is a key mediator in the primary control of pathogens within the epithelium. In this study, we examine the role of DNA damage repair regulators in the intracellular lifestyle of UPEC within superficial bladder epithelial cells. LexA and RecA coordinate various operons for repair of DNA damage due to exogenous and endogenous agents and are known regulators of sulA. UPEC strains defective in regulation of the SOS response mediated by RecA and LexA display attenuated virulence in immunocompetent mice within the first 6 h post infection. RecA and LexA regulation of the SOS regulon is dispensable in immunocompromised mice. These data suggest that epithelial cells produce sufficient levels of DNA damaging agents, such that the bacterial DNA damage repair response is essential, as a means to control invading bacteria. Since many pathogens interact with the epithelium before exposure to professional phagocytes, it is likely that adaptation to oxidative radicals during intracellular growth provides additional protection from killing by innate immune phagocytes.