Heligmosomoides polygyrus induces TLR4 on murine mucosal T cells that produce TGFbeta after lipopolysaccharide stimulation

Helminths are immune modulators that down-regulate colitis in inflammatory bowel disease. In animal models, intestinal bacteria drive colitis and in humans certain alleles of the LPS receptor protein TLR4 increase inflammatory bowel disease susceptibility. To understand helminthic immune modulation in the gut, we studied the influence of intestinal Heligmosomoides polygyrus colonization on LPS-induced lamina propria mononuclear cell (LPMC) cytokine responses in mice. LPS did not stimulate TGFbeta production from LPMC of uninfected mice. LPS strongly induced LPMC from worm-infected animals to secrete TGFbeta, but not TNF-alpha or IL-12. The TGFbeta derived from mucosal T cells. Helminth infection up-regulated TLR4 expression only in lamina propria T cells. LPMC from worm-infected TLR4 mutant animals did not respond to LPS, suggesting that LPS required TLR4 to stimulate TGFbeta secretion. Thus, during helminth infection, LPS challenge induces mucosal T cells to make TGFbeta through a TLR4-dependent process without promoting synthesis of proinflammatory cytokines.     

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.     

TLR-induced inflammation in cystic fibrosis and non-cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is a genetic disease characterized by severe neutrophil-dominated airway inflammation. An important cause of inflammation in CF is Pseudomonas aeruginosa infection. We have evaluated the importance of a number of P. aeruginosa components, namely lipopeptides, LPS, and unmethylated CpG DNA, as proinflammatory stimuli in CF by characterizing the expression and functional activity of their cognate receptors, TLR2/6 or TLR2/1, TLR4, and TLR9, respectively, in a human tracheal epithelial line, CFTE29o(-), which is homozygous for the DeltaF508 CF transmembrane conductance regulator mutation. We also characterized TLR expression and function in a non-CF airway epithelial cell line 16HBE14o(-). Using RT-PCR, we demonstrated TLR mRNA expression. TLR cell surface expression was assessed by fluorescence microscopy. Lipopeptides, LPS, and unmethylated CpG DNA induced IL-8 and IL-6 protein production in a time- and dose-dependent manner. The CF and non-CF cell lines were largely similar in their TLR expression and relative TLR responses. ICAM-1 expression was also up-regulated in CFTE29o(-) cells following stimulation with each agonist. CF bronchoalveolar lavage fluid, which contains LPS, bacterial DNA, and neutrophil elastase (a neutrophil-derived protease that can activate TLR4), up-regulated an NF-kappaB-linked reporter gene and increased IL-8 protein production in CFTE29o(-) cells. This effect was abrogated by expression of dominant-negative versions of MyD88 or Mal, key signal transducers for TLRs, thereby implicating them as potential anti-inflammatory agents for CF.     

TLR2, TLR3, and TLR4 activation specifically alters the oxidative status of intestinal epithelial cells

Intestinal inflammatory diseases are the result of multiple processes, including mucosal oxidative stress and perturbed homeostasis between commensal bacteria and mucosal immunity. Toll-like receptors (TLRs) recognize molecular-associated microorganisms' patterns and trigger innate immunity responses contributing to intestinal homeostasis and inflammatory responses. However, TLRs effects on redox balance in intestinal mucosa remain unknown. Therefore, the present study analyzes the effect of TLR2, TLR3, and TLR4 on both oxidative damage of lipids and proteins, and the activity of antioxidant enzymes in enterocyte-like Caco-2 cells. The results show that the activation of these TLRs increased lipid and protein oxidation levels; however, the effect on the antioxidant enzymes activity is different depending on the TLR activated. These results suggest that the activation of TLR2, TLR3, and TLR4 might affect intestinal inflammation by not only their inherent innate immunity responses, but also their pro-oxidative effects on intestinal epithelial cells.     

TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells

Thymic stromal lymphopoietin (TSLP) is elevated in asthma and triggers dendritic cell-mediated activation of Th2 inflammatory responses. Although TSLP has been shown to be produced mainly by airway epithelial cells, the regulation of epithelial TSLP expression has not been extensively studied. We investigated the expression of TSLP in cytokine- or TLR ligand-treated normal human bronchial epithelial cells (NHBE). The mRNA for TSLP was significantly up-regulated by stimulation with IL-4 (5.5-fold) and IL-13 (5.3-fold), weakly up-regulated by TNF-alpha, TGF-beta, and IFN-beta, and not affected by IFN-gamma in NHBE. TSLP mRNA was only significantly up-regulated by the TLR3 ligand (dsRNA) among the TLR ligands tested (66.8-fold). TSLP was also induced by in vitro infection with rhinovirus. TSLP protein was detected after stimulation with dsRNA (120 +/- 23 pg/ml). The combination of TNF-alpha and IL-4 produced detectable levels of TSLP protein (40 +/- 13 pg/ml). In addition, TSLP was synergistically enhanced by a combination of IL-4 and dsRNA (mRNA; 207-fold, protein; 325 +/- 75 pg/ml). The induction of TSLP by dsRNA was dependent upon NF-kappaB and IFN regulatory factor 3 (IRF-3) signaling via TLR3 as indicated by a study with small interfering RNA. The potent topical glucocorticoid fluticasone propionate significantly suppressed dsRNA-dependent TSLP production in NHBE. These results suggest that the expression of TSLP is induced in airway epithelial cells by stimulation with the TLR3 ligand and Th2 cytokines and that this response is suppressed by glucocorticoid treatment. This implies that respiratory viral infection and the recruitment of Th2 cytokine producing cells may amplify Th2 inflammation via the induction of TSLP in the asthmatic airway.     

多聚免疫球蛋白受体（pIgR）在粘膜免疫中的重要功能

多聚免疫球蛋白受体（pIgR）属于Ⅰ型跨膜糖蛋白,可与多聚免疫球蛋白A和多聚免疫球蛋白M特异性结合,通过穿胞转运,将它们从上皮细胞基底侧膜转运到顶膜,并最终分泌到外分泌液中去. 在此过程中,多聚免疫球蛋白受体的细胞外段被水解,释放出与多聚免疫球蛋白A或多聚免疫球蛋白M相结合的细胞外段（又称为分泌成分）. 分泌成分是sIgA分子的重要组成部分,直接参与sIgA的粘膜防御功能,而且在被动粘膜免疫中也有重要作用. 多聚免疫球蛋白受体通过介导细胞内多聚免疫球蛋白的转运,可以在粘膜的腔面阻止病原体粘附,在上皮细胞内中和病毒,也可以将固有层内的抗原分泌出去. 因此,多聚免疫球蛋白受体的有效分泌是多聚免疫球蛋白发挥粘膜防御功能的必要条件. 但在某些情况下,该受体也可以介导微生物对上皮屏障的入侵. 多聚免疫球蛋白受体是高度 N -糖基化的,其分子中独特的糖链结构,可能与受体的穿胞转运、sIgA在粘膜的正确定位,以及抗原对上皮细胞的粘附有关. 多聚免疫球蛋白受体和分泌成分参与的多重分子机制,使它们在粘膜免疫中起着举足轻重的作用.     

Cellular differentiation-induced attenuation of LPS response in HT-29 cells is related to the down-regulation of TLR4 expression

Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.     

Human intestinal epithelial cells are broadly unresponsive to Toll-like receptor 2-dependent bacterial ligands: implications for host-microbial interactions in the gut

Intestinal epithelial cells (IEC) interact with a high density of Gram-positive bacteria and are active participants in mucosal immune responses. Recognition of Gram-positive organisms by Toll-like receptor (TLR)2 induces proinflammatory gene expression by diverse cells. We hypothesized that IEC are unresponsive to Gram-positive pathogen-associated molecular patterns and sought to characterize the functional responses of IEC to TLR2-specific ligands. Human colonic epithelial cells isolated by laser capture microscopy and IEC lines (Caco-2, T84, HT-29) were analyzed for expression of TLR2, TLR6, TLR1, and Toll inhibitory protein (Tollip) mRNA by RT-PCR and quantitative real-time PCR. Response to Gram-positive bacterial ligands was measured by NF-kappa B reporter gene activation and IL-8 secretion. TLR2 protein expression was analyzed by immunofluorescence and flow cytometry. Colonic epithelial cells and lamina propria cells from both uninflamed and inflamed tissue demonstrate low expression of TLR2 mRNA compared with THP-1 monocytes. IECs were unresponsive to TLR2 ligands including the staphylococcal-derived Ags phenol soluble modulin, peptidoglycan, and lipotechoic acid and the mycobacterial-derived Ag soluble tuberculosis factor. Transgenic expression of TLR2 and TLR6 restored responsiveness to phenol soluble modulin and peptidoglycan in IEC. In addition to low levels of TLR2 protein expression, IEC also express high levels of the inhibitory molecule Tollip. We conclude that IEC are broadly unresponsive to TLR2 ligands secondary to deficient expression of TLR2 and TLR6. The relative absence of TLR2 protein expression by IEC and high level of Tollip expression may be important in preventing chronic proinflammatory cytokine secretion in response to commensal Gram-positive bacteria in the gut.     

Oral Administration of High Molecular Weight Hyaluronan (900 kDa) Controls Immune System via Toll-like Receptor 4 in the Intestinal Epithelium

Low molecular weight hyaluronan enhances or induces inflammation through toll-like receptor 4 (TLR-4).However, the effects of high molecular weight hyaluronan (HA900) on TLR-4 are unknown. In this study, HA900 (900 kDa) was administered orally to MRL-lpr/lpr mice, a Th-1-type autoimmune disease model. Lymphoaccumulation of double-negative T cells, which is enhanced by proinflammatory cytokines, was suppressed by HA900 treatment. Cytokine array analysis showed that HA900 treatment enhanced production of interleukin-10, an anti-inflammatory cytokine, and down-regulated chemokine production. HA900 colocalized with TLR-4 on the luminal surface of epithelial cells in the large intestine. These cells are parts of the immune system and express cytokines. DNA array analysis of the tissue from the large intestine showed that HA900 treatment up-regulated suppressor of cytokine signaling 3 (SOCS3) expression and down-regulated pleiotrophin expression. Treatment of cultured double-negative T cells from MRL-lpr/lpr mice with pleiotrophin rescued these cells. SOCS3, which is known to suppress inflammation, was enhanced by HA900 treatment. In TLR-4 knockdown HT29 cells (a cell line derived from large intestinal cells), HA900 did not bind to HT29 cells and did not up-regulate SOCS3 expression. Our results suggest that oral administration of HA900 modulates Th-1-type autoimmune disease and inflammation by up-regulating SOCS3 expression and down-regulating pleiotrophin expression via TLR-4 in intestinal epithelial cells.     

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.     

Recognition of double-stranded RNA by TLR3 induces severe small intestinal injury in mice

The role of TLRs on intestinal epithelial cells (IECs) is controversial, and the mechanisms by which TLRs influence mucosal homeostasis are obscure. In this study, we report that genomic dsRNA from rotavirus, and its synthetic analog polyinosinic-polycytidylic acid (poly(I:C)), induce severe mucosal injury in the small intestine. Upon engaging TLR3 on IECs, dsRNA triggers IECs to secrete IL-15, which functions to increase the percentage of CD3+NK1.1+ intestinal intraepithelial lymphocytes (IELs) and enhances the cytotoxicity of IELs. Moreover, The CD3+NK1.1+ IELs are proved as CD8alphaalpha+ IELs. These results provide direct evidence that abnormal TLR3 signaling contributes to breaking down mucosal homeostasis and the first evidence of pathogenic effects mediated by CD8alphaalpha+ IELs. The data also suggest that genomic dsRNA may be involved in the pathogenesis of acute rotavirus gastroenteritis.     

Depletion of gut commensal bacteria attenuates intestinal ischemia/reperfusion injury

Gut commensal bacteria play important roles in the development and homeostasis of intestinal immunity. However, the role of gut commensals in intestinal ischemia/reperfusion (I/R) injury is unclear. To determine the roles of gut commensal bacteria in intestinal IR injury, we depleted gut microbiota with a broad-spectrum antibiotic cocktail and performed mesenteric I/R (M I/R). First, we confirmed that antibiotic treatment completely depleted gut commensal bacteria and diminished the size of secondary lymphoid tissues such as the Peyer's patches. We next found that antibiotic treatment attenuated intestinal injury following M I/R. Depletion of gut commensal bacteria reduced the expression of Toll-like receptor (TLR)2 and TLR4 in the intestine. Both are well-known receptors for gram-positive and -negative bacteria. Decreased expression of TLR2 and TLR4 led to the reduction of inflammatory mediators, such as TNF, IL-6, and cyclooxygenase-2. Intestinal I/R injury is initiated when natural antibodies recognize neo-antigens that are revealed on ischemic cells and activate the complement pathway. Thus we evaluated complement and immunoglobulin (Ig) deposition in the damaged intestine and found that antibiotic treatment decreased the deposition of both C3 and IgM. Interestingly, we also found that the deposition of IgA also increased in the intestine following M I/R compared with control mice and that antibiotic treatment decreased the deposition of IgA in the damaged intestine. These results suggest that depletion of gut commensal bacteria decreases B cells, Igs, and TLR expression in the intestine, inhibits complement activation, and attenuates intestinal inflammation and injury following M I/R.     

Expression of TLR2 and TLR4 in murine small intestine during postnatal development

The important role played by the gut microbiota in host immunity is mediated, in part, through toll-like receptors (TLRs). We evaluated the postnatal changes in expression of TLR2 and TLR4 in the murine small intestine and assessed how expression is influenced by gut microbiota. The expression of TLR2 and TLR4 in the murine small intestine was highly dynamic during development. The changes were especially profound during the suckling period, with the maximal mRNA levels detected in the mid-suckling period. Immunohistochemical and flow-cytometric analyses indicated that the changes in TLR2 and TLR4 expression involve primarily epithelial cells. The germ-free mice showed minor changes in TLR2/TLR4 mRNA and TLR2 protein during the suckling period. This study demonstrated that the postnatal expression of TLR2 and TLR4 in small intestinal epithelial cells is dynamic and depends on the presence of commensal intestinal microbiota.     

Human intestinal epithelial cells express a novel receptor for IgA

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.