TLR4 Facilitates Translocation of Bacteria across Renal Collecting Duct Cells (original) (raw)
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Cellular Microbiology, 2014
Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4 −/− MCDs, but not in Tlr5 −/− MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5 −/− than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5 −/− kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 postinfected Tlr5 −/− kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.
Campylobacter jejuni induces transcellular translocation of commensal bacteria via lipid rafts
Gut Pathogens, 2009
Background: Campylobacter enteritis represents a risk factor for the development of inflammatory bowel disease (IBD) via unknown mechanisms. As IBD patients exhibit inflammatory responses to their commensal intestinal microflora, factors that induce translocation of commensal bacteria across the intestinal epithelium may contribute to IBD pathogenesis. This study sought to determine whether Campylobacter induces translocation of non-invasive intestinal bacteria, and characterize underlying mechanisms. Methods: Mice were infected with C. jejuni and translocation of intestinal bacteria was assessed by quantitative bacterial culture of mesenteric lymph nodes (MLNs), liver, and spleen. To examine mechanisms of Campylobacter-induced bacterial translocation, transwell-grown T84 monolayers were inoculated with non-invasive Escherichia coli HB101 ± wild-type Campylobacter or invasiondefective mutants, and bacterial internalization and translocation were measured. Epithelial permeability was assessed by measuring flux of a 3 kDa dextran probe. The role of lipid rafts was assessed by cholesterol depletion and caveolin co-localization. Results: C. jejuni 81-176 induced translocation of commensal intestinal bacteria to the MLNs, liver, and spleen of infected mice. In T84 monolayers, Campylobacter-induced internalization and translocation of E. coli occurred via a transcellular pathway, without increasing epithelial permeability, and was blocked by depletion of epithelial plasma membrane cholesterol. Invasiondefective mutants and Campylobacter-conditioned cell culture medium also induced E. coli translocation, indicating that C. jejuni does not directly 'shuttle' bacteria into enterocytes. In C. jejunitreated monolayers, translocating E. coli associated with lipid rafts, and this phenomenon was blocked by cholesterol depletion. Conclusion: Campylobacter, regardless of its own invasiveness, promotes the translocation of noninvasive bacteria across the intestinal epithelium via a lipid raft-mediated transcellular process. Background Patients with IBD appear to display aberrant inflammatory responses to their commensal intestinal microflora via unknown mechanisms [1]. Normally, the intestinal microflora is effectively confined to the lumen by the epithelium. However, intestinal epithelial barrier defects
TLR4-mediated expulsion of bacteria from infected bladder epithelial cells
Proceedings of the National Academy of Sciences, 2009
Uropathogenic Escherichia coli invade bladder epithelial cells (BECs) by direct entry into specialized cAMP regulated exocytic compartments. Remarkably, a significant number of these intracellular bacteria are subsequently expelled in a nonlytic and piecemeal fashion by infected BECs. Here, we report that expulsion of intracellular E. coli by infected BECs is initiated by the pattern recognition receptor, Toll-like receptor (TLR)4, after activation by LPS. Also, we reveal that caveolin-1, Rab27b, PKA, and MyRIP are components of the exocytic compartment, and that they form a complex involved in the exocytosis of bacteria. This capacity of TLR4 to mediate the expulsion of intracellular bacteria from infected cells represents a previously unrecognized function for this innate immune receptor.
Journal of cell …, 2004
Introduction The innate immune system has evolved the capacity to recognise a broad spectrum of pathogens and to discriminate between 'infectious non-self' and 'non-infectious self' (Medzhitov and Janeway, 2002). The basis of microbial recognition lies in the ability of the innate immune system to recognise conserved microbial components that are specific to the microorganisms but not to the host. These conserved microbial components are referred to as pathogen-associated molecular patterns (PAMPs). Most widely known PAMPs include lipopolysaccharide (LPS) from Gram-negative bacteria and lipoteichoic acids (LTA) as well as peptidoglycan from Gram-positive bacteria. Many different types of receptors participate in the microbial detection by the innate immune system and they are called pattern-recognition receptors (PRRs). Such receptors include CD14 (Wright et al., 1990), integrins such as CD11c/CD18 (Ingalls and Golenbock, 1995) and CD11b/CD18 (Perera et al., 2001), CD55 (Heine et al., 2001; Heine et al., 2003), heat shock proteins (Byrd et al., 1999; Triantafilou et al., 2001a), but most importantly the Tolllike receptor (TLR) family that has been shown to participate in the recognition of microbial pathogens in several organisms including humans, mice and flies (Underhill and Ozinsky, 2002). The best characterised members of the TLR family include TLR4 and TLR2. TLR4 has been shown to recognise LPS from Gram-negative bacteria (Poltorak et al., 1998), and TLR2 has been shown to mediate responses against various fungal, Gram-positive and mycobacterial components (Qureshi et al.
Infection and Immunity, 2006
Substantial data implicate the commensal flora as triggers for the initiation of enteric inflammation or inflammatory disease relapse. We have shown that enteric epithelia under metabolic stress respond to nonpathogenic bacteria by increases in epithelial paracellular permeability and bacterial translocation. Here we assessed the structural basis of these findings. Confluent filter-grown monolayers of the human colonic T84 epithelial cell line were treated with 0.1 mM dinitrophenol (which uncouples oxidative phosphorylation) and noninvasive, nonpathogenic Escherichia coli (strain HB101, 10 6 CFU) with or without pretreatment with various pharmacological agents. At 24 h later, apoptosis, tight-junction protein expression, transepithelial resistance (TER; a marker of paracellular permeability), and bacterial internalization and translocation were assessed. Treatment with stabilizers of microtubules (i.e., colchicine), microfilaments (i.e., jasplakinolide) and clathrin-coated pit endocytosis (i.e., phenylarsine oxide) all failed to block DNP؉E. coli HB101-induced reductions in TER but effectively prevented bacterial internalization and translocation. Neither the TER defect nor the enhanced bacterial translocations were a consequence of increased apoptosis. These data show that epithelial paracellular and transcellular (i.e., bacterial internalization) permeation pathways are controlled by different mechanisms. Thus, epithelia under metabolic stress increase their endocytotic activity that can result in a microtubule-, microfilament-dependent internalization and transcytosis of bacteria. We speculate that similar events in vivo would allow excess unprocessed antigen and bacteria into the mucosa and could evoke an inflammatory response by, for example, the activation of resident or recruited immune cells. on December 24, 2015 by guest http://iai.asm.org/ Downloaded from on December 24, 2015 by guest http://iai.asm.org/ Downloaded from on December 24, 2015 by guest http://iai.asm.org/ Downloaded from 196 NAZLI ET AL. INFECT. IMMUN. on December 24, 2015 by guest http://iai.asm.org/ Downloaded from 198 NAZLI ET AL. INFECT. IMMUN.
European Journal of Immunology, 2006
Detection of microorganisms through microbe-associated molecular patterns (MAMP) by Toll-like receptors (TLR) is crucial to trigger protective immunity. In the mucosa, sentinel cells are exposed to MAMP from both pathogens and commensals; however, the TLR response is tightly controlled to avoid inflammation in response to commensals. Uropathogenic Escherichia coli (UPEC) trigger innate responses during urinary tract infection in a TLR4-dependent and CD14-independent manner. UPEC express virulence factors, such as type 1 fimbriae and/or P fimbriae, allowing bacterial attachment to the epithelium. In this issue of the European Journal of Immunology, Fisher et al. show that fimbriae are required to induce a TLR4-specific epithelial response. Depending on the fimbriae expressed by UPEC, different adaptor molecules are involved in TLR4 signaling. These data add to the recent body of evidence suggesting that TLR responses are regulated by co-receptors, such as receptors for virulence factors. In conclusion, the "pathogenic" TLR stimulation provides a novel way for the host to ignore commensal bacteria.
Blood, 2002
Polarized migrating T cells possess 2 poles, the uropod protrusion at the rear and the leading edge at the front, with specific protein composition and function. The influenza virus hemagglutinin (HA) is a prototypical molecule that uses lipid rafts for biosynthetic transport to the apical surface in polarized epithelial Madin-Darby canine kidney (MDCK) cells. In this study, HA was used as a tool to investigate the role of lipid rafts in vectorial protein traffic in polarized T lymphocytes. Results show that newly synthesized HA becomes selectively targeted to the uropod subdomain in polarized T lymphoblasts. HA incorporates into rafts soon after biosynthesis, suggesting that delivery of HA to the uropod occurs through a pathway of transport reminiscent of that used for its specific targeting to the apical surface. HA and the adhesion molecules, intercellular adhesion molecule 3 (ICAM-3), CD44, and CD43, 3 endogenous uropod markers, were detected in surface rafts of T lymphoblasts. ...
Laboratory Investigation, 2009
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes outbreaks of bloody diarrhea and the hemolytic-uremic syndrome. EHEC intimately adheres to epithelial cells, effaces microvilli and induces attaching-effacing (AE) lesions. Detergent-resistant microdomains (lipid rafts) serve as membrane platforms for the recruitment of signaling complexes to mediate host responses to infection. The aim of this study was to define the role of lipid rafts in activating signal transduction pathways in response to AE bacterial pathogens. Epithelial cell monolayers were infected with EHEC (MOI 100:1, 3 h, 371C) and lipid rafts isolated by buoyant density ultracentrifugation. Phosphoinositide 3-kinase (PI3K) localization to lipid rafts was confirmed using PI3K and anti-caveolin-1 antibodies. Mice with cholesterol storage disease Niemann-Pick, type C were used as in vivo models to confirm the role of lipid rafts in mediating signaling response to AE organisms. In contrast to uninfected cells, PI3K was recruited to lipid rafts in response to EHEC infection. Metabolically active bacteria and cells with intact cholesterol-rich microdomains were necessary for the recruitment of second messengers to lipid rafts. Recruitment of PI3K to lipid rafts was independent of the intimin (eaeA) gene, type III secretion system, and production of Shiga-like toxins. Colonization of NPC À/À colonic mucosa by Citrobacter rodentium and AE lesion formation were both delayed, compared with wild-type mice infected with the murine-specific AE bacterial pathogen. C. rodentium-infected NPC À/À mice had reduced colonic epithelial hyperplasia (64 ± 8.251 vs 112 ± 2.958 mm; Po0.05) and decreased secretion of IFN-g (17.6 ± 17.6 vs 71 ± 26.3 pg/ml, Po0.001). Lipid rafts mediate host cell signal transduction responses to AE bacterial infections both in vitro and in vivo. These findings advance the current understanding of microbial-eukaryotic cell interactions in response to enteric pathogens that hijack signaling responses mediated through lipid rafts.
Enterocyte TLR4 Mediates Phagocytosis and Translocation of Bacteria Across the Intestinal Barrier
The Journal of Immunology, 2006
Translocation of bacteria across the intestinal barrier is important in the pathogenesis of systemic sepsis, although the mechanisms by which bacterial translocation occurs remain largely unknown. We hypothesized that bacterial translocation across the intact barrier occurs after internalization of the bacteria by enterocytes in a process resembling phagocytosis and that TLR4 is required for this process. We now show that Fc␥RIIa-transfected enterocytes can internalize IgG-opsonized erythrocytes into actin-rich cups, confirming that these enterocytes have the molecular machinery required for phagocytosis. We further show that enterocytes can internalize Escherichia coli into phagosomes, that the bacteria remain viable intracellularly, and that TLR4 is required for this process to occur. TLR4 signaling was found to be necessary and sufficient for phagocytosis by epithelial cells, because IEC-6 intestinal epithelial cells were able to internalize LPS-coated, but not uncoated, latex particles and because MD2/TLR4-transfected human endothelial kidney (HEK)-293 cells acquired the capacity to internalize E. coli, whereas nontransfected HEK-293 cells and HEK-293 cells transfected with dominant-negative TLR4 bearing a P712H mutation did not. LPS did not induce membrane ruffling or macropinocytosis in enterocytes, excluding their role in bacterial internalization. Strikingly, the internalization of Gram-negative bacteria into enterocytes in vivo and the translocation of bacteria across the intestinal epithelium to mesenteric lymph nodes were significantly greater in wild-type mice as compared with mice having mutations in TLR4. These data suggest a novel mechanism by which bacterial translocation occurs and suggest a critical role for TLR4 in the phagocytosis of bacteria by enterocytes in this process.