Infection of epithelial cells by Cryptosporidium parvum triggers a variety of host-cell innate and adaptive immune responses including release of cytokines/chemokines and up-regulation of antimicrobial peptides. The mechanisms that trigger these host-cell responses are unclear. Thus, we evaluated the role of TLRs in host-cell responses during C. parvum infection of cultured human biliary epithelia (i.e., cholangiocytes). We found that normal human cholangiocytes express all known TLRs. C. parvum infection of cultured cholangiocytes induces the selective recruitment of TLR2 and TLR4 to the infection sites. Activation of several downstream effectors of TLRs including IL-1R-associated kinase, p-38, and NF-κB was detected in infected cells. Transfection of cholangiocytes with dominant-negative mutants of TLR2 and TLR4, as well as the adaptor molecule myeloid differentiation protein 88 (MyD88), inhibited C. parvum-induced activation of IL-1R-associated kinase, p-38, and NF-κB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-κB activation. Moreover, C. parvum selectively up-regulated human β-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-κB activation were each associated with a reduction of C. parvum-induced human β-defensin-2 expression. A significantly higher number of parasites were detected in cells transfected with a MyD88 dominant-negative mutant than in the control cells at 48–96 h after initial exposure to parasites, suggesting MyD88-deficient cells were more susceptible to infection. These findings demonstrate that cholangiocytes express a variety of TLRs, and suggest that TLR2 and TLR4 mediate cholangiocyte defense responses to C. parvum via activation of NF-κB.

Cholangiocytes, epithelial cells lining the biliary tree, have primary cilia extending from their apical membrane into the ductal lumen. Although important in disease, cilia also play a vital role in normal cellular functions. We reported that cholangiocyte cilia are sensory organelles responding to mechanical stimuli (i.e., luminal fluid flow) by alterations in intracellular Ca(2+) and cAMP. Because cholangiocyte cilia are also ideally positioned to detect changes in composition and tonicity of bile, we hypothesized that cilia also function as osmosensors. TRPV4, a Ca(2+)-permeable ion channel, has been implicated in signal transduction of osmotic stimuli. Using purified rat cholangiocytes and perfused intrahepatic bile duct units (IBDUs), we found that TRPV4 is expressed on cholangiocyte cilia, and that hypotonicity induces an increase in intracellular Ca(2+) in a TRPV4-, ciliary-, and extracellular calcium-dependent manner. The osmosensation of luminal tonicity by ciliary TRPV4 induces bicarbonate secretion, the main determinant of ductal bile formation, by a mechanism involving apical ATP release. Furthermore, the activation of TRPV4 in vivo, by its specific agonist, 4alphaPDD, induces an increase in bile flow as well as ATP release and bicarbonate secretion. Our results suggest that cholangiocyte primary cilia play an important role in ductal bile formation by acting as osmosensors.

Previous work from our laboratory has implicated hormone-induced plasma membrane movement (i.e., endo- and exocytosis) in water and electrolyte transport by the epithelial cells that line the ducts in the liver (i.e., cholangiocytes). To further explore the cellular mechanisms regulating ductal bile secretion, we infused somatostatin and/or secretin intravenously into rats 2 wk after either bile duct ligation (BDL), a procedure that induces selective proliferation of cholangiocytes, or sham surgery and measured bile flow and biliary constituents. We also determined the effect of somatostatin on basal and secretin-induced exocytosis by purified cholangiocytes isolated from rat liver after BDL. Finally, we studied the expression of the somatostatin receptor gene by both ribonuclease (RNase) protection and nuclear run-on assays using cDNA encoding for two subtypes of the somatostatin receptor gene (i.e., SSTR1 and SSTR2). In vivo, somatostatin infusion caused a dose-dependent bicarbonate-poor decrease (57% maximal decrease below baseline; P < 0.05) in bile flow in BDL but not in sham-operated rats; in contrast, secretin caused a dose-dependent bicarbonate-rich choleresis (228% maximal increase above baseline; P < 0.05) in BDL but not in sham-operated rats. Simultaneous or prior infusion of somatostatin inhibited the secretin-induced hypercholeresis in BDL rats. In vitro, somatostatin had no effect on basal exocytosis by cholangiocytes isolated from BDL rats; however, somatostatin inhitibed (88% maximal inhibition; P < 0.05) secretin-induced exocytosis by cholangiocytes in a dose-dependent fashion. In addition, somatostatin inhibited secretin-induced increases in levels of adenosine 3',5'-cyclic monophosphate (cAMP) in cholangiocytes isolated from BDL rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear.

Introduction Primary sclerosing cholangitis (PSC) is a chronic, idiopathic, fibro-inflammatory cholangiopathy. The role of the microbiota in PSC etiopathogenesis may be fundamentally important yet remains obscure. We tested the hypothesis that germ-free (GF) mdr2−/− mice develop a distinct PSC phenotype compared to conventionally-housed (CV) mdr2−/− mice. Methods Mdr2−/− mice (n=12) were re-derived as GF by embryo transfer, maintained in isolators, and sacrificed at 60 days in parallel with age-matched CV mdr2−/− mice. Serum biochemistries, gallbladder bile acids, and liver sections were examined. Histologic findings were validated morphometrically, biochemically, and by immunofluorescence microscopy (IFM). Cholangiocyte senescence was assessed by p16INK4a in situ hybridization in liver tissue and by β-galactosidase (SA-β-gal) staining in a culture-based model of insult-induced senescence. Results Serum biochemistries, including alkaline phosphatase, aspartate aminotransferase, and bilirubin, were significantly higher in GF mdr2−/− (p<0.01). Primary bile acids were similar, while secondary bile acids were absent in GF mdr2−/− mice. Fibrosis, ductular reaction, and ductopenia were significantly more severe histopathologically in GF mdr2−/− mice (p<0.01) and were confirmed by hepatic morphometry, hydroxyproline assay, and IFM. Cholangiocyte senescence was significantly increased in GF mdr2−/− mice and abrogated in vitro by ursodeoxycholic acid treatment. Conclusions GF mdr2−/− mice exhibit exacerbated biochemical and histologic features of PSC and increased cholangiocyte senescence, a characteristic and potential mediator of progressive biliary disease. Ursodeoxycholic acid, a commensal microbial metabolite, abrogates senescence in vitro. These findings demonstrate the importance of the commensal microbiota and its metabolites in protecting against biliary injury and suggest avenues for future studies of biomarkers and therapeutic interventions in PSC.

Aquaporin-1 (AQP1) water channels are present in the apical and basolateral plasma membrane domains of bile duct epithelial cells, or cholangiocytes, and mediate the transport of water in these cells. We previously reported that secretin, a hormone known to stimulate ductal bile secretion, increases cholangiocyte osmotic water permeability and stimulates the redistribution of AQP1 from an intracellular vesicular pool to the cholangiocyte plasma membrane. Nevertheless, the target plasma membrane domain (i.e., basolateral or apical) for secretin-regulated trafficking of AQP1 in cholangiocytes is unknown, as is the functional significance of this process for the secretion of ductal bile. In this study, we used primarily an in vivo model (i.e., rats with cholangiocyte hyperplasia induced by bile duct ligation) to address these issues. AQP1 was quantitated by immunoblotting in apical and basolateral plasma membranes prepared from cholangiocytes isolated from rats 20 min after intravenous infusion of secretin. Secretin increased bile flow (78%, P < 0.01) as well as the amount of AQP1 in the apical cholangiocyte plasma membrane (127%, P < 0.05). In contrast, the amount of AQP1 in the basolateral cholangiocyte membrane and the specific activity of an apical cholangiocyte marker enzyme (i.e., γ-glutamyltranspeptidase) were unaffected by secretin. Similar observations were made when freshly isolated cholangiocytes were directly exposed to secretin. Immunohistochemistry for AQP1 in liver sections from secretin-treated rats showed intensified staining at the apical region of cholangiocytes. Pretreatment of rats with colchicine (but not with its inactive analog β-lumicolchicine) inhibited both the increases of AQP1 in the cholangiocyte plasma membrane (94%, P < 0.05) and the bile flow induced by secretin (54%, P < 0.05). Our results in vivo indicate that secretin induces the microtubule-dependent insertion of AQP1 exclusively into the secretory pole (i.e., apical membrane domain) of rat cholangiocytes, a process that likely accounts for the ability of secretin to stimulate ductal bile secretion.

These observations support the concept that, while C. parvum triggers host cell apoptosis in bystander uninfected biliary epithelial cells, which may limit spread of the infection, it directly activates the NF-kappaB/I(kappa)B system in infected biliary epithelia thus protecting infected cells from death and facilitating parasite survival and propagation.

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl ؊ channel and a Cl ؊ /HCO 3 ؊ exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 M) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane. After administration of secretin to rats in vivo, bile flow increased, and AQP1, CFTR, and AE2 co-redistributed to the apical cholangiocyte membrane; both events were blocked by pharmacologic disassembly of microtubules. Based on these in vitro and in vivo observations utilizing independent and complementary approaches, we propose that cholangiocytes contain an organelle that sequesters functionally related proteins that can account for ion-driven water transport, that this organelle moves to the apical cholangiocyte membrane in response to secretory agonists, and that these events account for ductal bile secretion at a molecular level.Cholangiocytes are cells that line intrahepatic bile ducts and, like other epithelia, possess discrete, specialized apical and basolateral membranes. Each cholangiocyte membrane contains specific receptors and flux molecules (i.e. channels, exchangers, and transporters) that accomplish the vectorial movement of solutes, ions, and water across the biliary epithelial barrier (1-3), resulting in ductal bile formation by as yet unclear molecular mechanisms. Recently, we proposed a molecular model for hormone-induced bile secretion by cholangiocytes. The key feature of this model is the agonist-induced, coordinated, exocytic insertion into and endocytic retrieval from the apical cholangiocyte plasma membrane of key flux molecules that, in the unstimulated state, are sequestered in an intracytoplasmic vesicular compartment (4). We also proposed that one of these flux proteins was AQP1, a member of the aquaporin (AQP) 1 family of water channels that mediate the bidirectional, passive movement of water molecules across epithelial cells in response to osmotic gradients established by ions and solutes (1, 5-7). In support of this model are data showing that secretin, a hormone that stimulates ductal bile secretion, can also trigger the exocytic insertion of AQP1 into the apical cholangiocyte plasma membrane (6). More recently, we also provided data in hepatocytes, the other epithelial cell in the liver involved in bile formation, indicating that recycling of AQP8 may account for agonist-induced canalicular bile secreti...