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TRPV4 is a type of nonselective cation channel, and activation of TRPV4 in the gastrointestinal tract causes experimental colitis in mice. A previous study found that tyrosine-phosphorylated claudin-7 is increased in experimental colitis. The relationship between tyrosine-phosphorylated claudin-7 and TRPV4 remains undefined. In the present study, we developed a claudin-7 mutant by replacing tyrosine with glutamic acid at position 210, named cld7-Y210E colonic cells. We found that activation of TRPV4 by GSK1016790A increased the permeability of control colonic cell monolayers, which was decreased by the TRPV4 antagonist HC067047. In monolayers of cld7-Y210E colonic cells, no differences in permeability were found between GSK1016790A and HC067047 treatments. GSK1016790A increased the aggregation of claudin-7 at the cell membrane in control colonic cells, and the effect was diminished by HC067047. In cld7-Y210E colonic cells, neither GSK1016790A nor HC067047 apparently changed the aggregation of claudin-7. Neither GSK1016790A nor HC067047 altered the TRPV4 protein level in vector colonic cells. In cld7-wild colonic cells, GSK1016790A did not alter the TRPV4 protein level, while HC067047 increased the TRPV4 protein level. The TRPV4 protein level was increased in cld7-Y210E colonic cells, decreased by GSK1016790A and further decreased by HC067047. Calcium influx was not significantly changed in the control colonic cells treated with GSK1016790A. However, GSK1016790A significantly increased calcium influx in cld7-Y210E colonic cells. We concluded that tyrosine-phosphorylated claudin-7 affects the TRPV4-modulated intestinal epithelial barrier, TRPV4-mediated calcium influx, and the protein expression of TRPV4 in human colonic cells. We suggest that tyrosine-phosphorylated claudin-7 affects the TRPV4-modulated intestinal epithelial barrier, which might be related to TRPV4 expression and TRPV4-mediated calcium influx., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Cryptosporidium , a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and animals, is an important opportunistic pathogen in AIDS patients and one of the most common enteric pathogens affecting young children in developing regions. This parasite is referred to as a "minimally invasive" mucosal pathogen, and epithelial cells play a central role in activating and orchestrating host immune responses. We previously demonstrated that Cryptosporidium parvum infection stimulates host epithelial cells to release exosomes, and these released exosomes shuttle several antimicrobial peptides to carry out anti- C. parvum activity. In this study, we detected the upregulation of inflammatory genes in the liver and spleen following C. parvum intestinal infection in neonatal mice. Interestingly, exosomes released from intestinal epithelial cells following C. parvum infection could activate the nuclear factor kappa B signaling pathway and trigger inflammatory gene transcription in isolated primary splenocytes. Several epithelial cell-derived proteins and a subset of parasite RNAs were detected in the exosomes released from C. parvum -infected intestinal epithelial cells. Shuttling of these effector molecules, including the high mobility group box 1 protein, was involved in the induction of inflammatory responses in splenocytes induced by the exosomes released from infected cells. Our data indicate that exosomes released from intestinal epithelial cells upon C. parvum infection can activate immune cells by shuttling various effector molecules, a process that may be relevant to host systemic responses to Cryptosporidium infection., (Copyright © 2019 American Society for Microbiology.)

Abnormal phosphorylation of claudins changes the interaction and aggregation of tight junction proteins, affecting the intestinal epithelial barrier. Selective blockade of transient receptor potential vanilloid 4 (TRPV4) alleviated experimental colitis. Whether TRPV4 affects the intestinal epithelial barrier and the relationship to claudin-7 phosphorylation remain unknown. In the present study, we investigated the TRPV4 expression in human colonic tissues and colonic cells. Using the site-directed mutagenesis approach, we also identified the roles of claudin-7 phosphorylation in the epithelial barrier and the relationship between TRPV4 and claudin-7 phosphorylation. Increased TRPV4 expression was found in the colonic mucosa from IBD patients. In colonic cells, the mutation of claudin-7 at position 204 decreased the TRPV4 expression. Mutation of claudin-7 at position 204 significantly decreased the FD20 permeability in monolayer colonic cells, while mutations of claudin-7 at positions S206 and S207 increased the FD20 permeability. Meanwhile, mutations of claudin-7 at positions S204 and S207 increased the TER in monolayer colonic cells. TRPV4 agonist GSK1016790 A increased the FD20 permeability in the control group, cld7-wild group, cld7-S206A group and cld7-S207 A group, while the TRPV4 antagonist HC067047 decreased the FD20 permeability in the same groups. HC067047 treatment increased the TER in vector cells, cld7-wild cells and cld7-S206 A cells compared to the respective cells in GSK1016790A-treated groups. HC067047 treatment decreased the migration in vector cells, cld7-wild cells and cld7-S206 A cells compared to the respective cells in the GSK1016790A-treated groups. These results indicated that TRPV4 might be a target for the maintenance of the intestinal epithelial barrier and indicated the mechanism involved in the modulation of serine phosphorylated claudin-7., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Intestinal infection by Cryptosporidium is known to cause epithelial cell migration disorder but the underlying mechanisms are unclear. Previous studies demonstrated that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using multiple models of intestinal cryptosporidiosis, we report here that C. parvum infection induces expression and release of the dickkopf protein 1 (Dkk1) from intestinal epithelial cells. Delivery of parasite Cdg7_FLc_1030 RNA to intestinal epithelial cells triggers transactivation of host Dkk1 gene during C. parvum infection. Release of Dkk1 is involved in C. parvum-induced inhibition of cell migration of epithelial cells, including noninfected bystander cells. Moreover, Dkk1-mediated suppression of host cell migration during C. parvum infection involves inhibition of Cdc42/Par6 signaling. Our data support the hypothesis that attenuation of intestinal epithelial cell migration during Cryptosporidium infection involves parasite Cdg7_FLc_1030 RNA-mediated induction and release of Dkk1 from infected cells.

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3., (© The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Cryptosporidium parvum is an important opportunistic parasite pathogen for immunocompromised individuals and a common cause of diarrhea in young children. Previous studies have identified a panel of RNA transcripts of very low protein-coding potential in C. parvum. Using an in vitro model of human intestinal cryptosporidiosis, we report here that some of these C. parvum RNA transcripts were selectively delivered into the nuclei of host epithelial cells during C. parvum infection. Nuclear delivery of several such parasitic RNAs, including Cdg7_FLc_0990, involved heat-shock protein 70-mediated nuclear importing mechanism. Overexpression of Cdg7_FLc_0990 in intestinal epithelial cells resulted in significant changes in expression levels of specific genes, with significant overlapping with alterations in gene expression profile detected in host cells after C. parvum infection. Our data demonstrate that C. parvum transcripts of low protein-coding potential are selectively delivered into epithelial cells during infection and may modulate gene transcription in infected host cells., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

The NF-kB pathway is key to epithelial immune defense and has been implicated in secretion of antimicrobial peptides, release of cytokines/chemokines to mobilize immune effector cells, and activation of adaptive immunity. The expression of many inflammatory genes following infection involves the remodeling of the chromatin structure. We reported here that histone deacetylases (HDACs) and NF-kB signaling coordinate expression of CX3CL1 in epithelial cells following Cryptosporidium parvum infection. Upregulation of CX3CL1 was detected in cultured human biliary epithelial cells following infection. Expression of miR-424 and miR-503 was downregulated, and was involved in the induction of CX3CL1 in infected cells. C. parvum infection suppressed transcription of the mir-424-503 gene in a NF-kB- and HDAC-dependent manner. Increased promoter recruitment of NF-kB p50 and HDACs, and decreased promoter H3 acetylation associated with the mir-424-503 gene were observed in infected cells. Upregulation of CX3CL1 in biliary epithelial cells and increased infiltration of CX3CR1(+) cells were detected during C. parvum infection in vivo. Induction of CX3CL1 and downregulation of miR-424 and miR-503 were also detected in epithelial cells in response to LPS stimulation. The above results indicate that HDACs and NF-kB signaling coordinate epithelial expression of CX3CL1 to promote mucosal antimicrobial defense through suppression of the mir-424-503 gene.

Toleroge nic DCs and Tregs are believed to play a critical role in oral tolerance. However, the mechanisms of the generation of tolerogenic DCs and activation of Tregs in the gut remain poorly understood. This study aims to dissect the molecular mechanisms by which IECs and protein antigen induce functional tolerogenic DCs and Tregs. Expression of αvβ6 by gut epithelial cell-derived exosomes, its coupling with food antigen, and their relationship with the development of functional tolerogenic DCs and Tregs were examined by using in vitro and in vivo approaches. The results show that IECs up-regulated the integrin αvβ6 upon uptake of antigens. The epithelial cell-derived exosomes entrapped and transported αvβ6 and antigens to the extracellular environment. The uptake of antigens alone induced DCs to produce LTGFβ, whereas exosomes carrying αvβ6/antigen resulted in the production of abundant, active TGF-β in DCs that conferred to DCs the tolerogenic properties. Furthermore, αvβ6/OVA-carrying, exosome-primed DCs were found to promote the production of active TGF-β in Tregs. Thus, in vivo administration of αvβ6/OVA-laden exosomes induced the generation of Tregs and suppressed skewed Th2 responses toward food antigen in the intestine. Our study provides important molecular insights into the molecular mechanisms of Treg development by demonstrating an important role of IEC-derived exosomes carrying αvβ6 and food antigen in the induction of tolerogenic DCs and antigen-specific Tregs.

Mucosal surface epithelial cells are equipped with several defense mechanisms that guard against pathogens. Recent studies indicate that microRNAs (miRNAs) mediate post-transcriptional gene suppression and may be a critical component of the complex regulatory networks in epithelial immune responses. Transcription of miRNA genes in epithelial cells can be elaborately controlled through pathogen recognition receptors, such as Toll-like receptors (TLRs), and associated nuclear factor kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways, and ultimately nuclear transcription factor associated-transactivation and transrepression. Activation of these intracellular signaling pathways may also modulate the process of miRNA maturation. Functionally, miRNAs may modulate epithelial immune responses at every step of the innate immune network, including production and release of cytokines/chemokines, expression of adhesion and costimulatory molecules, shuttling of miRNAs through release of exosomes and feedback regulation of immune homeostasis. Therefore, miRNAs act as critical regulators to the fine-tuning of epithelial immune responses.

Cryptosporidium parvum is a protozoan parasite that infects gastrointestinal epithelial cells and causes diarrhoeal disease in humans and animals globally. Pathological changes following C. parvum infection include crypt hyperplasia and a modest inflammatory reaction with increased infiltration of lymphocytes into intestinal mucosa. Expression of adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), on infected epithelial cell surfaces may facilitate adhesion and recognition of lymphocytes at infection sites. MicroRNAs (miRNAs) are small RNA molecules of 23 nucleotides that negatively regulate protein-coding gene expression via translational suppression or mRNA degradation. We recently reported that microRNA-221 (miR-221) regulates ICAM-1 translation through targeting the ICAM-1 3'-untranslated region (UTR). In this study, we tested the role of miR-221 in regulating ICAM-1 expression in epithelial cells in response to C. parvum infection using an in vitro model of human biliary cryptosporidiosis. Up-regulation of ICAM-1 at both message and protein levels was detected in epithelial cells following C. parvum infection. Inhibition of ICAM-1 transcription with actinomycin D could only partially block C. parvum-induced ICAM-1 expression at the protein level. Cryptosporidium parvum infection decreased miR-221 expression in infected epithelial cells. When cells were transfected with a luciferase reporter construct covering the miR-221 binding site in the ICAM-1 3'-UTR and then exposed to C. parvum, an enhanced luciferase activity was detected. Transfection of miR-221 precursor abolished C. parvum-stimulated ICAM-1 protein expression. In addition, expression of ICAM-1 on infected epithelial cells facilitated epithelial adherence of co-cultured Jurkat cells. These results indicate that miR-221-mediated translational suppression controls ICAM-1 expression in epithelial cells in response to C. parvum infection., (Copyright © 2011 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Expression of the cytokine-inducible Src homology 2 (CIS) protein and suppressors of cytokine signaling (SOCS) proteins represents an important element of host cell reactions in response to infection. We have demonstrated previously that Cryptosporidium parvum infection down-regulates microRNA-98 (miR-98) and let-7 to induce CIS expression in biliary epithelial cells. We report here that down-regulation of miR-98 and let-7 also coordinates epithelial expression of SOCS4 after C. parvum infection. Targeting of the SOCS4 3' untranslated region by miR-98 or let-7 resulted in translational repression. Functional manipulation of miR-98 caused reciprocal alterations in SOCS4 protein expression. Transfection of miR-98 precursor abolished C. parvum-stimulated SOCS4 up-regulation. Moreover, expression of SOCS4 in epithelial cells showed an inhibitory effect on phosphorylation of signal transducers and activators of transcription proteins induced by C. parvum. These data suggest that miRNAs play an important role in the coordinated regulation of CIS and SOCS expression in epithelial cells in response to C. parvum infection.

The majority of human miRNA genes is transcribed by polymerase II and can be classified as class II genes similar to protein-coding genes. Whereas current research on miRNAs has focused on the physiological and pathological functions, the molecular mechanisms underlying their transcriptional regulation are largely unknown. We recently reported that lipopolysaccharide (LPS) alters mature miRNA expression profile in human biliary epithelial cells. In this study, we tested the role of transcription factor NF-kappaB in LPS-induced transcription of select miRNA genes. Of the majority of LPS-up-regulated mature miRNAs in cultured human biliary epithelial cells, potential NF-kappaB binding sites were identified in the putative promoter elements of their corresponding genes. Inhibition of NF-kappaB activation by SC-514, an IKK2 inhibitor, blocked LPS-induced up-regulation of a subset of pri-miRNAs, including pri-miR-17-92, pri-miR-125b-1, pri-miR-21, pri-miR-23b-27b-24-1, pri-miR-30b, pri-miR-130a and pri-miR-29a. Moreover, direct binding of NF-kappaB p65 subunit to the promoter elements of mir-17-92, mir-125b-1, mir-21, mir-23b-27b-24-1, mir-30b and mir-130a genes was identified by chromatin immunoprecipitation analysis and confirmed by the luciferase reporter assay. Thus, a subset of miRNA genes is regulated in human biliary epithelial cells through NF-kappaB activation induced by LPS, suggesting a role of the NF-kappaB pathway in the transcriptional regulation of miRNA genes.

Aberrant cholangiocyte reactions in response to inflammatory stimuli are important pathogenic factors for the persistent biliary inflammation in patients with cholangiopathies. Overexpression of intercellular cell adhesion molecule-1 (ICAM-1) in cholangiocytes is a common pathological feature in inflammatory cholangiopathies and can promote cholangiocyte interactions with effector lymphocytes in the portal region. In this study, we tested the involvement of miRNA-mediated posttranscriptional regulation in IFN-gamma-induced ICAM-1 expression in cholangiocytes. Using both immortalized and nonimmortalized human cholangiocyte cell lines, we found that IFN-gamma activated ICAM-1 transcription and increased ICAM-1 protein expression. Inhibition of ICAM-1 transcription could only partially block IFN-gamma-induced ICAM-1 expression at the protein level. In silico target prediction analysis revealed complementarity of miR-221 to the 3'-untranslated region of ICAM-1 mRNA. Targeting of ICAM-1 3'-untranslated region by miR-221 resulted in translational repression in cholangiocytes but not ICAM-1 mRNA degradation. Functional inhibition of miR-221 with anti-miR-221 induced ICAM-1 protein expression. Moreover, IFN-gamma stimulation decreased miR-221 expression in cholangiocytes in a signal transducer and activator of transcription 1-dependent manner. Transfection of miR-221 precursor abolished IFN-gamma-stimulated ICAM-1 protein expression. In addition, miR-221-mediated expression of ICAM-1 on cholangiocytes showed a significant influence on the adherence of cocultured T cells. These findings indicate that both transcriptional and miRNA-mediated posttranscriptional mechanisms are involved in IFN-gamma-induced ICAM-1 expression in human cholangiocytes, suggesting an important role for miRNAs in the regulation of cholangiocyte inflammatory responses.

Expression of B7 costimulatory molecules represents an important compartment of immune response of epithelial cells after microbial infection. We report here that the protozoan parasite Cryptosporidium parvum induced B7-H1 expression in cultured human cholangiocytes. Induced expression of B7-H1 was identified in cells after exposure to infective C. parvum parasite or parasite lysate. Interestingly, the level of microRNA-513 (miR-513) was reduced in cells after exposure to C. parvum, which resulted in a relief of 3' untranslated region-mediated translational suppression of B7-H1. Overexpression of miR-513 through transfection of miR-513 precursor inhibited C. parvum-induced B7-H1 protein expression. Moreover, enhanced apoptotic cell death was identified in activated human T cells after coculture with C. parvum-infected cholangiocytes. The apoptosis of activated T cells was partially blocked by a neutralizing antibody to B7-H1 or transfection of cholangiocytes with miR-513 precursor. These data suggest a role of miR-513 in regulating B7-H1 expression by cholangiocytes in response to C. parvum infection.

Cholangiocytes, the epithelial cells lining bile ducts, provide the first line of defense against lumenal microbes in the biliary system. Recent advances in biliary immunity indicate that cholangiocytes express a variety of pathogen-recognition receptors and can activate a set of intracellular signaling cascades to initiate a profound antimicrobial defense, including release of proinflammatory cytokines and chemokines, production of antimicrobial peptides and maintenance of biliary epithelial integrity. Cholangiocytes also interact with other cell types in the liver (for example, lymphocytes and Kupffer cells) through expression and release of adhesion molecules and immune mediators. Subsequently, through an intricate feedback mechanism involving both epithelial and other liver cells, a set of intracellular signaling pathways are activated to regulate the functional state of cholangiocyte responses during microbial infection. Thus, cholangiocytes are actively involved in mucosal immunity of the biliary system and represent a fine-tuned, integral component of liver immunity.

Toll-like receptors (TLRs) are important pathogen recognition molecules and are key to epithelial immune responses to microbial infection. However, the molecular mechanisms that regulate TLR expression in epithelia are obscure. Micro-RNAs play important roles in a wide range of biological events through post-transcriptional suppression of target mRNAs. Here we report that human biliary epithelial cells (cholangiocytes) express let-7 family members, micro-RNAs with complementarity to TLR4 mRNA. We found that let-7 regulates TLR4 expression via post-transcriptional suppression in cultured human cholangiocytes. Infection of cultured human cholangiocytes with Cryptosporidium parvum, a parasite that causes intestinal and biliary disease, results in decreased expression of primary let-7i and mature let-7 in a MyD88/NF-kappaB-dependent manner. The decreased let-7 expression is associated with C. parvum-induced up-regulation of TLR4 in infected cells. Moreover, experimentally induced suppression or forced expression of let-7i causes reciprocal alterations in C. parvum-induced TLR4 protein expression, and consequently, infection dynamics of C. parvum in vitro. These results indicate that let-7i regulates TLR4 expression in cholangiocytes and contributes to epithelial immune responses against C. parvum infection. Furthermore, the data raise the possibility that micro-RNA-mediated post-transcriptional pathways may be critical to host-cell regulatory responses to microbial infection in general.

While Cryptosporidium parvum infection of the intestine has been reported in both immunocompetent and immunocompromised individuals, biliary infection is seen primarily in adult AIDS patients and is associated with development of AIDS cholangiopathy. However, the mechanisms of pathogen-induced AIDS cholangiopathy remain unclear. Since we previously demonstrated that the Fas/Fas ligand (FasL) system is involved in paracrine-mediated C. parvum cytopathicity in cholangiocytes, we also tested the potential synergistic effects of human immunodeficiency virus type 1 (HIV-1) transactivator of transcription (Tat)-mediated FasL regulation on C. parvum-induced apoptosis in cholangiocytes by semiquantitative reverse transcription-PCR, immunoblotting, immunofluorescence analysis, and immunogold electron microscopy. H69 cells do not express CXCR4 and CCR5, which are receptors required for direct HIV-1 viral infection. However, recombinant biologically active HIV-1-associated Tat protein increased FasL expression in the cytoplasm of cholangiocytes without a significant increase in apoptosis. We found that C. parvum-induced apoptosis was associated with translocation of intracellular FasL to the cell membrane surface and release of full-length FasL from infected H69 cells. Tat significantly (P < 0.05) increased C. parvum-induced apoptosis in bystander cells in a dose-dependent manner. Moreover, Tat enhanced both C. parvum-induced FasL membrane translocation and release of full-length FasL. In addition, the FasL neutralizing antibody NOK-1 and the caspase-8 inhibitor Z-IETD-fmk both blocked C. parvum-induced apoptosis in cholangiocytes. The data demonstrated that HIV-1 Tat enhances C. parvum-induced cholangiocyte apoptosis via a paracrine-mediated, FasL-dependent mechanism. Our results suggest that concurrent active HIV replication, with associated production of Tat protein, and C. parvum infection synergistically increase cholangiocyte apoptosis and thus jointly contribute to AIDS-related cholangiopathies.

Cryptosporidium parvum attaches to intestinal and biliary epithelial cells via specific molecules on host-cell surface membranes including Gal/GalNAc-associated glycoproteins. Subsequent cellular entry of this parasite depends on host-cell membrane alterations to form a parasitophorous vacuole via activation of phosphatidylinositol 3-kinase (PI-3K)/Cdc42-associated actin remodelling. How C. parvum hijacks these host-cell processes to facilitate its infection of target epithelia is unclear. Using specific probes to known components of sphingolipid-enriched membrane microdomains (SEMs), we detected aggregation of host-cell SEM components at infection sites during C. parvum infection of cultured human biliary epithelial cells (i.e. cholangiocytes). Activation and membrane translocation of acid-sphingomyelinase (ASM), an enzyme involved in SEM membrane aggregation, were also observed in infected cells. Pharmacological disruption of SEMs and knockdown of ASM via a specific small interfering RNA (siRNA) significantly decreased C. parvum attachment (by approximately 84%) and cellular invasion (by approximately 88%). Importantly, knockdown of ASM and disruption of SEMs significantly blocked C. parvum-induced accumulation of Gal/GalNAc-associated glycoproteins at infection sites by approximately 90%. Disruption of SEMs and knockdown of ASM also significantly blocked C. parvum-induced activation of host-cell PI-3K and subsequent accumulation of Cdc42 and actin by up to 75%. Our results suggest an important role of SEMs for C. parvum attachment to and entry of host cells, likely via clustering of membrane-binding molecules and facilitating of C. parvum-induced actin remodelling at infection sites through activation of the PI-3K/Cdc42 signalling pathway.

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-kappaB 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-kappaB. Short-interfering RNA to TLR2, TLR4, and MyD88 also blocked C. parvum-induced NF-kappaB activation. Moreover, C. parvum selectively up-regulated human beta-defensin-2 in directly infected cells, and inhibition of TLR2 and TLR4 signals or NF-kappaB activation were each associated with a reduction of C. parvum-induced human beta-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-kappaB.

Background: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH., Methods: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H2O2. Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05., Results: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O2 toxicity did lead to a reduced endogenous expression of hNTH in A549 cells., Conclusion: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O2 and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O2 and its metabolites.

Background & Aims: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia., Methods: In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion., Results: c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion., Conclusions: C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

Cholangiocytes-the epithelial cells which line the bile ducts-are increasingly recognized as important transporting epithelia actively involved in the absorption and secretion of water, ions, and solutes. This recognition is due in part to the recent development of new experimental models. New biologic concepts have emerged including the identification and topography of receptors and flux proteins on the apical and/or basolateral membrane which are involved in the molecular mechanisms of ductal bile secretion. Individually isolated and/or perfused bile duct units from livers of rats and mice serve as new,physiologically relevant in vitro models to study cholangiocyte transport. Biliary tree dimensions and novel insights into anatomic remodeling of proliferating bile ducts have emerged from three-dimensional reconstruction using CT scanning and sophisticated software. Moreover, new pathologic concepts have arisen regarding the interaction of cholangiocytes with pathogens such as Cryptosporidium parvum. These concepts and associated methodologies may provide the framework to develop new therapies for the cholangiopathies, a group of important hepatobiliary diseases in which cholangiocytes are the target cell.

Cryptosporidium is a zoonotic apicomplexan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans. It is an important opportunistic pathogen in AIDS patients and a leading cause of infectious diarrhea and diarrheal-related death in children worldwide. The intestinal epithelial cells provide the first line of defense against Cryptosporidium infection and play a central role in activating and regulating the host's antiparasitic response. Increasing evidence suggests that long noncoding RNAs (lncRNAs) participate in host-pathogen interactions and play a regulatory role in the pathogenesis of diseases but the underlying molecular mechanisms are not fully understood. We previously identified a panel of host lncRNAs that are upregulated in murine intestinal epithelial cells following Cryptosporidium infection, including U90926. We demonstrate here that U90926 is acting in a pro-parasitic manner in regulating intestinal epithelial cell-autonomous antiparasitic defense. Inhibition of U90926 resulted in a decreased infection burden of the parasite while overexpression of U90926 showed an increase in infection burden in cultured murine intestinal epithelial cells. Induction of U90926 suppressed transcription of epithelial defense genes involved in controlling Cryptosporidium infection through epigenetic mechanisms. Specifically, transcription of Aebp1, which encodes the Aebp1 protein, a potent modulator of inflammation and NF-κB signaling, was suppressed by U90926. Gain- or loss-of-function of Aebp1 in the host's epithelial cells caused reciprocal alterations in the infection burden of the parasite. Interestingly, Cryptosporidium carries the Cryptosporidium virus 1 (CSpV1), a double-stranded (ds) RNA virus coding two dsRNA fragments, CSpV1-dsRdRp and CSpV1-dsCA. Both CSpV1-dsRdRp and CSpV1-dsCA can be delivered into infected cells as previously reported. We found that cells transfected with in vitro transcribed CSpV1-dsCA or CSpV1-dsRdRp displayed an increased level of U90926, suggesting that CSpV1 is involved in the upregulation of U90926 during Cryptosporidium infection. Our study highlights a new strategy by Cryptosporidium to hijack a host lncRNA to suppress epithelial cell-autonomous antiparasitic defense and allow for a robust infection. [ABSTRACT FROM AUTHOR]

The gastrointestinal epithelium guides the immune system to differentiate between commensal and pathogenic microbiota, which relies on intimate links with the type I IFN signal pathway. Epithelial cells along the epithelium provide the front line of host defense against pathogen infection in the gastrointestinal tract. Increasing evidence supports the regulatory potential of long noncoding RNAs (lncRNAs) in immune defense but their role in regulating intestinal epithelial antimicrobial responses is still unclear. Cryptosporidium, a protozoan parasite that infects intestinal epithelial cells, is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelial cells. We previously identified a panel of host cell lncRNAs that are upregulated in murine intestinal epithelial cells following microbial challenge. One of these lncRNAs, NR_033736, is upregulated in intestinal epithelial cells following Cryptosporidium infection and displays a significant suppressive effect on type I IFN-controlled gene transcription in infected host cells. NR_033736 can be assembled into the ISGF3 complex and suppresses type I IFN-mediated gene transcription. Interestingly, upregulation of NR_033736 itself is triggered by the type I IFN signaling. Moreover, NR_033736 modulates epithelial anti-Cryptosporidium defense. Our data suggest that upregulation of NR_033736 provides negative feedback regulation of type I IFN signaling through suppression of type I IFN-controlled gene transcription, and consequently, contributing to fine-tuning of epithelial innate defense against microbial infection. Author summary: Intestinal epithelial cells are an important component of gastrointestinal mucosal immunity. They generate various types of barriers to protect the intestinal mucosa from commensal microbes or invading pathogenic microorganisms. Upon microbial infection, intestinal epithelial cells quickly initiate a series of innate immune reactions, including production of antimicrobial molecules and release of inflammatory chemokines and cytokines. These chemokines and cytokines of epithelial cell origin may mobilize and activate immune effector cells to the infection sites. One family of these cytokines are type I IFNs (e.g., IFN-α/β), which not only activate other immune effector cells at gastrointestinal mucosa but also stimulate epithelial function through ligation of receptors expressed on intestinal epithelial cells themselves in an autocrine manner. Cryptosporidium is an important opportunistic pathogen in AIDS patients and a common cause of diarrhea in young children in developing countries. Recent advances in Cryptosporidium research have revealed a strong type I IFN response in infected intestinal epithelium. In this study, we provide data demonstrating an important role for long noncoding RNAs (lncRNAs) in regulating type I IFN signaling in intestinal epithelial cells in response to Cryptosporidium infection. LncRNAs are recently identified long non-coding transcripts (>200 nt) that are not translated into protein. We found that upregulation of one host cell lncRNA, NR_033736, displays a significant suppressive effect on type I IFN-controlled gene transcription in intestinal epithelial cells following infection, contributing to the fine-tuning of epithelial anti-Cryptosporidium defense. Our data suggest a new arm of feedback regulation for type I IFN signaling through lncRNA-mediated gene transcription, contributing to fine-tuning of epithelial innate defense against microbial infection in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]

Intestinal infection by Cryptosporidium parvum causes inhibition of epithelial turnover, but underlying mechanisms are unclear. Previous studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected epithelial cells. Using in vitro and in vivo models of intestinal cryptosporidiosis, we report here that host delivery of parasite Cdg7_FLc_1000 RNA results in inhibition of epithelial cell migration through suppression of the gene encoding sphingomyelinase 3 (SMPD3). Delivery of Cdg7_FLc_1000 into infected cells promotes the histone methyltransferase G9a-mediated H3K9 methylation in the SMPD3 locus. The DNA-binding transcriptional repressor, PR domain zinc finger protein 1, is required for the assembly of Cdg7_FLc_1000 into the G9a complex and associated with the enrichment of H3K9 methylation at the gene locus. Pathologically, nuclear transfer of Cryptosporidium parvum Cdg7_FLc_1000 RNA is involved in the attenuation of intestinal epithelial cell migration via trans-suppression of host cell SMPD3. [ABSTRACT FROM AUTHOR]

Cryptosporidium parvum is an important opportunistic parasite pathogen for immunocompromised individuals and a common cause of diarrhea in young children. Previous studies have identified a panel of RNA transcripts of very low protein-coding potential in C. parvum. Using an in vitro model of human intestinal cryptosporidiosis, we report here that some of these C. parvum RNA transcripts were selectively delivered into the nuclei of host epithelial cells during C. parvum infection. Nuclear delivery of several such parasitic RNAs, including Cdg7_FLc_0990, involved heat-shock protein 70-mediated nuclear importing mechanism. Overexpression of Cdg7_FLc_0990 in intestinal epithelial cells resulted in significant changes in expression levels of specific genes, with significant overlapping with alterations in gene expression profile detected in host cells after C. parvum infection. Our data demonstrate that C. parvum transcripts of low protein-coding potential are selectively delivered into epithelial cells during infection and may modulate gene transcription in infected host cells. [ABSTRACT FROM AUTHOR]