SHP-2 Mediates Cryptosporidium parvum Infectivity in Human Intestinal Epithelial Cells

The parasite, Cryptosporidium parvum, induces human gastroenteritis through infection of host epithelial cells in the small intestine. During the initial stage of infection, C. parvum is reported to engage host mechanisms at the host cell-parasite interface to form a parasitophorous vacuole. We determined that upon infection, the larger molecular weight proteins in human small intestinal epithelial host cells (FHs 74 Int) appeared to globally undergo tyrosine dephosphorylation. In parallel, expression of the cytoplasmic protein tyrosine phosphatase Src homology-2 domain-containing phosphatase 2 (SHP-2) increased in a time-dependent manner. SHP-2 co-localized with the C. parvum sporozoite and this interaction increased the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Furthermore, we show that one potential target that SHP-2 acts upon is the focal adhesion protein, paxillin, which undergoes moderate dephosphorylation following infection, with inhibition of SHP-2 rescuing paxillin phosphorylation. Importantly, treatment with an inhibitor to SHP-2 and with an inhibitor to paxillin and Src family kinases, effectively decreased the multiplicity of C. parvum infection in a dose-dependent manner. Thus, our study reveals an important role for SHP-2 in the pathogenesis of C. parvum. Furthermore, while host proteins can be recruited to participate in the development of the electron dense band at the host cell-parasite interface, our study implies for the first time that SHP-2 appears to be recruited by the C. parvum sporozoite to regulate infectivity. Taken together, these findings suggest that SHP-2 and its down-stream target paxillin could serve as targets for intervention.     

Interaction with Intestinal Epithelial Cells Promotes an Immunosuppressive Phenotype in Lactobacillus casei

Maintenance of the immunological tolerance and homeostasis in the gut is associated with the composition of the intestinal microbiota. We here report that cultivation of Lactobacillus casei ATCC 334 in the presence of human intestinal epithelial cells promotes functional changes in bacteria. In particular, the interaction enhanced the immunosuppressive phenotype of L. casei as demonstrated by the ability of L. casei to generate functional regulatory T cells (CD4+CD25+FoxP3+) and production of the anti-inflammatory cytokine interleukin-10 by human peripheral blood mononuclear cells. The results indicate microbe-host cross-talk that changes features of microbes, and suggest that in vitro simulation of epithelial cell interaction can reveal functional properties of gut microbes more accurately than conventional cultivation.     

TLR9 and RIG-I Signaling in Human Endocervical Epithelial Cells Modulates Inflammatory Responses of Macrophages and Dendritic Cells In Vitro

The innate immune system has evolved to recognize invading pathogens through pattern recognition receptors (PRRs).Among PRRs, Toll like receptors (TLRs 3, 7/8,9) and RIG-I like receptors (RLRs) have been shown to recognize viral components. Mucosal immune responses to viral infections require coordinated actions from epithelial as well as immune cells. In this respect, endocervical epithelial cells (EEC''s) play an important role in initiating innate immune responses via PRRs. It is unknown whether EEC''s can alter immune responses of macrophages and dendritic cells (DC''s) like its counterparts in intestinal and respiratory systems. In this study, we show that endocervical epithelial cells (End1/E6E7) express two key receptors, TLR9 and RIG-I involved in anti-viral immunity. Stimulation of End1/E6E7 cells lead to the activation of NF-κB and increased secretion of pro-inflammatory cytokines, IL-6 and IL-8. Polarized End1/E6E7 cells responded to apical stimulation with ligands of TLR9 and RIG-I, CpG-ODN and Poly(I:C)LL respectively, without compromising End1/E6E7 cell integrity. At steady state, spent medium from End1/E6E7 cells significantly reduced secretion of pro-inflammatory cytokines from LPS treated human primary monocyte derived macrophages (MDMs) and DC:T cell co-cultures. Spent medium from End1/E6E7 cells stimulated with ligands of TLR9/RIG-I restored secretion of pro-inflammatory cytokines as well as enhanced phagocytosis and chemotaxis of monocytic U937 cells. Spent medium from CpG-ODN and Poly(I:C)LL stimulated End1/E6E7 cells showed significant increased secretion of IL-12p70 from DC:T cell co-cultures. The anti-inflammatory effect of spent media of End1/E6E7 cell was observed to be TGF-β dependent. In summary, the results of our study indicate that EEC''s play an indispensable role in modulating anti-viral immune responses at the female lower genital tract.     

Sugars Increase Non-Heme Iron Bioavailability in Human Epithelial Intestinal and Liver Cells

Previous studies have suggested that sugars enhance iron bioavailability, possibly through either chelation or altering the oxidation state of the metal, however, results have been inconclusive. Sugar intake in the last 20 years has increased dramatically, and iron status disorders are significant public health problems worldwide; therefore understanding the nutritional implications of iron-sugar interactions is particularly relevant. In this study we measured the effects of sugars on non-heme iron bioavailability in human intestinal Caco-2 cells and HepG2 hepatoma cells using ferritin formation as a surrogate marker for iron uptake. The effect of sugars on iron oxidation state was examined by measuring ferrous iron formation in different sugar-iron solutions with a ferrozine-based assay. Fructose significantly increased iron-induced ferritin formation in both Caco-2 and HepG2 cells. In addition, high-fructose corn syrup (HFCS-55) increased Caco-2 cell iron-induced ferritin; these effects were negated by the addition of either tannic acid or phytic acid. Fructose combined with FeCl₃ increased ferrozine-chelatable ferrous iron levels by approximately 300%. In conclusion, fructose increases iron bioavailability in human intestinal Caco-2 and HepG2 cells. Given the large amount of simple and rapidly digestible sugars in the modern diet their effects on iron bioavailability may have important patho-physiological consequences. Further studies are warranted to characterize these interactions.     

Human Rhinovirus Induced Cytokine/Chemokine Responses in Human Airway Epithelial and Immune Cells

Infections with human rhinovirus (HRV) are commonly associated with acute upper and lower respiratory tract disease and asthma exacerbations. The role that HRVs play in these diseases suggests it is important to understand host-specific or virus-specific factors that contribute to pathogenesis. Since species A HRVs are often associated with more serious HRV disease than species B HRVs, differences in immune responses they induce should inform disease pathogenesis. To identify species differences in induced responses, we evaluated 3 species A viruses, HRV 25, 31 and 36 and 3 species B viruses, HRV 4, 35 and 48 by exposing human PBMCs to HRV infected Calu-3 cells. To evaluate the potential effect of memory induced by previous HRV infection on study responses, we tested cord blood mononuclear cells that should be HRV naïve. There were HRV-associated increases (significant increase compared to mock-infected cells) for one or more HRVs for IP-10 and IL-15 that was unaffected by addition of PBMCs, for MIP-1α, MIP-1β, IFN-α, and HGF only with addition of PBMCs, and for ENA-78 only without addition of PBMCs. All three species B HRVs induced higher levels, compared to A HRVs, of MIP-1α and MIP-1β with PBMCs and ENA-78 without PBMCs. In contrast, addition of CBMCs had less effect and did not induce MIP-1α, MIP-1β, or IFN-α nor block ENA-78 production. Addition of CBMCs did, however, increase IP-10 levels for HRV 35 and HRV 36 infection. The presence of an effect with PBMCs and no effect with CBMCs for some responses suggest differences between the two types of cells possibly because of the presence of HRV memory responses in PBMCs and not CBMCs or limited response capacity for the immature CBMCs relative to PBMCs. Thus, our results indicate that different HRV strains can induce different patterns of cytokines and chemokines; some of these differences may be due to differences in memory responses induced by past HRV infections, and other differences related to virus factors that can inform disease pathogenesis.     

Alpha-Lipoic Acid Modulates the Oxidative and Inflammatory Responses Induced by Traditional and Novel Tobacco Products in Human Liver Epithelial Cells

Smoking has been associated with NAFLD recently, thus might be a contributing factor for liver disease progression. In this study, we identified the modulative action of α-lipoic acid (α-LA), an organosulphur compound, towards heated tobacco product (HTP) and cigarette smoke extract (CSE)-induced oxidative stress and inflammation in human liver HepG2 cells. The cells were pre-treated with α-LA and exposed to tobacco extracts, and cytotoxicity, oxidative response (SOD, CAT activities and GSH, MDA levels), inflammation (nitrite, IL-6, AhR levels), and liver function (AST/ALT) were assessed. According to the results, a notable increase in oxidative response was observed with CSE, whereas GSH depletion and decreased SOD activity were the key toxicological events induced by HTP (p<0.05). The oxidative and inflammatory responses were ameliorated with α-LA treatment, particularly through GSH restoration and IL-6 modulation. To conclude, these findings on α-LA might contribute to the design of novel adjuvant therapies for people exposed to tobacco smoke.     

Lineage-Specific Expression of Bestrophin-2 and Bestrophin-4 in Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3^- or Cl^-. Bestrophin genes represent a newly identified group of calcium-activated Cl^- channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

A Mucus Adhesion Promoting Protein,MapA, Mediates the Adhesion of Lactobacillus reuteri to Caco-2 Human Intestinal Epithelial Cells

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.     

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia

Lysophosphatidic acid (LPA) acts on LPA₂ receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA₂ attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA₂ alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA₂ in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA₂; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA₂ colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA₂ compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA₂ overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA₂ alone can lead to intestinal dysplasia.     

Vibrio cholerae Cytolysin Causes an Inflammatory Response in Human Intestinal Epithelial Cells That Is Modulated by the PrtV Protease

Background

Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s) to induce interleukin-8 (IL-8) production by human intestinal epithelial cells. The aim was to identify the secreted component(s) of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV.

Methodology/Principal Findings

Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC) were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-α (TNF-α) secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis.

Conclusions/Significance

We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-α in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.     

GLP-1(1~37) 诱导人类胚胎小肠 上皮细胞表达胰岛素

胶原酶消化法分离培养人类胚胎小肠的上皮细胞,应用胰高血糖素样肽 1 (glucagon-like peptide 1 (1~37),GLP-1) 诱导小肠上皮细胞向胰岛素分泌细胞分化,免疫组化方法对分化的和未分化的细胞进行鉴定, RT-PCR 检测胰岛内分泌细胞相关基因的表达 . 结果成功分离培养出人类小肠上皮细胞,免疫组化证明细胞表达小肠上皮的标志物细胞角蛋白 18 和 19 ,同时细胞也表达胰高血糖素和生长抑素,但无胰岛素表达 . GLP-1(1~37) 诱导小肠上皮细胞 6 天, RT-PCR 显示胰十二指肠同源异型基因盒 1 (pancreatic duodenal homeobox-1 , PDX-1) 、葡萄糖转运蛋白 2 (glucose transporter-2 , GLUT-2) 和胰岛素基因均有表达,免疫组化也检测到胰岛素阳性小肠上皮细胞 . 未用 GLP-1(1~37) 诱导小肠上皮细胞为对照的 RT-PCR 显示 PDX-1 、 GLUT-2 也表达,但无胰岛素 mRNA 和蛋白质的表达 . 研究表明 GLP-1(1~37) 能够诱导人类胚胎小肠上皮细胞向胰岛素分泌细胞分化 .     

Lactobacillus casei NY1301 Increases the Adhesion of Lactobacillus gasseri NY0509 to Human Intestinal Caco-2 Cells

In the presence of Lactobacillus casei NY1301, the adhesion of Lactobacillus gasseri NY0509 to cultured human intestinal Caco-2 cells was significantly increased (P<0.01). In contrast, L. gasseri NY0509 did not affect the adhesion of L. casei NY1301. A heat-stable cell component of L. casei NY1301 was involved in this increase of adhesion. These results suggest that a combination of these strains may have synergistic effects of adhesion to human intestinal mucosa.     

Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection.