The sialidase NEU1 directly interacts with the juxtamembranous segment of the cytoplasmic domain of mucin-1 to inhibit downstream PI3K-Akt signaling.

The extracellular domain (ED) of the membrane-spanning sialoglycoprotein, mucin-1 (MUC1), is an in vivo substrate for the lysosomal sialidase, neuraminidase-1 (NEU1). Engagement of the MUC1-ED by its cognate ligand, Pseudomonas aeruginosa-expressed flagellin, increases NEU1-MUC1 association and NEU1-mediated MUC1-ED desialylation to unmask cryptic binding sites for its ligand. However, the mechanism(s) through which intracellular NEU1 might physically interact with its surface-expressed MUC1-ED substrate are unclear. Using reciprocal coimmunoprecipitation and in vitro binding assays in a human airway epithelial cell system, we show here that NEU1 associates with the MUC1-cytoplasmic domain (CD) but not with the MUC1-ED. Prior pharmacologic inhibition of the NEU1 catalytic activity using the NEU1-selective sialidase inhibitor, C9-butyl amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid, did not diminish NEU1-MUC1-CD association. In addition, glutathione-S-transferase (GST) pull-down assays using the deletion mutants of the MUC1-CD mapped the NEU1-binding site to the membrane-proximal 36 aa of the MUC1-CD. In a cell-free system, we found that the purified NEU1 interacted with the immobilized GST-MUC1-CD and the purified MUC1-CD associated with the immobilized 6XHis-NEU1, indicating that the NEU1-MUC1-CD interaction was direct and independent of its chaperone protein, protective protein/cathepsin A. However, the NEU1-MUC1-CD interaction was not required for the NEU1-mediated MUC1-ED desialylation. Finally, we demonstrated that overexpression of either WT NEU1 or a catalytically dead NEU1 G68V mutant diminished the association of the established MUC1-CD binding partner, PI3K, to MUC1-CD and reduced downstream Akt kinase phosphorylation. These results indicate that NEU1 associates with the juxtamembranous region of the MUC1-CD to inhibit PI3K-Akt signaling independent of NEU1 catalytic activity.

Neuraminidase 1-mediated desialylation of the mucin 1 ectodomain releases a decoy receptor that protects against Pseudomonas aeruginosa lung infection.

Pseudomonas aeruginosa (Pa) expresses an adhesin, flagellin, that engages the mucin 1 (MUC1) ectodomain (ED) expressed on airway epithelia, increasing association of MUC1-ED with neuraminidase 1 (NEU1) and MUC1-ED desialylation. The MUC1-ED desialylation unmasks both cryptic binding sites for Pa and a protease recognition site, permitting its proteolytic release as a hyperadhesive decoy receptor for Pa. We found here that intranasal administration of Pa strain K (PAK) to BALB/c mice increases MUC1-ED shedding into the bronchoalveolar compartment. MUC1-ED levels increased as early as 12 h, peaked at 24-48 h with a 7.8-fold increase, and decreased by 72 h. The a-type flagellin-expressing PAK strain and the b-type flagellin-expressing PAO1 strain stimulated comparable levels of MUC1-ED shedding. A flagellin-deficient PAK mutant provoked dramatically reduced MUC1-ED shedding compared with the WT strain, and purified flagellin recapitulated the WT effect. In lung tissues, Pa increased association of NEU1 and protective protein/cathepsin A with MUC1-ED in reciprocal co-immunoprecipitation assays and stimulated MUC1-ED desialylation. NEU1-selective sialidase inhibition protected against Pa-induced MUC1-ED desialylation and shedding. In Pa-challenged mice, MUC1-ED-enriched bronchoalveolar lavage fluid (BALF) inhibited flagellin binding and Pa adhesion to human airway epithelia by up to 44% and flagellin-driven motility by >30%. Finally, Pa co-administration with recombinant human MUC1-ED dramatically diminished lung and BALF bacterial burden, proinflammatory cytokine levels, and pulmonary leukostasis and increased 5-day survival from 0% to 75%. We conclude that Pa flagellin provokes NEU1-mediated airway shedding of MUC1-ED, which functions as a decoy receptor protecting against lethal Pa lung infection.

Growth-Promoting and Antioxidant Effects of Magnolia Bark Extract in Chickens Uninfected or Co-Infected with Clostridium perfringens and Eimeria maxima as an Experimental Model of Necrotic Enteritis.

BACKGROUND: Magnolia tree bark has been widely used in traditional Asian medicine. However, to our knowledge, no studies have been reported investigating the effects of dietary supplementation with magnolia bark extract in chickens. OBJECTIVE: We tested the hypothesis that dietary supplementation of chickens with a Magnolia officinalis bark extract would increase growth performance in uninfected and Eimeria maxima/Clostridium perfringens co-infected chickens. METHODS: A total of 168 chickens were fed from hatch either a standard diet or a diet supplemented with 0.33 mg or 0.56 mg M. officinalis bark extract/kg (M/H low or M/H high, respectively) from days 1 to 35. At day 14, half of the chickens were orally infected with E. maxima, followed by C. perfringens infection at day 18 to induce experimental avian necrotic enteritis. Daily feed intake, feed conversion ratio, body weight gain, and final body weight were measured as indicators of growth performance. Serum α1-acid glycoprotein (AGP) concentrations were measured as an indicator of systemic inflammation, and intestinal lesion scores were determined as a marker of disease progression. Transcript levels for catalase, heme oxygenase 1, and superoxide dismutase in the intestine, liver, spleen, and skeletal muscle were measured as indicators of antioxidant status. RESULTS: Growth performance increased between days 1 and 35 in uninfected and E. maxima/C. perfringens co-infected chickens fed M/H-low or M/H-high diets compared with unsupplemented controls. Gut lesion scores were decreased, whereas AGP concentrations were unchanged, in co-infected chickens fed magnolia-supplemented diets compared with unsupplemented controls. In general, transcripts for antioxidant enzymes increased in chickens fed magnolia-supplemented diets compared with unsupplemented controls, and significant interactions between dietary supplementation and co-infection were observed for all antioxidant enzyme transcript levels. CONCLUSION: Magnolia bark extract might be useful for future development of dietary strategies to improve poultry health, disease resistance, and productivity without the use of antibiotic growth promoters.

Pseudomonas aeruginosa increases MUC1 expression in macrophages through the TLR4-p38 pathway.

Alveolar macrophages (AMs) play a critical role in the clearance of Pseudomonas aeruginosa (Pa) from the airways. However, hyper-activation of macrophages can impair bacterial clearance and contribute to morbidity and mortality. MUC1 mucin is a membrane-tethered, high molecular mass glycoprotein expressed on the apical surface of mucosal epithelial cells and some hematopoietic cells, including macrophages, where it counter-regulates inflammation. We recently reported that Pa up-regulates the expression of MUC1 in primary human AMs and THP-1 macrophages, and that increased MUC1 expression in these cells prevents hyper-activation of macrophages that appears to be important for host defense against severe pathology of Pa lung infection. The aims of this study were to elucidate the mechanism by which Pa increases MUC1 expression in macrophages. The results showed that: (a) Pa stimulation of THP-1 macrophages increased MUC1 expression both at transcriptional and protein levels in a dose-dependent manner; (b) Both Pa- and LPS-induced MUC1 expression in THP-1 cells were significantly diminished by an inhibitory peptide of TLR4; and (c) LPS-stimulated MUC1 expression was diminished at both the mRNA and protein levels by an inhibitor of the p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2, JNK, or IKK. We conclude that Pa-stimulated MUC1 expression in THP-1 macrophages is regulated mainly through the TLR4-p38 signaling pathway.

As human lung microvascular endothelia achieve confluence, src family kinases are activated, and tyrosine-phosphorylated p120 catenin physically couples NEU1 sialidase to CD31.

In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation. No increases in tyrosine phosphorylation of NEU1 or CD31 were detected. However, in postconfluent cells, we found increased tyrosine phosphorylation of a 120 kDa protein that was identified as p120 catenin (p120ctn). Prior silencing of c-src, fyn, or yes each reduced p120ctn phosphorylation. Prior knockdown of p120ctn prevented NEU1-CD31 association in both co-immunoprecipitation and pull-down assays. In these same assays, p120ctn associated with each of the four HPMEC-expressed SFKs as well as CD31 and NEU1. The CD31-p120ctn interaction was SFK-dependent whereas the NEU1-p120ctn interaction was not. Using purified recombinant binding partners in a cell-free system, direct protein-protein interactions between NEU1, CD31, and p120ctn were detected. Our combined data indicate that as HPMECs achieve confluence and CD31 ectodomains become homophilically engaged, multiple SFKs are activated to increase tyrosine phosphorylation of p120ctn, which in turn, functions as a cross-bridging adaptor molecule that physically couples NEU1 to CD31, permitting NEU1-mediated desialylation of CD31. These findings establish a SFK-driven, p120ctn-dependent mechanism for NEU1 recruitment to CD31.

The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50 > 800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the ß-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

Elevated expression of NEU1 sialidase in idiopathic pulmonary fibrosis provokes pulmonary collagen deposition, lymphocytosis, and fibrosis.

Idiopathic pulmonary fibrosis (IPF) poses challenges to understanding its underlying cellular and molecular mechanisms and the development of better therapies. Previous studies suggest a pathophysiological role for neuraminidase 1 (NEU1), an enzyme that removes terminal sialic acid from glycoproteins. We observed increased NEU1 expression in epithelial and endothelial cells, as well as fibroblasts, in the lungs of patients with IPF compared with healthy control lungs. Recombinant adenovirus-mediated gene delivery of NEU1 to cultured primary human cells elicited profound changes in cellular phenotypes. Small airway epithelial cell migration was impaired in wounding assays, whereas, in pulmonary microvascular endothelial cells, NEU1 overexpression strongly impacted global gene expression, increased T cell adhesion to endothelial monolayers, and disrupted endothelial capillary-like tube formation. NEU1 overexpression in fibroblasts provoked increased levels of collagen types I and III, substantial changes in global gene expression, and accelerated degradation of matrix metalloproteinase-14. Intratracheal instillation of NEU1 encoding, but not control adenovirus, induced lymphocyte accumulation in bronchoalveolar lavage samples and lung tissues and elevations of pulmonary transforming growth factor-ß and collagen. The lymphocytes were predominantly T cells, with CD8(+) cells exceeding CD4(+) cells by nearly twofold. These combined data indicate that elevated NEU1 expression alters functional activities of distinct lung cell types in vitro and recapitulates lymphocytic infiltration and collagen accumulation in vivo, consistent with mechanisms implicated in lung fibrosis.

Early Molecular Events in Murine Gastric Epithelial Cells Mediated by Helicobacter pylori CagA.

BACKGROUND: Murine models of Helicobacter pylori infection are used to study host-pathogen interactions, but lack of severe gastritis in this model has limited its usefulness in studying pathogenesis. We compared the murine gastric epithelial cell line GSM06 to the human gastric epithelial AGS cell line to determine whether similar events occur when cultured with H. pylori. MATERIALS AND METHODS: The lysates of cells infected with H. pylori isolates or an isogenic cagA-deficient mutant were assessed for translocation and phosphorylation of CagA and for activation of stress pathway kinases by immunoblot. RESULTS: Phosphorylated CagA was detected in both cell lines within 60 minutes. Phospho-ERK 1/2 was present within several minutes and distinctly present in GSM06 cells at 60 minutes. Similar results were obtained for phospho-JNK, although the 54 kDa phosphoprotein signal was dominant in AGS, whereas the lower molecular weight band was dominant in GSM06 cells. CONCLUSION: These results demonstrate that early events in H. pylori pathogenesis occur within mouse epithelial cells similar to human cells and therefore support the use of the mouse model for the study of acute CagA-associated host cell responses. These results also indicate that reduced disease in H. pylori-infected mice may be due to lack of the Cag PAI, or by differences in the mouse response downstream of the initial activation events.

Membrane-Tethered MUC1 Mucin Counter-Regulates the Phagocytic Activity of Macrophages.

MUC1 (MUC in human; Muc in animals) is a transmembrane mucin glycoprotein expressed in mucosal epithelial cells and hematopoietic cells. MUC1 is involved in the resolution of inflammation during airway Pseudomonas aeruginosa (Pa) infection by suppressing Toll-like receptor signaling in airway epithelial cells. Although alveolar macrophages are recognized as critical mediators of cell-mediated immunity against microorganisms inhaled into the airways, the role of MUC1 in regulating their response is unknown. The aims of this study were to determine whether macrophages express MUC1, and, if so, whether MUC1 expression might be associated with macrophage M0/M1/M2 differentiation or phagocytic activity. Human and mouse MUC1/Muc1 expression was drastically up-regulated in classically activated (M1) macrophages compared with nonactivated (M0) and alternatively activated (M2) macrophages. M1 polarization and Pa stimulation each increased MUC1 ectodomain shedding from the macrophage surface in a TNF-α-converting enzyme-dependent manner. MUC1/Muc1 deficiency in M0 macrophages increased adhesion and phagocytosis of Pa and Escherichia coli compared with MUC1/Muc1-expressing cells, and attenuation of phagocytosis by MUC1 was augmented after polarization into M1 macrophages compared with M0 macrophages. Finally, MUC1/Muc1 deficiency in macrophages increased reactive oxygen species production and TNF-α release in response to Pa compared with MUC1/Muc1-sufficient cells. These results indicate that MUC1/Muc1 expression by macrophages is predominantly in the M1 subtype, and that MUC1/Muc1 expression in these cells decreases their phagocytic activity in an antiinflammatory manner.

Pseudomonas aeruginosa stimulates tyrosine phosphorylation of and TLR5 association with the MUC1 cytoplasmic tail through EGFR activation.

BACKGROUND: MUC1 is a membrane-tethered mucin expressed on the surface of epithelial and hematopoietic cells. Previous studies have established that MUC1 attenuates airway inflammation in response to Pseudomonas aeruginosa (Pa) through suppression of Toll-like receptor (TLR) signaling. Here, we elucidate the mechanism through which the MUC1 cytoplasmic tail (CT) inhibits TLR5 signaling in response to Pa and its flagellin in primary normal human bronchial epithelial (NHBE) cells. METHODS: NHBE and human and mouse macrophages were stimulated with Pa or flagellin and transforming growth factor-α (TGF-α) and tumor necrosis factor-α (TNF-α) levels in cell culture supernatants were measured by ELISA. NHBE cells were stimulated with Pa, flagellin, or TNF-α and MUC1-CT, and epidermal growth factor receptor (EGFR) levels were measured by immunoblotting. NHBE cells were stimulated with Pa and MUC1-CT/TLR5 and MUC1-CT/EGFR association were detected by co-immunoprecipitation. RESULTS: Stimulation of NHBE cells with Pa and flagellin each increased release of the EGFR ligand, TGF-α, from NHBE cells. Both stimuli also activated EGFR tyrosine phosphorylation in these same cells. By contrast, stimulation of NHBE cells with Pa failed to induce TNF-α release, whereas stimulation of human or mouse macrophages with Pa promoted TNF-α release. Stimulation of NHBE cells with recombinant TNF-α increased both MUC1 and EGFR protein levels, and stimulation of these cells with Pa enhanced MUC1-CT tyrosine phosphorylation and increased MUC1-CT/TLR5 and MUC1-CT/EGFR protein association, in an EGFR-dependent manner. CONCLUSIONS: These results indicate that in response to Pa or flagellin, EGFR associates with and tyrosine phosphorylates MUC1-CT in primary NHBE cells, leading to increased MUC1-CT association with TLR5. Based on prior studies in tumor cells, increased MUC1-CT/TLR5 association in NHBE cells is predicted to competitively inhibit Pa/flagellin-stimulated TLR5 activation, reduce TLR5-dependent cell signaling, and down-regulate airway inflammation. Given that MUC1 is a universal suppressor of TLR signaling, the results from this study suggest that abnormal interactions between MUC1 and EGFR or TLRs may lead to the development of chronic inflammatory diseases. Thus, this is an important finding from the clinical point of view.

Dietary Capsicum and Curcuma longa oleoresins increase intestinal microbiome and necrotic enteritis in three commercial broiler breeds.

Three commercial broiler breeds were fed from hatch with a diet supplemented with Capsicum and Curcuma longa oleoresins, and co-infected with Eimeria maxima and Clostridium perfringens to induce necrotic enteritis (NE). Pyrotag deep sequencing of bacterial 16S rRNA showed that gut microbiota compositions were quite distinct depending on the broiler breed type. In the absence of oleoresin diet, the number of operational taxonomic units (OTUs), was decreased in infected Cobb, and increased in Ross and Hubbard, compared with the uninfected. In the absence of oleoresin diet, all chicken breeds had a decreased Candidatus Arthromitus, while the proportion of Lactobacillus was increased in Cobb, but decreased in Hubbard and Ross. Oleoresin supplementation of infected chickens increased OTUs in Cobb and Ross, but decreased OTUs in Hubbard, compared with unsupplemented/infected controls. Oleoresin supplementation of infected Cobb and Hubbard was associated with an increased percentage of gut Lactobacillus and decreased Selenihalanaerobacter, while Ross had a decreased fraction of Lactobacillus and increased Selenihalanaerobacter, Clostridium, Calothrix, and Geitlerinema. These results suggest that dietary Capsicum/Curcuma oleoresins reduced the negative consequences of NE on body weight and intestinal lesion, in part, through alteration of the gut microbiome in 3 commercial broiler breeds.

MUC1 regulates epithelial inflammation and apoptosis by PolyI:C through inhibition of Toll/IL-1 receptor-domain-containing adapter-inducing IFN-ß (TRIF) recruitment to Toll-like receptor 3.

MUC1/Muc1 (MUC1 in humans, Muc1 in animals) is a membrane-tethered mucin expressed by airway epithelial cells and plays an antiinflammatory role during airway bacterial infection. We previously demonstrated that MUC1/Muc1 is a negative regulator of Toll-like receptor (TLR) inflammatory signaling mediated through the myeloid differentiation primary response gene 88 (MyD88) adaptor protein. In the present study, we determined whether MUC1 regulates MyD88-independent TLR signaling mediated through the TLR3-Toll/IL-1 receptor-domain-containing adapter-inducing IFN-ß (TRIF) pathway in response to poly(I:C). Compared with MUC1/Muc1-expressing controls, cells deficient in MUC1/Muc1 were more prone to poly(I:C)-induced apoptosis; had increased poly(I:C)-driven activation of caspase-3, caspase-8, IFN regulatory factor-3, and NF-κB; and displayed heightened IFN-ß gene expression. MUC1 overexpression by these cells had the opposite effects. Reciprocal coimmunoprecipitation experiments established constitutive TLR3/MUC1-CT (cytoplasmic tail) protein interaction in human embryonic kidney (HEK)293T cells overexpressing the two proteins and in lung epithelial cells expressing the endogenous proteins, the latter of which was confirmed by immunofluorescence colocalization of TLR3 with MUC1-CT. Coimmunoprecipitation studies also revealed that MUC1 overexpression by HEK293T cells reduced poly(I:C)-induced TLR3/TRIF protein interaction. Finally, MUC1 overexpression had no effect on TRIF-dependent auto-activation of TLR3 signaling, suggesting that the site of action of the MUC1-CT in TLR3 signaling is not downstream of TRIF. These data indicate that MUC1-CT counter-regulates apoptotic and inflammatory responses of airway epithelial cell through constitutive association with TLR3, thereby inhibiting poly(I:C)-induced recruitment of TRIF to TLR3.

IL-17A regulates Eimeria tenella schizont maturation and migration in avian coccidiosis.

Although IL17A is associated with the immunological control of various infectious diseases, its role in host response to Eimeria infections is not well understood. In an effort to better dissect the role of IL17A in host-pathogen interactions in avian coccidiosis, a neutralizing antibody (Ab) to chicken IL17A was used to counteract IL17A bioactivity in vivo. Chickens infected with Eimeria tenella and treated intravenously with IL17A Ab, exhibited reduced intracellular schizont and merozoite development, diminished lesion score, compared with untreated controls. Immunohistological evaluation of cecal lesions in the parasitized tissues indicated reduced migration and maturation of second-generation schizonts and reduced lesions in lamina propria and submucosa. In contrast, untreated and infected chickens had epithelial cells harboring second-generation schizonts, which extend into the submucosa through muscularis mucosa disruptions, maturing into second generation merozoites. Furthermore, IL17A Ab treatment was associated with increased parameters of Th1 immunity (IL2- and IFNγ- producing cells), reduced levels of reactive oxygen species (ROS), and diminished levels of serum matrix metalloproteinase-9 (MMP-9). Finally, schizonts from untreated and infected chickens expressed S100, Wiskott-Aldrich syndrome protein family member 3 (WASF3), and heat shock protein-70 (HSP70) proteins as merozoites matured, whereas the expression of these proteins was absent in IL17A Ab-treated chickens. These results provide the first evidence that the administration of an IL17A neutralizing Ab to E. tenella-infected chickens inhibits the migration of parasitized epithelial cells, markedly reduces the production of ROS and MMP-9, and decreases cecal lesions, suggesting that IL17A might be a potential therapeutic target for coccidiosis control.

NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.

Genetic regulation of MUC1 expression by Helicobacter pylori in gastric cancer cells.

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.

Immune effects of dietary anethole on Eimeria acervulina infection.

The effects of anethole on in vitro and in vivo parameters of chicken immunity during experimental avian coccidiosis were evaluated. Anethole reduced the viability of invasive Eimeria acervulina sporozoites after 2 or 4 h of treatment in vitro by 45 and 42%, respectively, and stimulated 6.0-fold greater chicken spleen cell proliferation compared with controls. Broiler chickens continuously fed from hatch with an anethole-supplemented diet and orally challenged with live E. acervulina oocysts showed enhanced BW gain, decreased fecal oocyst excretion, and greater E. acervulina profilin antibody responses compared with infected chickens given an unsupplemented standard diet. The levels of transcripts encoding the immune mediators IL6, IL8, IL10, and tumor necrosis factor ligand superfamily member 15 (TNFSF15) in intestinal lymphocytes were increased in E. acervulina-infected chickens fed the anethole-containing diet compared with untreated controls. Global gene expression analysis by microarray hybridization identified 1,810 transcripts (677 upregulated, 1,133 downregulated) whose levels were significantly altered in intestinal lymphocytes of anethole-fed birds compared with unsupplemented controls. From this transcriptome, 576 corresponding genes were identified. The most significant biological function associated with these genes was "Inflammatory Response" in the "Disease and Disorders" category. This new information documents the immunologic and genomic changes that occur in chickens following anethole dietary supplementation that may be relevant to host protective immune response to avian coccidiosis.

Parasiticidal activity of a novel synthetic peptide from the core α-helical region of NK-lysin.

NK-lysin is an anti-microbial peptide that plays a critical role in innate immunity against infectious pathogens through its selective membrane disruptive property. We previously expressed and purified a full-length chicken NK-lysin (cNKL) recombinant protein, and demonstrated its in vitro anti-parasitic activity against the apicomplexan protozoan, Eimeria, the etiologic agent of avian coccidiosis. This study evaluated the in vitro and in vivo anti-parasitic properties of a synthetic peptide (cNK-2) incorporating a predicted membrane-permeating, amphipathic α-helix of the full-length cNKL protein. The cNK-2 peptide exhibited dose- and time-dependent in vitro cytotoxic activity against E. acervulina and E. tenella sporozoites. The cytotoxic activity of 1.5 µM of cNK-2 peptide against E. acervulina following 6h incubation was equal to that of 2.5 µM of melittin, the principal active component of apitoxin (bee venom) that also exhibits anti-microbial activity. Even greater activity was detected against E. tenella, where 0.3 µM of cNK-2 peptide was equivalent to 2.5 µM of melittin. Against Neospora caninum tacyzoites, however, the cytotoxic activity of cNK-2 peptide was inferior to that of melittin. Transmission electron microscopy of peptide-treated E. tenella sporozoites revealed disruption of the outer plasma membrane and loss of intracellular contents. In vivo administration of 1.5 µM of cNK-2 peptide increased protection against experimental E. acervulina infection, as measured by greater body weight gain and reduced fecal oocyst shedding, compared with saline controls. These results suggest that the cNK-2 synthetic peptide is a novel anti-infective peptide that can be used for protection against avian coccidiosis during commercial poultry production.

Dietary supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis.

The Clostridium-related poultry disease, necrotic enteritis (NE), causes substantial economic losses on a global scale. In the present study, a mixture of two plant-derived phytonutrients, Capsicum oleoresin and turmeric oleoresin (XT), was evaluated for its effects on local and systemic immune responses using a co-infection model of experimental NE in commercial broilers. Chickens were fed from hatch with a diet supplemented with XT, or with a non-supplemented control diet, and either uninfected or orally challenged with virulent Eimeria maxima oocysts at 14 d and Clostridium perfringens at 18 d of age. Parameters of protective immunity were as follows: (1) body weight; (2) gut lesions; (3) serum levels of C. perfringens α-toxin and NE B-like (NetB) toxin; (4) serum levels of antibodies to α-toxin and NetB toxin; (5) levels of gene transcripts encoding pro-inflammatory cytokines and chemokines in the intestine and spleen. Infected chickens fed the XT-supplemented diet had increased body weight and reduced gut lesion scores compared with infected birds given the non-supplemented diet. The XT-fed group also displayed decreased serum α-toxin levels and reduced intestinal IL-8, lipopolysaccharide-induced TNF-α factor (LITAF), IL-17A and IL-17F mRNA levels, while cytokine/chemokine levels in splenocytes increased in the XT-fed group, compared with the animals fed the control diet. In conclusion, the present study documents the molecular and cellular immune changes following dietary supplementation with extracts of Capsicum and turmeric that may be relevant to protective immunity against avian NE.

Comparison of live Eimeria vaccination with in-feed salinomycin on growth and immune status in broiler chickens.

Coccidiosis vaccines and anticoccidial drugs are commonly used to control Eimeria infection during commercial poultry production. The present study was conducted to compare the relative effectiveness of these two disease control strategies in broiler chickens in an experimental research facility. Birds were orally vaccinated with a live, attenuated vaccine (Inovocox), or were provided with in-feed salinomycin (Bio-Cox), and body weights, serum levels of nitric oxide (NO) and antibodies against Eimeria profilin and Clostridium perfringens PFO proteins, and intestinal levels of cytokine gene transcripts were measured. Vaccinated chickens had increased body weights, greater NO levels, and higher profilin and PFO antibody levels compared with salinomycin-fed birds. Transcripts for interleukin-6 (IL-6), tumor necrosis factor superfamily 15, and interferon-γ were increased, while mRNAs for IL-4 and IL-10 were decreased, in immunized chickens compared with salinomycin-treated chickens. In conclusion, vaccination against avian coccidiosis may be more effective compared with dietary salinomycin for increasing body weight and augmenting pro-inflammatory immune status during commercial poultry production.

Suppression of IL-8 production in gastric epithelial cells by MUC1 mucin and peroxisome proliferator-associated receptor-γ.

MUC1 is a membrane-tethered mucin expressed on the apical surface of epithelial cells. Our previous report (Guang W, Ding H, Czinn SJ, Kim KC, Blanchard TG, Lillehoj EP. J Biol Chem 285: 20547-20557, 2010) demonstrated that expression of MUC1 in AGS gastric epithelial cells limits Helicobacter pylori infection and reduces bacterial-driven IL-8 production. In this study, we identified the peroxisome proliferator-associated receptor-γ (PPARγ) upstream of MUC1 in the anti-inflammatory pathway suppressing H. pylori- and phorbol 12-myristate 13-acetate (PMA)-stimulated IL-8 production. Treatment of AGS cells with H. pylori or PMA increased IL-8 levels in cell culture supernatants compared with cells treated with the respective vehicle controls. Prior small interfering (si)RNA-induced MUC1 silencing further increased H. pylori- and PMA-stimulated IL-8 levels compared with a negative control siRNA. MUC1-expressing AGS cells pretreated with the PPARγ agonist troglitazone (TGN) had reduced H. pylori- and PMA-stimulated IL-8 levels compared with cells treated with H. pylori or PMA alone. However, following MUC1 siRNA knockdown, no differences in IL-8 levels were seen between TGN/H. pylori and H. pylori-only cells or between TGN/PMA and PMA-only cells. Finally, TGN-treated AGS cells had increased Muc1 promoter activity, as measured using a Muc1-luciferase reporter gene, and greater MUC1 protein levels by Western blot analysis, compared with vehicle controls. These results support the hypothesis that PPARγ stimulates MUC1 expression by AGS cells, thereby attenuating H. pylori- and PMA-induced IL-8 production.