Veterinary Parasitology 165 (2009) 281–289

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Veterinary Parasitology

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Mucin biosynthesis in the bovine goblet cell induced by oncophora infection

Robert W. Li a,*, Congjun Li a, Theodore H. Elsasser a, George Liu a, Wesley M. Garrett b, Louis C. Gasbarre a a Bovine Functional Genomics Laboratory, and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705, USA b Animal Biosciences and Biotechnology, Animal and Natural Resources Institute, USDA-ARS, Beltsville, MD 20705, USA

ARTICLE INFO ABSTRACT

Article history: hypersecretion is considered to be one of the most common components of the Received 26 March 2009 immune response to gastrointestinal infection. However, investigations have not Received in revised form 6 July 2009 been conducted in the Cattle–Cooperia oncophora system to verify the findings largely Accepted 7 July 2009 derived from murine models. In this study, we examined the expression of seven and seven enzymes in the mucin biosynthesis pathway involved in O-linked glycosylation in the Keywords: bovine small intestine including goblet cells enriched using laser capture microdissection Laser capture microdissection (LCM) during a primary C. oncophora infection. At the mRNA level, MUC2 expression was Goblet cells significantly higher in both lamina propria and goblet cells at 28 days post-infection Mucin Cooperia oncophora compared to the naı¨ve control. MUC5B expression at the mRNA level was also higher in Cattle lamina propria at 28 dpi. Expression of MUC1, MUC4, MUC5AC, and MUC6 was extremely Nematode low or not detectable in goblet cells, columnar epithelial cells, and lamina propria from both (GI) naı¨ve control and infected . Among the seven enzymes involved in post-translational O-linked glycosylation of mucins, GCNT3, which may represent one of the key rate-limiting steps in mucin biosynthesis, was up-regulated in goblet cells, columnar epithelial cells, lamina propria, and gross small intestine tissue during the course of infection. Western blot analysis revealed that MUC2 was strongly induced by infection in both gross small intestine tissue and its mucosal layer. In contrast, the higher MUC5B expression was observed only in the mucosal layer. Immunohistochemistry provided further evidence of the mucin glycoprotein production and localization. Our results provided insight into regulation of mucin biosynthesis in various cell types in the bovine small intestine during gastrointestinal nematode infection and will facilitate our understanding of mucins and their role in immune response against parasitic . Published by Elsevier B.V.

1. Introduction industry with an estimated annual cost in excess of $2 billion per year in lost productivity and increased operating Gastrointestinal nematodes have been ranked among expenses. At least 41 different species of nematodes were the top three diseases or conditions that may have a reported to reside in the bovine gastrointestinal (GI) tract significant economic impact on the American cattle with predilection sites in the abomasum, small intestine, and large intestine. Cooperia oncophora is one of the most important gastrointestinal nematodes infecting cattle in the * Corresponding author at: Bovine Functional Genomics Laboratory, temperate regions of the world and its infection has been Animal and Natural Resources Institute, USDA-ARS, BARC East, Building shown to reduce weight gain up to 13.5% of total cattle 200, Room 16, Beltsville, MD 20705, USA. Tel.: +1 301 504 5185; fax: +1 301 504 8414. bodyweights (Coop et al., 1979). The manifestations of host E-mail address: [email protected] (R.W. Li). immunity against C. oncophora infection have long been

0304-4017/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.vetpar.2009.07.008 282 R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 observed and include reduced female fecundity and arrested 2. Materials and methods parasite development. Additionally, certain Cooperia species are becoming more important to American cattle operations 2.1. Animals and parasitology due to the increasing reports of resistance to macrocyclic Six Holstein bull calves used in this study were obtained lactone by this parasite genus (unpublished). within 2–3 days after birth; and after weaning, these calves C. oncophora infection results in local inflammation and were fed ad lib with standard calf diet supplemented with elicits a Th2-like immune response, characterized by up- hay and maintained on concrete for the duration of the regulation of IL-4 and the involvement of both eosinophils experiment. Oral infection with C. oncophora infective L3 and mucosal IgA (Gasbarre et al., 2001; Kanobana et al., larvae (105 larvae per animal) was initiated after calves 2002). Host serological response to C. oncophora experi- reached approximately 3 months of age and infection was mental infection has been extensively studied (Kanobana allowed to progress for 28 days. Age-matched, uninfected et al., 2001, 2002; Nieuwland et al., 1995). Expulsion of the naı¨ve animals were used as controls. The animal main- adult Cooperia worm appeared to be associated with a tenance and handling were based on the protocol approved significant increase in mucosal IgA and an influx of by The USDA-ARS Animal Care and Use Committee eosinophils (Kanobana et al., 2002). PIGR, a responsible following Institutional Animal Care and Use Committees for trans-epithelial transport of polymeric immunoglobu- (IACUC) guidelines. At the end of the experiment, small lins, such as IgA dimers, into mucosal secretions, was intestine samples (SI) were collected from the jejunum strongly induced in the heifers resistant to parasitic approximately 3 m from the pyloric sphincter and snap nematodes after experimental challenge (Li et al., 2007). frozen in liquid nitrogen prior to storage at 80 8C until As a group of high molecular weight total RNA was extracted. Tissues were also stored in OCT consisting of a mucin peptide backbone and O-linked compound (Sakura Inc., Torrance, CA, USA) for cryo- oligosaccharides, mucins are the major protein compo- sectioning. Fecal egg count (EPG) was determined by nents of the protective mucus barrier that cover epithelial double centrifugation using zinc sulfate and parasite surfaces in the GI tract. This barrier is considered a first line burden from the entire small intestine was determined of defense against colonization by gut pathogens. Success- as previously described (Li et al., 2007). ful penetration of the mucus barrier allows pathogens to invade the host. By contrast, the hosts could alter the 2.2. Histology, eosinophil counts, and laser capture structure of their mucins as a form of active immune microdissection (LCM) response against pathogens. Gastrointestinal nematode infection has been shown to induce goblet cell hyperplasia The tissues were preserved in OCT and kept at 80 8C and mucus hypersecretion. Expression of (MUC2) until cryo-sectioning. The tissues were sectioned at 6– and trefoil factor family 3 (TFF3) at mRNA levels was up- 7 mM and eosinophils were enumerated on H&E stained regulated as early as 2–4 dpi in the rat intestinal slides. A total of 10 image fields (400) were counted for epithelium during Nippostrongylus brasiliensis infection, each sample. The eosinophil data were analyzed using the suggesting these may be associated with an early unpaired t-test. innate protective response (Yamauchi et al., 2006). The Laser capture microdissection was carried out using a expression of these two genes was induced similarly in Leica AS LMD system (Leica Microsystems, Wetzlar, both euthymic and athymic rats at 7 and/or 10 dpi, Germany). Briefly, frozen tissues embedded in OCT blocks indicating the regulation of these genes was independent were sectioned at 7 mM; and the sections were then of thymus-derived T cells (Kawai et al., 2007). Immune- mounted on laser-penetrable PEN-Membrane slides mediated changes in secretion of gastric mucins were (Leica). After H&E staining, the cells of interest were responsible, at least partially, for expulsion of this dissected and collected using the Leica AS LMD system. nematode species. Enhanced goblet cell hyperplasia and This system allows the dissected individual cells to fall into increased mucin production were also observed in the rat– the lid of a collection tube by gravity. On average, a total of Hymenolepis diminuta (Webb et al., 2007), sheep–Hae- 900–1000 individual cells were collected per sample for monchus contortus (Newlands et al., 1990), and mouse– subsequent RNA extraction and amplification. Trichinella spiralis as well as many other host–parasite systems (Theodoropoulos et al., 2001). However, much of 2.3. RNA extraction, amplification, and real-time RT-PCR our knowledge about mucins and their regulation during parasitic nematode infection is derived from murine Total RNA extraction from the gross tissue was pre- models. Mucins and their roles in innate immune response viously described (Li and Li, 2006). Briefly, total RNA was in cattle during C. oncophora infection have not been extracted using Trizol (Invitrogen, Carlsbad, CA) and further characterized. In this study, we examined cell-specific purified using an RNeasy Mini kit (Qiagen, Valenica, CA). expression of seven mucins and seven enzymes involved in After cells of interest were successfully isolated using O-linked glycosylation in mucin biosynthesis at mRNA the Leica LCM system from frozen sections, total RNA was levels using laser capture microdissection (LCM). Some extracted using a RNeasy Micro Kit (Qiagen). RNA integrity results were further verified by Western blot analysis and was verified using a Bioanalyzer and RNA Pico chips immunohistochemistry. These studies may provide insight (Agilent, Palo Alto, CA). We were generally able to obtain into the roles that mucins play in protective immunity 22–24 ng of high-quality total RNA from 900 to 1000 LCM against nematode infection in the bovine gastrointestinal isolated cells, i.e., approximately 24–26 pg of total RNA per tract. cell, in good agreement with previously published reports R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 283 that a typical mammalian cell contains 10–30 pg of scraping frozen small intestine surfaces with a microscope total RNA. Extracted total RNA from the LCM isolated glass slide and immediately freezing in liquid nitrogen. cells was subjected to RNA amplification using the Ground gross tissue powder and frozen mucosal layers WT-OvationTM Pico System (NuGen Technologies, San were placed (1:5, w/v) in Mammalian Protein Extraction Carlos, CA). This system uses a linear isothermal protocol Reagent (M-PER, Pierce, Rockford, IL, USA) with a protease and amplification is initiated both at the 30 end and inhibitor cocktail (Sigma, St. Louis, MO) added prior to use. randomly throughout the whole transcriptome to enable Samples were then homogenized using a Polytron (Fisher amplification of non-poly(A) transcripts and compro- Scientific) for 20 s at 4 8C and the obtained homogenates mised RNA samples. Using this system, we typically were briefly centrifuged at 4 8C for 2 min at 16,100 g to obtained 6–10 mg of amplified cDNA from 6 to 8 ng of remove debris. initial input total RNA for subsequent real-time RT-PCR Protein quantification, SDS-PAGE and Western Blot analysis. analysis were described previously (Li and Li, 2006). For real-time RT-PCR, cDNA synthesis was performed Briefly, the protein from different samples was separated with an iScript cDNA Synthesis kit (BioRad, Hercules, CA). by SDS-PAGE on two identical 4–20% polyacrylamide Real-time RT-PCR analysis was carried out with the iQ gradient gels. One gel was stained with SimpleBlue SYBR Green Supermix kit (Biorad) using 200 nM of each (Invitrogen) and one was transferred to a membrane and amplification primer (Table 1) and 20 ng of amplified probed with anti-MUC2 (sc-15334), anti-MUC5AC (sc- cDNA in a 25 ml reaction volume as described (Li et al., 59951), and anti-MUC5B (sc-20119) antibodies (Santa 2006). The amplification was carried out on an iCycler Cruz Biotechnology, Santa Cruz, CA, USA). The relative iQTM Real Time PCR Detection System (BioRad) with the densities of the target bands on the Western Blots following profile: 95 8C60s;40cyclesof948C15s,608C were quantified using the imaging software UN-SCAN-IT 30 s, and 72 8C 30 s. A melting curve analysis was (Silk Scientific, Orem, UT, USA). The data were then performed for each primer pair. Standards for each gene statistically analyzed using one-way analysis of variance were prepared from PCR products purified using the (ANOVA). QIAquick purification kit (Qiagen). The expression levels were determined from a standard curve of known target 2.5. Immunohistochemistry cDNA copy numbers (1.0 102 to 1.0 107 molecules), which was analyzed simultaneously with the experi- Protein expression of three secreted mucins, MUC2, mental samples. MUC5AC, and MUC5B was assessed by immunohisto- chemistry using rabbit or mouse ABC Staining System 2.4. Western blot analysis (Santa Cruz Biotechnology). Small intestine samples from the jejunum approximately 3 m from the pyloric sphincter Crude were extracted from bovine small were collected and fixed in 10% formaldehyde and intestine (gross tissue) and its mucosal layer. Small embedded in paraffin. Tissue sections (5 mM) were intestine samples from the jejunum approximately 3 m processed according to a standard protocol, and depar- from the pyloric sphincter were flushed through with affinized and hydrated. The slides were then incubated for chilled PBS, and the mucosal surface was exposed by 10 min in 1% H2O2 to quench endogenous peroxidase longitudinal dissection. The mucosal layer was obtained by activity. After an hour incubation in 1.5% blocking serum,

Table 1 Primers used in the study.

Accession Annotation Gene Forward primer Reverse Primer symbol

XM_613170 Alpha-1,4-N-acetylglucosaminyltransferase A4GNT CCAGCCCTTTCCCTCCTCTC GACACCGCCATACTTCCAGATG NM_001098473 UDP-GlcNAc:betaGal B3GNT3 GGAGGGCTTGATGAGTGAATC GGAATGGATGTAGCAGCAGTAG beta-1,3-N-acetylglucosaminyltransferase 3 NM_001103307 UDP-GlcNAc:betaGal B3GNT6 TTGTGAACCGAACCTCCATTG GCTCAGTCCTTCTTGTCCATC beta-1,3-N-acetylglucosaminyltransferase 6 (core 3 synthase) NM_177510 Glucosaminyl (N-acetyl) transferase 1, GCNT1 TGTTTGGACGAGCATCTAAGG TGGATTCTGGAGGCGGAAG core 2 (beta-1,6-N-acetylglucosaminyltransferase) NM_001101227 Glucosaminyl (N-acetyl) transferase 2, GCNT2 CCTTCACCGATGCGTTCC CCCAAGTGTCTGAGTTATTTAGC I-branching enzyme (I blood group) NM_205809 Glucosaminyl (N-acetyl) transferase 3, mucin type GCNT3 CCTGGCTCTGTTCCTTAC GAGGTGATGGTTCTGGAG XM_001250805 Glucosaminyl (N-acetyl) transferase 4, GCNT4 ACTTCTGGGCTACCTTAATTCG CCGCTGCTCCATAGATACAC core 2 (beta-1,6-N-acetylglucosaminyltransferase) NM_174115 Mucin 1 MUC1 AAGGCACAGTTCAGTCAG GGCAATGAGATAGATGATGG XM_001256289 Mucin 2 MUC2 CCGTCGTGAGGGAGATTTC CGAGCACTTGTGGTCCAG XR_028426 MUC4 GACACTAAGGACATCACATTCAC CCTGGTCACTGCTTGCTAG XM_604045 Mucin 5AC MUC5AC CTCTGCCCACCTCTTCTACC CACCACCACGAGCCCATC XM_001256219 MUC5B ACTGTGCGGTGTGAGGAG GTTGTCCACGAGGCTGTTG XM_870967 MUC6 CGCACAGAGGGAAGCAAAG CGTTTATTTGTCACCCGTTCAC NM_176631 Mucin 15 MUC15 TGGTTCTAGCACTCTAAGGAAG CCAGCACTCTGATACCTCTC 284 R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 the sections were incubated overnight at 4 8C with the following primary antibodies: Mucin 2 (H-300), sc-15334; Mucin 5B (H-300): sc-20119; and Mucin 5AC (2-11M1): sc-59951 (Santa Cruz Biotechnology). After three washes in PBS, the slides were incubated for 30min at room temperature with biotinylated secondary antibodies (anti-rabbit or anti-mouse IgG-B). Following three 5 min washes in PBS, slides were incubated for 30 min with AB enzyme reagent. The slides were washed three times in PBS and subsequently incubated in 1–3 drops of peroxide substrate for 5 min for optimal stain intensities. Slides were then counterstained with hematoxylin for 5 s, dehydrated and mounted with permanent mounting medium under cover slips for observation using light microscopy.

3. Results

3.1. Parasite burden and eosinophil counts

Total parasites including both male and female as well as larvae recovered from the small intestine at 28 dpi ranged from 22,950 to 29,450. These numbers represented approximately 23–29% of the total L3 larvae innoculated, which is consistent with previous published observations (Coop et al., 1979). The number of eosinophils in the bovine small intestine followed the general trend observed during Fig. 1. The expression profiles of mucins in goblet and columnar epithelial helminth parasite infection. The naı¨ve control group cells in the bovine small intestine detected using real-time RT-PCR during Cooperia oncophora infection. Mean fold changes (N = 4) were presented averaged 23 eosinophils per graticule field in the lamina with SEM as error bars. The expression value of the naı¨ve control samples propria while the 28 dpi group was significantly higher was set as 1.0. (A) MUC2 and (B) MUC5B. *Statistically significant at (P = 0.02) at 54 eosinophils per field. As expected, P < 0.05. expression of both IL-4 and IL-13 were up-regulated whereas TNFa and IFN-g were unchanged in the bovine control (Fig. 1). The highest up-regulation of MUC2 (15- small intestine by infection (data not shown), which is fold) and MUC5B (3-fold) mRNA was observed in lamina consistent with previously published data observed in propria at 28 dpi, which suggested that other cell types other host–parasite systems (Webb et al., 2007). The (rather than epithelial cells) contributed to significant up- pattern of IL-5 expression was almost identical to that of regulation of these two mucins by infection. In the gross eosinophil infiltration during the course of infection. The small intestine tissue, these mucins were detectable but number of eosinophils and IL-5 expression was strongly remained unchanged from uninfected naı¨ve controls. correlated with R2 = 0.734. 3.3. Expression of the genes involved in O-linked 3.2. Gene expression of mucins glycosylation

Expression of seven mucin genes was examined using The mRNA levels of seven enzymes involved in real-time RT-PCR in various cell types during C. oncophora glycosylation of mucin core peptides, including A4GNT, infection. These mucins included four secretory mucins, B3GNT3, B3GNT6, GCNT1, GCNT2, GCNT3, and GCNT4, MUC2, MUC5AC, MUC5B, and MUC6, as well as three cell were examined in this study. Expression of A4GNT was not surface-associated mucins, MUC1, MUC4, and MUC15. In detectable in both columnar epithelial cells and lamina goblet cells, mRNA levels of MUC1, MUC5AC, MUC4, MUC6, propria and was extremely low in goblet cells and whole and MUC15 were extremely low or not detectable (40Ct) in small intestine tissue. Expression of B3GNT3 was up- both naı¨ve and infected animals. MUC2 transcripts were regulated approximately fivefold in columnar epithelial abundant in goblet cells and up-regulated approximately cells whereas it was otherwise down-regulated in goblet twofold during infection (Fig. 1). Similarly, MUC5B expres- cells, lamina propria, and gross tissue by infection sion was low but detectable in goblet cells; however, its (Table 2). GCNT3 expression was strongly up-regulated expression was largely unchanged or slightly down- by infection in both columnar epithelial cells (38-fold) regulated by infection. and goblet cells (20-fold) (Table 2) as well as in the gross The expression patterns of these seven mucins were tissue. However, its mRNA level was unchanged in lamina similar in columnar epithelial cells, with only MUC2 and propria, suggesting the regulation of this gene may be MUC5B being detectable. MUC2 mRNA expression was epithelium-specific. Strong up-regulation of GCNT3 by induced whereas MUC5B was slightly down-regulated in infection started as early as 7 dpi and remained throughout columnar epithelial cells at 28 dpi comparing to the naı¨ve the infection until 42 dpi (Li and Gasbarre, 2009), when the R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 285

Table 2 Copy numbers detected using real-time RT-PCR (mean SEM)a.

B3GNT3 GCNT3

Control 28 dpi Control 28 dpi

Columnar epithelial 3231 3047 15,327 14,243 903 896 34,191 33,380 Goblet cell 36,045 29,850 21,688 19,479 535 483 10,736 10,335 Lamina propria 3679 2890 3015 1680 996 991 1360 1359 SI tissue 5168 2613 3415 1237 1046 345 8550 5961 a Copy numbers in 20 ng of both amplified RNA and total RNA from small intestine (SI, gross tissue). Assuming that the typical amount of total RNA in mammalian cells is 30 pg/cell, 20 ng of total RNA 667 cells.

vast majority of the parasites were already expulsed from failure of gene expression due solely to technical aspects the GI tract. Transcript levels of the remaining four (e.g., primers unable to work) can be ruled out. enzymes including GCNT4 were detected in epithelia, lamina propria, as well as the gross tissue but were not 3.4. Protein expression and immunohistochemistry inducible by infection. All primers used in this experiment were specifically designed based on published bovine gene Protein expression of three secretory mucins, MUC2, sequences. RNA samples in which these genes are known MUC5AC, and MUC5B, was examined using Western blot to be expressed were included on the same real-time PCR analysis (Figs. 2 and 3) and immunohistochemistry (Fig. 4). plates as positive controls. Therefore, the possibility that a Consistent with the real-time RT-PCR results, MUC5AC expression at the protein level was not detectable by Western blot or immunohistochemistry. As depicted in Fig. 2, MUC2 protein was higher in gross small intestine tissue as well as in its mucosal layer at 28 dpi compared to

Fig. 2. Western blot analysis of MUC2 in the whole small intestine tissue Fig. 3. Protein expression of MUC5B in the whole small intestine tissue (jejunum) and the mucosal layer after Cooperia oncophora infection for 28 (jejunum) and the mucosal layer after Cooperia oncophora infection for 28 days. The antibody used was rabbit anti-MUC2 (sc-15334) from Santa days by dot blot analysis. The antibody used was rabbit anti-MUC5B (sc- Cruz Biotechnology. The relative densities of the target bands were 20119) from Santa Cruz Biotechnology. The relative densities of the target qualified using UN-SCAN-IT from Silk Scientific. bands were qualified using UN-SCAN-IT from Silk Scientific. 286 R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289

Fig. 4. Immunoperoxidase staining of formalin fixed, paraffin-embedded jejunum (3 m from the pyloric sphincter) of Holstein calves after Cooperia oncophora infection (magnification: 20). (A) Naı¨ve control, MUC2, (B) infected for 28 days, MUC2, (C) naı¨ve control, MUC5B and (D) infected for 28 days, MUC5B. the naı¨ve control. Similarly, MUC5B protein expression to clump in vitro and may cause a significant reduction in was slightly up-regulated in the mucosal layer at 28 dpi larval establishment in naive sheep (Harrison et al., 1999), (Fig. 3). although the molecular nature of this activity remains unclear. 4. Discussion MUC6, MUC2, MUC5AC, and MUC5B, which encode secreted gel-forming mucins, are clustered on Mucins are the major protein components of the 11p15 (Vincent et al., 2007) and appear to be protective mucus barrier that cover epithelial surfaces in evolutionarily related. This orientation of localization the GI tract and have long been implicated in human health appears to be preserved in cattle on bovine chromosome and diseases. Mucins and their O-linked oligosaccharides BTA29. As a major mucin in the GI tract, MUC2 production also have important normal physiological functions. was strongly up-regulated by parasite T. spiralis in mice Secretory and membrane mucins contribute to mucocilli- (Shekels et al., 2001) and the amount of MUC2 mRNA ary defense, an innate immune defense system that synthesized displayed a strong significant negative corre- protects the airways against pathogens and environmental lation with the biomass of the cestode H. diminuta (Webb toxins (Rose and Voynow, 2006). It has long been et al., 2007). It has long been known that the amount of suggested that mucins may be responsible for accelerated MUC2 synthesized was correlated with altered biological intestinal nematode expulsion in rats by enveloping the properties and tumor status such as metastatic potentials parasites and interrupting cell adhesion (Ishikawa et al., in colon carcinoma cells (Bresalier et al., 1991). In the 1994). Initial host immune response resulted in adult present study, we were able to detect only two of the seven parasites being damaged, which in turn induced the mucins tested, MUC2 and MUC5B, in the bovine small alteration of oligosaccharide residues of mucins. The intestine of both normal and infected cattle. At the mRNA expression of such altered mucins was highly effective level, MUC2 was strongly induced in the lamina propria not only in causing expulsion of established damaged after infection. The induction was mild (twofold) but still worms but also in preventing establishment of normal statistically significant (P < 0.05) in goblet cells. This cell- worms as a selective barrier with the ability to distinguish specific expression pattern suggested that while MUC2 parasite species. In addition, mucus obtained from sheep protein (MUC2 apomucin or peptide backbone plus O- immunized by multiple truncated infections caused the linked glycans) may be secreted from goblet cells, the bulk parasitic nematode Trichostrongylus colubriformis L3 larvae of MUC2 apomucin could be synthesized elsewhere and R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 287 transported to goblet cells in order to complete O-linked glycoproteins (Shiraishi et al., 2001). As one of many glycosylation and secretion. The only enzyme responsible genes involved in poly-N-acetyllactosamine biosynthesis, for synthesis of all known b6 N-acetylglucosaminides in B3GNT3 is located mainly in the GI tract, colon, jejunum, mucins, GCNT3, was strongly up-regulated in goblet cells stomach, and esophagus, as well as pancreas and placenta as well as columnar epithelial cells (Table 2). At the protein in (Shiraishi et al., 2001). B3GNT6 encodes an level, MUC2 was also strongly induced by infection. enzyme synthesizing the core 3 structure of O-glycans, MUC5B mRNA levels were largely unchanged in goblet which can be found in mucins from tissues mainly in the cells; and its expression was actually down-regulated GI tract (Iwai et al., 2002). Moderate mRNA abundance in slightly in columnar epithelial cells. MUC5B protein was goblet cells and their readiness to be induced by parasitic also slightly down-regulated in the tissue by infection, nematode C. oncophora suggest that both B3GNT3 and suggesting that normal physiological functions of MUC5B B3GNT6 are likely to be responsible for O-linked could be impaired during infection. glycosylation of secreted mucins in the bovine small O-Linked glycan biosynthesis is a very complicated intestine. process, which starts with the biosynthesis of N-acetylga- GCNT3, via catalyzing formation of all three branching lactosamine (GalNAc). Various glycosyltransferases cata- structures, including core 2, core 4, and blood group I, lyze the formation of the four main O-glycan core structures, plays a critical role in O-linked glycosylation in mucin core 1, core 2, core 3, and core 4, which can be further biosynthesis. This gene has a tissue-specific expression extended, branched and terminated to form complex O- pattern and is heavily expressed in colon, testis, stomach, glycans. For example, GalNAc can be converted to core 1 by and small intestine as well as trachea in both humans (Yeh core 1 b3-Gal-transferase, which can then be branched to et al., 1999) and cattle (Choi et al., 2004), distinguishable form core 2. Alternatively, GalNAc can be extended to form from the other eight GlcNAc transferases cloned so far. core 3, which can in turn be branched to form core 4. A4GNT Expression of GCNT3 and its enzymatic activity were both gene encodes a-1,4-N-acetylglucosaminyltransferase, an up-regulated by Th2 cytokines such as IL-4 and IL-13 in enzyme responsible for the transfer of N-acetylglucosamine human airway epithelial cell line and by retinoic acid (RA) (GlcNAc) to core 2 branched O-glycans to form a unique in a dose-dependent manner (Beum et al., 2005)aswellas structure, GlcNAca1 ! 4Galb ! R. A4GNT is essential for by lipopolysacharide (Yanagihara et al., 2001). Indeed, the biosynthesis of so-called type III mucins, such as two cis-regulatory elements along with putative RA and MUC5AC and MUC6. In this study, A4GNT mRNA was not STAT response elements were identified in its promoter detectable; neither were MUC5AC and MUC6, suggesting region (Tan and Cheng, 2007). In this study, GCNT3 gene that these mucins may not be of importance in the bovine was up-regulated during C. oncophora infection as early as small intestine. In humans, previous reports showed that 7 dpi. It is possible that IL-13 may be involved in the both MUC5AC and MUC6, carrying GlcNAca1 ! 4Galb ! R regulation of GCNT3 expression since both IL-4 and IL-13 structures, were present in gastric glands and A4GNT played were also induced in the bovine small intestine by a critical role in the biosynthesis of these mucous infection. GCNT3 was up-regulated in the bovine aboma- glycoproteins (Zhang et al., 2001). Indeed, in various human sum during O. ostertagi infection as well (unpublished tissues, A4GNT-positive cells uniformly co-expressed data). This is the first time evidence was presented that GlcNAca1 ! 4Galb ! R, irrespective of their transforming parasitic nematodes were able to induce GCNT3. At both 7 status; and this co-expression has a more restricted and 14 dpi, a pathway called LPS/IL-1b mediated inhibi- distribution than that of MUC6 (Nakajima et al., 2003). tion of RXR functions was regulated. TR/RXR activation Recent data suggested a unique glycan structure, a1,4- was significantly impacted at 14 and 28 dpi. The relation- GlcNAc-capped core 2-branched O-glycans, existed exclu- ship between these pathways and host immune response sively in MUC6 and absent in MUC5AC, acted as an to GI nematodes remains unclear at this point. However, it antimicrobial agent and suppressed Helicobacter pylori is known that retinoids inhibit LPS-stimulated production growth by inhibiting the synthesis of a-glucosyl cholesterol, of IL-12 (a Th1 cytokine) in a dose-dependent manner via a major component of its cell walls (Kawakubo et al., 2004; ligand-independent binding of NF-kBtoRXR(Na et al., Lee et al., 2008). We are currently investigating expression 1999). In addition, retinoic acid can inhibit cytokine and regulation of these genes in the bovine abomasum expression in hepatic macrophage including reduction of during infection. mRNA levels of TNFa, iNOS, IL-6, and IL-1b (Motomura Expression of glycosyltransferases, B3GNT3 and et al., 2001). Recently, it has been observed that LPS- B3GNT6, was also investigated in this study. These specific regulatory networks in which NF-kBplayeda glyocsyltransferases are structurally related to the focal role in the mouse submucosa overlapped with the b1,3-galactosyltransferase family but do not possess LPS/IL-1b mediated inhibition of RXR functions (Saban b1,3-galactosyltransferase activity. B3GNT3 encodes a et al., 2008). RXR is known to be capable of mediating member of the beta-1,3-N-acetylglucosaminyltransferase retinoid signaling and may play critical roles in develop- family and is involved in the biosynthesis of poly-N- ment, adult physiology, and metabolic processes by acetyllactosamine chains in addition to playing a dominant partnering with a number of nuclear receptors to form role in L-selectin ligand biosynthesis, lymphocyte homing functional heterodimers (Szanto et al., 2004). Also, 9-cis and lymphocyte trafficking. The poly-N-acetyllactosamine retinoic acid and docosahexaenoic acid (DHA) were structure exists in many molecules including glycolipids, suggested to be endogenous RXR ligands (de Urquiza keratin sulfate , and human milk oligosac- et al., 2000). It is possible that regulation of RXR functions charides, in addition to N- and O-linked glycans of was an indirect effect resulting from the host lipid 288 R.W. Li et al. / Veterinary Parasitology 165 (2009) 281–289 metabolism, which was significantly impaired during C. Kawai, Y., Yamauchi, J., Soga, K., Yamada, M., Uchikawa, R., Tegoshi, T., Arizono, N., 2007. T cell-dependent and -independent expression of oncophora infection. intestinal epithelial cell-related molecules in rats infected with the In summary, we investigated cell-specific expression of nematode Nippostrongylus brasiliensis. 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