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Am J Physiol Gastrointest Liver Physiol 290: G164-G174, 2006. First published August 25, 2005; doi:10.1152/ajpgi.00460.2004
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MUCOSAL BIOLOGY

NF-{kappa}B-mediated expression of iNOS promotes epithelial defense against infection by Cryptosporidium parvum in neonatal piglets

Jody L. Gookin,1 Sophia Chiang,1 Jessica Allen,1 Martha U. Armstrong,1 Stephen H. Stauffer,1 Colleen Finnegan,2 and Michael P. Murtaugh2

1Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; and 2Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, Minnesota

Submitted 13 October 2004 ; accepted in final form 20 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cryptosporidium sp. parasitizes intestinal epithelium, resulting in enterocyte loss, villous atrophy, and malabsorptive diarrhea. We have shown that mucosal expression of inducible nitric oxide (NO) synthase (iNOS) is increased in infected piglets and that inhibition of iNOS in vitro has no short-term effect on barrier function. NO exerts inhibitory effects on a variety of pathogens; nevertheless, the specific sites of iNOS expression, pathways of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear. Using an in vivo model of Cryptosporidium parvum infection, we have examined the location, mechanism of induction, specificity, and consequence of iNOS expression in neonatal piglets. In acute C. parvum infection, iNOS expression predominated in the villous epithelium, was NF-{kappa}B dependent, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion but was not detrimental to maintenance of mucosal barrier function. Intensified parasitism could not be attributed to attenuated fluid loss or changes in epithelial proliferation or replacement rate, inasmuch as iNOS inhibition did not alter severity of diarrhea, piglet hydration, Cl– secretion, or kinetics of bromodeoxyuridine-labeled enterocytes. These findings suggest that induction of iNOS represents a nonspecific response of the epithelium that mediates enterocyte defense against C. parvum infection. iNOS did not contribute to the pathogenic sequelae of C. parvum infection.

nitric oxide; barrier function; diarrhea; cryptosporidiosis

A NONINVASIVE Protozoan, Cryptosporidium, parasitizes the small intestinal epithelium. Infection results in accelerated loss of villous enterocytes, leading to severe villous atrophy and a malabsorptive and secretory diarrhea. Acute epithelial defense against infection requires the elimination of parasitized enterocytes with preservation of epithelial continuity, as provided by enterocytes generated in the crypt and migrating onto the villus. These mechanisms must keep pace with ongoing infection until specific immune mechanisms result in clearance of the organisms. Surprisingly, little is known regarding the mechanisms and mediators of epithelial defense that predominate at the time of acute epithelial infection and diarrhea in the naturally susceptible host. Although studies in mice with defined immunodeficiency have identified a number of mediators (e.g., IFN-{gamma}, CD40, and the CD4-positive T cell) that are necessary for onset of acquired immunity to chronic Cryptosporidium parvum infection, these animals do not develop villous atrophy or diarrhea, which characterize the acute clinical infection. Similarly, epithelial cell culture models of Cryptosporidium infection are unable to recapitulate the mechanisms of epithelial loss and replacement that characterize in vivo infection.

Synthesis of nitric oxide (NO) is consistently elevated in patients with infectious diarrhea of a variety of causes (26), although its physiological value is unresolved. Similarly, expression of inducible NO synthase (iNOS) and synthesis of NO are significantly increased in intestinal mucosa from Cryptosporidium-infected piglets and mice (14, 26, 30). Induction of iNOS by or exposure of cell monolayers to high concentrations of NO promotes cytotoxicity and barrier disruption (4, 36, 43, 44, 47). Conversely, NO exerts inhibitory effects on numerous microbial pathogens (23, 38); NO donors reduce in vitro viability of C. parvum sporozoites (29), and iNOS-knockout mice or mice treated with iNOS inhibitors exhibit more severe cryptosporidial infection and delayed parasite clearance (29, 30). Nevertheless, the specific sites of iNOS expression, pathway of iNOS induction, and mechanism of NO action in cryptosporidiosis remain unclear.

iNOS is constitutively expressed by intestinal villous epithelium in vivo (15, 19, 37, 50). Epithelium is also capable of rapid synthesis of iNOS and NO after acute gut injury (4, 15, 24, 37). The greater vulnerability of villous epithelium to injury and infection, coupled with the ability to produce rapid and sustained levels of NO, suggests that iNOS may serve an important role in epithelial defense. The present studies investigate the mechanism, specificity, and consequence of iNOS expression in an in vivo model of cryptosporidial infection. In acute C. parvum infection of neonatal piglets, iNOS expression was NF-{kappa}B dependent, predominated in the villous epithelium, and was not restricted to infected enterocytes. Ongoing treatment of infected piglets with a selective iNOS inhibitor resulted in significant increases in villous epithelial parasitism and oocyst excretion and was not detrimental to maintenance of mucosal barrier function. Crypt cell proliferation and replacement rate of the infected epithelium and severity of diarrhea and fluid loss were not altered by iNOS inhibition, suggesting that, in vivo, iNOS mediates defense against C. parvum infection at the level of the enterocyte.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. One-day-old crossbred piglets were obtained from the College of Agriculture, randomly allocated to infection-and-control isolation units, and fed a liquid diet by an automated delivery system. An inoculum of 108 C. parvum oocysts was given by orogastric tube on day 3 of life. Control and infected piglets were euthanized with intravenous pentobarbital sodium 3–5 days after inoculation (at 6–8 days of age), a time that was shown previously to be inclusive of peak C. parvum infection (3). Sections of ileum, beginning 10 cm above the ileocecal junction, were taken sequentially for histology, immunoblotting, RNA extraction, and in vitro barrier function studies in Ussing chambers. Because of limitations on the availability of piglets and number of piglets that can be studied simultaneously, experiments were performed every 2 wk until sufficient animals were obtained for statistical comparison. To account for variability in severity of C. parvum infection between litters of piglets, treatment comparisons (as fold difference) were made between individual siblings that were first matched by body weight and infected with the same lot of C. parvum oocysts. The North Carolina State University Institutional Animal Care and Use Committee approved all studies.

NO analysis. Total NO2 + NO3 concentration was measured in urine by conversion of NO3 to NO2 by nitrate reductase and detection of NO2 with a commercial kit (Griess assay; Cayman Chemical, Ann Arbor, MI).

Epithelial exfoliation for RNA and protein extraction. Sheets of ileal mucosa (2.5 cm2) were obtained at 3–5 days of infection, and epithelium was exfoliated (28). Isolated mucosae were immersed in oxygenated citrate-phosphate buffer containing (mmol/l) 137 NaCl, 2.7 KCl, 8.0 Na2HPO4, 1.5 KH2PO4, 30 EDTA, and 2.5 glucose (pH 7.4) for 20 min at 37°C. To dislodge epithelial cells from the lamina propria, we grasped the submerged mucosa with forceps, which were then vibrated by contact with a vortex. Removal of epithelial cells was confirmed by histological examination. The epithelial cells were pelleted by centrifugation at 200 g for 10 min at 4°C. A glass slide was used to scrape the lamina propria from the muscularis. Each fraction was placed in 10 volumes of RNAlater or RLT buffer containing {beta}-mercaptoethanol (Qiagen, Valencia, CA) and stored at –20°C.

RNA isolation and real-time RT-PCR. Samples of epithelium and lamina propria (30 mg) were homogenized (QIAshredder, Qiagen), and total RNA was extracted using an RNeasy Mini Kit (Qiagen) with on-column DNase digestion (Qiagen). Concentration of RNA was determined by absorbance at 260 nm (Biophotometer, Eppendorf, Westbury, NY) and evaluated for RNA integrity using a bioanalyzer (model 2100, Agilent Technologies, Palo Alto, CA). Four micrograms of total RNA were reverse transcribed with 200 µg of random hexamers in a 40-µl total volume using an Invitrogen RT kit with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Intron-spanning primers for PCR amplification of cDNA were designed using Primer 3 software (Whitehead Institute, Cambridge, MA). Specific primer sequences and sources used in real-time RT-PCR and resulting amplification product sizes and melting temperatures are shown in Table 1.


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  		Table 1. Specific primer sequences and sources used in real time RT-PCR and their resulting amplification product sizes and temperatures

 
RNA integrity and cDNA production were verified by amplification of cyclophilin. Real-time PCR was performed in a 15-µl reaction volume with each primer at 50 nM, 60 ng of cDNA, and 2x SYBR Green Mastermix kit (Applied Biosystems, Foster City, CA) using an ABI Prism 7900 thermocycler or Bio-Rad iCycler for 50 cycles at 95°C for 15 s and 60°C for 1 min after an initial incubation for 10 min at 95°C. PCR products also were analyzed on a 1.5% agarose gel stained with ethidium bromide and sequenced. Comparative threshold cycle (CT) analysis was used as described in the ABI Prism 7700 Sequence Detection System User Bulletin 2 to calculate relative fold changes (2).

Immunoblotting. Samples of epithelium and lamina propria were thawed on ice in RIPA buffer [0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid, 1% Triton X-100, and 0.1% SDS] containing aprotinin, leupeptin, bestatin, antipain, EDTA, NaF, sodium orthovanadate, and PMSF. The mixture was sonicated and centrifuged at 10,000 g for 10 min at 4°C. The supernatants were saved, and the protein concentration was determined (Dc protein assay, Bio-Rad, Hercules, CA). Samples were mixed with an equal volume of 2x SDS-PAGE sample buffer, boiled for 4 min, and loaded at equal protein concentrations in SDS-polyacrylamide gels. Proteins were electroblotted to a nitrocellulose membrane (Hybond ECL, Amersham Life Science, Birmingham, UK). Membranes were blocked overnight at 4°C in Tris-buffered saline (TBS) containing 5% powdered milk (iNOS) or 2% bovine serum albumin (I{kappa}B{alpha}). Membranes were incubated overnight at 22°C in primary antibody (rabbit anti-iNOS, 1:10,000 dilution; Cayman Chemical, Ann Arbor, MI) or rabbit anti-I{kappa}B{alpha} (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). After they were washed three times with TBS + 0.05% Tween 20, the membranes were incubated for 45 min with horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; Santa Cruz Biotechnology). After three additional washes with TBS + 0.05% Tween 20, the membranes were developed by addition of enhanced chemiluminescence reagent according to the manufacturer's instructions (Amersham, Princeton, NJ).

Immunohistochemistry. Immunohistochemistry for iNOS was performed using formalin-fixed 5-µm sections of mucosa. Tissues were deparaffinized by immersion in xylene, rehydrated in a graded series of ethanol, and hydrated to buffer (PBS, pH 7.4). Tissues were treated with 3% H2O2 in methanol for 10 min at 4°C to quench endogenous peroxidase and blocked for 30 min at room temperature with nonimmune goat serum. A commercial kit was used for blocking endogenous avidin and biotin activity (Avidin/Biotin Blocking Kit, Zymed Laboratories, San Francisco, CA). Tissue sections were incubated with antibody (rabbit anti-iNOS primary antibody, 1:100 dilution; Transduction Laboratories, Lexington, KY) for 1 h. Sections were immunostained using a streptavidin-biotin-peroxidase system with 3,3'-diaminobenzidine as the chromogen (Santa Cruz Biotechnology). Sections were counterstained with hematoxylin and eosin. Negative control sections were incubated with isotype control rabbit IgG.

Immunofluorescence imaging of live enterocytes for colocalization of NO synthesis and C. parvum infection. Epithelial cells were exfoliated from control and infected ileal mucosa using the method previously described for RNA and protein extraction, filtered (BD Falcon 100-µm cell strainer), and pelleted by centrifugation at 1,500 rpm for 5 min. Cells representing epithelial cells and intraepithelial lymphocytes were resuspended in PBS containing 2.5 mM glucose, rabbit polyclonal anti-C. parvum antibody (a kind gift from Dr. Guan Zhu, 1:100 dilution) or rabbit IgG isotype control antibody (10 µg/ml), and 10 µM 4-amino-5-methylamino-2',7'-difluorescein (DAF-FM) diacetate (Molecular Probes, Eugene, OR) for 30 min at room temperature. DAF-FM diacetate is cell permeable, reacts with intracellular NO to form a green-fluorescent benzotriazole, and has been used to measure NO production by colorectal and lung carcinoma cell lines (DLD-1 and A549), human bronchial epithelial cells, endothelial cells, and neurons of the myenteric plexus (27, 42, 52). Fluorescence intensity of DAF-FM is linearly related to NO concentrations in the range 2–200 nM (21). Detection of NO synthesis in situ using DAF-FM requires the use of living cells. Demonstration of C. parvum infection by means of immunofluorescence of live epithelial cells has been previously described (35). Cells were subsequently washed once with PBS containing 2.5 mM glucose and incubated in the dark with Cy3-labeled goat anti-rabbit IgG (Zymed Laboratories; 1:150 dilution), 10 µM DAF-FM diacetate, and the nuclear stain Hoechst 33342 (Molecular Probes; 1 µM) for 15 min at room temperature. Cells were washed and resuspended with PBS containing 2.5 mM glucose and transferred to glass slides, coverslips were applied, and the cells were immediately imaged with a fluorescence microscope.

Selective inhibition of NF-{kappa}B and JAK-STAT pathways. Ileal mucosa from control and infected piglets (n = 3 each) obtained at peak infection were incubated for 5 h in Ussing chambers in the presence of 1) normal Ringer solution, 2) tyrphostin B42 (AG-490), a selective Jak-2 protein tyrosine kinase inhibitor (100 µM; Sigma Chemical), or 3) lactacystin, a cell-permeable and irreversible proteosome inhibitor (20 µM; Peptides International, Louisville, KY). Mucosal scrapings were preserved in RLT buffer and processed for quantification of iNOS mRNA expression by real time RT-PCR as previously described. Mucosal samples from control and infected piglets were also incubated in Ussing chambers with the protein synthesis inhibitor cycloheximide (100 µM; Sigma Chemical) for 1 and 3 h.

Activated NF-{kappa}B p65 ELISA. Superficial scrapings of ileal mucosa were obtained from three control and three infected piglets and frozen at –80°C. Whole cell protein was extracted from each sample (Nuclear Extraction Kit, Active Motif, Carlsbad, CA) and assayed for protein concentration using the bicinchoninic acid method (Pierce, Rockford, IL). Twenty micrograms of total protein from each sample were assayed for the presence of activated p65 by ELISA using antibodies specific for activated p65 after binding to NF-{kappa}B consensus sequence (TransAM NF-{kappa}B p65 ELISA kit, Active Motif).

In vivo iNOS inhibition studies. One-day-old littermate piglets were paired by body weight and treated daily with a selective iNOS inhibitor, L-N6-(1-iminoethyl)-lysine (L-NIL; Cayman Chemical; 3 mg/kg ip) or an equivalent volume of saline vehicle (2 ml ip) beginning at the time of orogastric inoculation with 108 oocysts and continuing until euthanasia 4 days after infection. Timing of euthanasia was chosen to correspond to the time of peak mucosal iNOS expression. The dose of L-NIL chosen for iNOS inhibition selectively reduces iNOS-mediated NO synthesis in a variety of in vivo models (20, 33, 45) and significantly inhibited the increase in NO2 + NO3 levels resulting from infection in the present study. Potential adverse effects of iNOS inhibition or saline (sham) administration were assessed by similar treatment of uninfected piglets (n = 9 each) for 4–11 days and daily observation for untoward effects on appetite, body weight, and fecal consistency.

Quantitation of oocyst excretion and severity of epithelial C. parvum infection. Thin fecal smears were prepared daily for each piglet from rectal swabs with a cotton-tipped applicator. Fecal consistencies were recorded as formed or diarrheic. Smears were stained by the auramine-O technique (3). Total numbers of oocysts were counted within a 64-mm2 grid with use of a fluorescence microscope (Zeiss, Welwyn Garden City, UK). Sections of ileal mucosa from each piglet were fixed in formalin, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin for examination by light microscopy. All infected piglets showed evidence of villous atrophy and organisms adherent to villus enterocytes, whereas control piglets showed normal villous architecture with no evidence of infection. Three sections from each tissue were examined. Average villus height (from the crypt opening to the villus tip) and crypt depth of five well-oriented villi were measured using an ocular micrometer, and the percentage of epithelialized villus surface was calculated from linear measurements of epithelialized vs. denuded villus perimeter. For each of the selected villi, the number of villous epithelial cells and the number of adherent parasites were counted. All measurements were performed by an examiner blinded to treatment category.

Scanning electron microscopy. Sheets of ileal mucosa from control piglets and infected piglets treated with saline or L-NIL were fixed in Trump's 4F:1G fixative at 4°C. Samples were rinsed twice for 15 min each with 1.0 M Sorenson's phosphate buffer (pH 7.2–7.4) and dehydrated in an ascending series of ethanol (50%, 75%, 95%, and 95%) for 15 min each, culminating in two washes in 100% ethanol for 30 min each. Samples were then dried in a Ladd critical-point dryer, mounted with carbon tape on aluminum specimen stubs, and sputter coated with ~20 nm of gold-palladium using an Anatch Hummer VI sputter coater. Samples were viewed using a JEOL 6360 LV scanning electron microscope.

Barrier function studies. Ussing chamber studies were performed as described elsewhere (15). Briefly, a 20-cm segment of ileum obtained from saline- or L-NIL-treated piglets at the time of euthanasia was opened along the antimesenteric border in an oxygenated Ringer solution, and the seromuscular layers were removed. Mucosal sheets were mounted in 1.13-cm2-aperture Ussing chambers and bathed on both surfaces with a Ringer solution containing 10 mM glucose (serosal) and 10 mM mannitol (mucosal) for 120 min. Solutions were oxygenated and circulated by gas lift (95% O2-5% CO2) and maintained at 37°C by water-jacketed reservoirs. The spontaneous potential difference (PD) was measured using Ringer-agar bridges connected to calomel electrodes, and the PD was short-circuited through Ag-AgCl electrodes using a voltage clamp that corrected for fluid resistance. If the spontaneous PD was between –1.0 and 1.0 mV, tissues were current clamped at ±100 µA for 5 s and the PD was recorded. Transepithelial electrical resistance (TER, {Omega}·cm2) was calculated from the spontaneous PD and short-circuit current (Isc), which were recorded at 5- to 15-min intervals over a 210-min period. Isotopic flux assessment of transmucosal permeability was performed using [3H]mannitol (2 µCi, 6.6 mM; Dupont, Boston, MA). Isotope was added to the mucosal reservoir immediately after the sample was mounted on the chamber. After 1 h, a 60-min flux period was recorded by taking paired samples from the serosal reservoir. Samples were counted for 3H in a liquid scintillation counter (LKB Wallac, Turku, Finland). Flux of [3H]mannitol from mucosa to serosa was calculated using standard equations.

Assessment of iNOS effects on epithelial replacement rate. The epithelial cell replacement rate of piglets treated with saline or L-NIL was determined by injecting each piglet on day 4 of infection with 5-bromo-2'-deoxyuridine (BrdU, 100 mg/kg in DMSO ip; Sigma). Piglets were euthanized 12 h (n = 8 each) or 36 h (n = 3 each) after injection. Immunohistochemical detection of BrdU (1:500 dilution of mouse anti-BrdU monoclonal antibody; Sigma) was performed using formalin-fixed 5-µm sections of ileal mucosa. Antigen unmasking was performed by incubation in 2 N HCl and 0.4% pepsin (each for 30 min at 37°C). Tissue sections were incubated with primary antibody for 2 h at 37°C, immunostained using a streptavidin-biotin-peroxidase system with 3,3'-diaminobenzidine as the chromogen (Mouse ABC Staining System, Santa Cruz Biotechnology), and counterstained with dilute hematoxylin. To determine the migration rate of the epithelium, the distance from the base of the crypt to the foremost cohort of labeled epithelial cells and the total distance from the base of the crypt to the villus tip were used to calculate the percentage of the total crypt-villus length populated by labeled epithelial cells and expressed for each piglet as an average representing three well-oriented crypt-villus units, as previously described (5, 11). Differences in epithelial cell proliferation rate were determined by counting the number of labeled cells generated in a discrete time interval after BrdU injection.

Data analysis. Values are means ± SE. For all analyses, P < 0.05 was considered significant. One-way ANOVA and Tukey's post hoc test or Student's paired t-test were used to compare differences between treated and control tissues (SigmaStat, Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NO synthesis is increased in piglets with C. parvum infection. The oxidative metabolites of NO (NO2 + NO3) were measured in urine samples obtained from control and infected animals at the time of peak diarrhea (3–5 days after infection). A significantly higher concentration of NO metabolites was excreted into the urine of piglets infected with C. parvum: 5 ± 1 and 22 ± 4 NO2 + NO3 (µM)/creatinine (mg/dl) for uninfected control (n = 6) and C. parvum (n = 5), respectively (P < 0.001 by 1-way ANOVA).

iNOS is expressed by parasitized epithelium in C. parvum infection. We previously showed that C. parvum infection increases mucosal expression of iNOS (14). To identify the specific location of iNOS, mRNA and protein expression were examined in epithelium and lamina propria fractions of ileal mucosa obtained from control and infected animals (days 3–5). Immunohistochemistry was also performed to localize iNOS expression along the crypt-villus axis. iNOS mRNA, with minimal protein expression, was present constitutively in epithelium and lamina propria fractions of ileal mucosa from control piglets. At peak infection, there was a significant increase in mRNA expression in the epithelium and lamina propria (Fig. 1A), with greater protein expression in the epithelium (Fig. 1B). In contrast, neither endothelial nor neuronal NOS mRNA was observed in the epithelium, and lamina propria levels did not vary with infection. Immunohistochemistry disclosed intense staining for iNOS along the apical villi of C. parvum-infected piglets, with less expression in the lamina propria (Fig. 2).



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  		Fig. 1. Localization of inducible nitric oxide synthase (iNOS) mRNA (A) and protein (B) expression to epithelial and lamina propria fractions of ileal mucosa from control and Cryptosporidium parvum-infected piglets. Data in A represent degree of difference in mRNA levels adjusted for differences in housekeeping cyclophilin expression among samples. Selectivity of iNOS induction was demonstrated by real-time RT-PCR analysis of endothelial and neuronal NOS (eNOS and nNOS) expression. *P < 0.05; **P < 0.01 vs. control (1-way ANOVA).

 


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  		Fig. 2. Immunohistochemical detection of iNOS expression by control and C. parvum-infected piglet ileal mucosa on days 3–5 of infection. In infected mucosa, iNOS was expressed by parasitized enterocytes along tips of villi, with lesser amounts in the lamina propria of the apical villi. Left: mucosa treated with 1:100 dilution of polyclonal rabbit anti-NOS II. Right: mucosa treated with isotype control primary antibody. Images are representative of 5 control and 5 infected piglets. Scale bars, 100 µm (top) and 50 µm (bottom).

 
Expression of iNOS was not observed in the crypts, despite the presence of infected epithelial cells. To determine whether NO synthesis was restricted to infected epithelial cells or was also present in noninfected cells in C. parvum infection, we isolated live epithelial cells from infected piglets and identified C. parvum-infected cells by labeling with an anti-C. parvum antibody. Cells were simultaneously incubated with a cell-permeable fluorochrome (DAF-FM diacetate) that is converted in the presence of NO to a green-fluorescent benzotriazole. These studies were performed on isolated epithelial cells, rather than histological sections, to assess active synthesis of NO and to examine the entirety of each cell for the presence of organisms. In piglets with C. parvum, infected and uninfected epithelial cells synthesized NO. Consistent with our observation of organisms, but not iNOS staining in the crypts, epithelial cell infection in the absence of NO synthesis was also observed (Fig. 3). In uninfected piglets, neither C. parvum nor NO synthesis by the epithelium could be demonstrated.



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  		Fig. 3. Immunofluorescent imaging of live epithelial cells extracted from ileal mucosa of a piglet with C. parvum infection. Epithelial infection was identified by labeling with rabbit anti-C. parvum antibody followed by Cy3-labeled goat anti-rabbit IgG (red). Nitric oxide (NO) formation was visualized by simultaneous incubation of cells with 4-amino-5-methylamino-2',7'-difluorescein (DAF-FM) diacetate, which is converted to a fluorescent compound on exposure to NO (green). Nuclei were visualized by counterstaining with Hoechst 33342 (blue). A: epithelial cells treated with anti-C. parvum antibody, DAF-FM, and Hoechst 33342. a, Nominal NO synthesis by C. parvum-infected enterocyte; b, abundant NO synthesis (green) by C. parvum (red)-infected enterocyte; c, NO synthesis by uninfected enterocyte; d, absence of NO synthesis by uninfected enterocyte. B: infected epithelial cells treated with isotype control primary antibody, DAF-FM diacetate, and Hoechst 33342. Images are representative of experiments performed on 3 piglets. No fluorescence was observed when epithelial cells from piglets without C. parvum infection were incubated with anti-C. parvum antibody and DAF-FM (data not shown).

 
iNOS mediates the increase in NO synthesis resulting from C. parvum infection. To determine whether iNOS is the source of increased NO synthesis in C. parvum infection, the oxidative metabolites of NO were measured in urine samples taken on day 4 from infected piglets treated daily with saline vehicle or the selective iNOS inhibitor L-NIL (3 mg/kg ip). The increased synthesis of NO metabolites in urine of infected piglets was significantly inhibited in pigs treated with L-NIL: 36 ± 10 and 17 ± 5 NO2 + NO3 (µM)/creatinine (mg/dl) for C. parvum + PBS and C. parvum + L-NIL (n = 8 each), respectively (P < 0.05 by Student's paired t-test).

Epithelial expression of iNOS is mediated by NF-{kappa}B in C. parvum infection. To identify the underlying mechanism of iNOS induction by C. parvum infection in vivo, we performed inhibitor studies of the two key signaling pathways for epithelial iNOS induction: NF-{kappa}B and JAK-STAT. Maintenance of transcription of iNOS mRNA by ileal mucosa from C. parvum-infected piglets was significantly inhibited by lactacystin, a proteosome inhibitor that prevents degradation of I{kappa}B{alpha}. Tyrphostin B42, a selective Jak-2 protein tyrosine kinase inhibitor, had no effect on iNOS transcription (Fig. 4). Synthesis of I{kappa}B{alpha} was significantly increased in epithelial cells from piglets infected with C. parvum (Fig. 5A), suggesting ongoing stimulation by NF-{kappa}B, the major regulator of I{kappa}B{alpha} expression. Active I{kappa}B{alpha} synthesis and degradation were further demonstrated by rapid disappearance of I{kappa}B{alpha} after incubation of infected mucosa with the protein synthesis inhibitor cycloheximide (Fig. 5B). Finally, ongoing activation of NF-{kappa}B signaling in the infected epithelium was specifically demonstrated by the binding of active p65 to NF-{kappa}B consensus sequence using an ELISA: optical density = 0.390 ± 0.017 and 0.116 ± 0.018 for C. parvum-infected and uninfected control (n = 3 each), respectively (P < 0.001 by Student's t-test).



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  		Fig. 4. Change in expression of iNOS mRNA of C. parvum-infected ileal mucosa in response to treatment with a selective NF-{kappa}B (lactacystin) or Jak-2 protein tyrosine kinase inhibitor (tyrphostin B42) in Ussing chambers. Data (means ± SE) represent degree of difference in mRNA levels adjusted for differences in housekeeping cyclophilin expression among samples (n = 3 piglets each). **P < 0.01 vs. normal Ringer (NR) by Student's t-test.

 


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  		Fig. 5. Expression of I{kappa}B{alpha} by intestinal epithelial cells. A: immunoblot of I{kappa}B{alpha} in epithelial cells from uninfected (control, n = 6) and infected (C. parvum, n = 6) piglets. B: effect of incubation with the protein synthesis inhibitor cycloheximide (CHX) on expression of I{kappa}B{alpha} by control and infected mucosa after incubation for 1 or 3 h in Ussing chambers. I{kappa}B{alpha} lane is positive control consisting of TNF-{alpha}-stimulated HeLa cell lysate.

 
iNOS counters epithelial parasitism and oocyst excretion in C. parvum infection without detriment to barrier function. To determine whether iNOS promotes epithelial defense against infection by C. parvum in vivo, littermate-matched piglets were treated with a selective iNOS inhibitor or an equivalent volume of saline vehicle beginning at the time of oral inoculation with oocysts and continuing until euthanasia 4 days after infection. The resulting burden of infection was determined by daily enumeration of excreted oocysts and quantitative light microscopy of ileal mucosa. For individual piglets, oocysts were excreted in waves consistent with cycles of replication of the parasites within the small intestine. Coincident with the typical onset of diarrhea (day 3 of infection), piglets treated with L-NIL excreted significantly greater numbers of oocysts in feces: 17 ± 13 and 80 ± 30 for C. parvum + PBS and C. parvum + L-NIL, respectively (P < 0.05 by Student's t-test; Fig. 6). When examined after euthanasia (day 4 of infection), ileal mucosa from L-NIL-treated piglets had greater villous atrophy (see Fig. 9 for representative histological section) and significantly greater numbers of parasites per villus enterocyte than ileal mucosa from infected littermates treated with vehicle alone (Figs. 7 and 8). There was no difference in severity of epithelial disruption, which was minimal in both groups of piglets: 98.0 ± 1.99 and 99.6 ± 0.34% of villous linear surface epithelialized for saline and L-NIL (n = 8 each), respectively. Treatment of uninfected piglets with saline or iNOS inhibitor (n = 3 each) at an equivalent dose and route for 11 days did not result in diarrhea, change in appetite, or intestinal mucosal pathology.



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  		Fig. 6. Time course of oocyst excretion in pigs treated with the iNOS inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL). Data (means ± SE) are expressed as fold increase of oocysts shed by treated piglets relative to littermate-matched controls infected with the same batch of C. parvum and treated daily with vehicle (PBS). *P < 0.05 by Student's paired t-test.

 


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  		Fig. 9. Lack of effect of iNOS inhibition on epithelial cell proliferation and replacement. Ileal mucosa from 1 uninfected control (A) and 2 C. parvum-infected piglets (B and C) 36 h after 5-bromo-2'-deoxyuridine injection (100 mg/kg ip). Infected piglets were treated with saline vehicle (PBS, 2 ml ip once daily; B) or an iNOS-selective inhibitor (L-NIL; 3 mg/kg ip once daily; C). There was no significant difference between infected treatment groups in epithelial migration rate along the crypt-villus axis. Scale bar, 50 µm.

 


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  		Fig. 7. Villus height and crypt depth of control and C. parvum-infected ileal mucosa. Littermate-matched piglets were infected with C. parvum and treated daily with a selective iNOS inhibitor (L-NIL, 3 mg/kg ip) or vehicle (PBS, 2 ml ip) and killed 4 days after infection. For each animal, 5 well-oriented villus-crypt units were measured using an ocular micrometer and averaged. For each of the selected villi, the number of villous epithelial cells and the number of adherent parasites were counted. Infected piglets treated with the iNOS inhibitor had greater villous atrophy and greater numbers of villous epithelial parasites than infected piglets treated with vehicle alone. *P < 0.05 by Student's paired t-test.

 


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  		Fig. 8. Effect of iNOS inhibition on morphology of C. parvum-infected ileum. Light microscopic (A–C) and scanning electron microscopic (D–F) images of ileal mucosa obtained from uninfected control (A and D) and C. parvum-infected piglets treated daily with saline vehicle (B and E; PBS, 2 ml ip) or a selective iNOS inhibitor (C and F; L-NIL, 3 mg/kg ip). Piglets were killed on day 4 of infection. Scale bars, 50 µm (A) and 10 µm (B and C).

 
We previously showed that C. parvum results in significant loss of barrier function in infected piglets. Treatment of infected ileal mucosa ex vivo with a selective iNOS inhibitor (L-NIL) results in a modest, but not significant, decrease in maintenance of barrier function (14). To determine whether inhibition of iNOS in vivo resulted in a decrease in barrier function of infected piglets, sheets of ileal mucosa were obtained from L-NIL- and saline-treated piglets on day 4 of infection and mounted in Ussing chambers for measurement of TER and mucosal-to-serosal flux of [3H]mannitol. There was no significant difference in TER or permeability to [3H]mannitol between L-NIL- and saline-treated piglets: 25 ± 0.9 and 25 ± 1.2 {Omega}·cm2 mucosa at 120 min for infected + PBS and infected + L-NIL (n = 12), respectively, and 0.13 ± 0.01 and 0.14 ± 0.02 mucosal-to-serosal flux of [3H]mannitol for infected + PBS and infected + L-NIL (n = 8), respectively. Similarly, TER of ileal mucosa was not diminished by treatment of uninfected control piglets in vivo with the iNOS inhibitor: 47 ± 6 and 58 ± 8 {Omega}·cm2 mucosa at 120 min for control + PBS and control + L-NIL (n = 6), respectively.

iNOS does not promote diarrhea or epithelial cell replacement. iNOS has been implicated in mediating a myriad of electrolyte transport events (22). To determine whether iNOS mediates epithelial defense against C. parvum by promoting fluid loss and, thus, evacuation of infective stages of the parasite from the intestine, severity of diarrhea and piglet hydration were quantified for animals treated with L-NIL or saline in vivo. Also, sheets of ileal mucosa were obtained at peak infection for measurement of Isc, an indirect measurement of Cl– secretion in this tissue (2). Infection with C. parvum alone resulted in significant diarrhea, loss of body weight, and increase in epithelial Isc compared with uninfected piglets (Table 2). Inhibition of iNOS did not alter these parameters in infected or uninfected piglets compared with littermates treated with vehicle alone. Thus iNOS is unlikely to have mediated epithelial defense against C. parvum by promoting any gross alteration in intestinal water balance.


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  		Table 2. Severity of diarrhea, body weight, and epithelial Cl– secretion in piglets infected with C. parvum and treated in vivo with L-NIL or PBS

 
To determine whether iNOS could promote defense against C. parvum infection by enhancing the replacement rate of the epithelium, the proliferation and migration rate of infected epithelium in piglets treated with saline or L-NIL were quantified by BrdU incorporation. There was no difference in crypt cell proliferation rate between L-NIL- and saline-treated piglets as determined by the in vivo nuclear incorporation of BrdU: 3.2 ± 0.3 and 3.8 ± 0.3 BrdU-positive cells/h for saline (n = 7) and L-NIL (n = 8), respectively. There were also no differences between L-NIL- and saline-treated piglets in migration rate of BrdU-labeled epithelial cells along the crypt-villus axis 12 or 36 h after injection of BrdU: 39 ± 5 and 41 ± 5% crypt-villus length labeled at 12 h for saline and L-NIL (n = 7 each), respectively, and 50 ± 11 and 81 ± 10% crypt-villus length labeled at 36 h for saline and L-NIL, respectively (Fig. 9). Thus the increase in number of epithelial parasites infecting piglets treated with the iNOS inhibitor was independent of the epithelial cell replacement rate.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We previously showed that C. parvum infection results in expression of iNOS and synthesis of NO by ileal mucosa from experimentally infected piglets. The present study demonstrates that the villous epithelium is the predominant site of iNOS expression. Subepithelial expression of iNOS was minor, perhaps because of the mild degree of inflammatory infiltrate in C. parvum infection (3) and the lack of involvement of iNOS in porcine inflammatory responses (39). Epithelial iNOS expression has likewise been reported in mice experimentally infected with C. parvum (30). Novel to the present study is our finding that iNOS expression is induced primarily along the villus, despite the presence of organisms in the crypts. NO synthesis was observed in infected and uninfected enterocytes. This observation suggests that induction of NOS represents an innate or nonspecific response of the villus to epithelial infection. This conclusion is also consistent with the early time frame of our studies (≤4 days) and prior reports that iNOS-knockout mice and mice treated with iNOS inhibitors are capable of eventual recovery from Cryptosporidium infection (17, 25).

The present study is the first to identify NF-{kappa}B as the pathway responsible for maintenance of iNOS expression in C. parvum-infected epithelium. Others have shown that infection of biliary or epithelial monolayers with C. parvum directly results in NF-{kappa}B activation, which protects infected epithelial cells from apoptosis (6, 34). These findings differ from those of Vallance et al. (49), who found, in an in vivo study of Citrobacter rodentium colitis, that iNOS is selectively expressed by uninfected, but not infected, epithelial cells. In the latter study, iNOS expression was attributed to bacterial LPS and was dependent on an intact type III secretory mechanism. Thus these differences likely reflect diversity in pathogenesis between bacterial and protozoal infection. In contrast to NF-{kappa}B signaling, JAK-STAT signaling was not required for maintenance of iNOS expression by C. parvum-infected epithelium. These findings indirectly suggest that IFN-{gamma} is not a major mediator of iNOS expression in C. parvum infection. In support of this suggestion, we previously showed normal induction of epithelial NOS and synthesis of NO in IFN-{gamma}-knockout or anti-IFN-{gamma}-treated mice after infection with C. parvum (12). Furthermore, iNOS does not appear to mediate IFN-{gamma}-induced resistance of enterocytes to infection by C. parvum in vitro (41).

Synthesis of NO is consistently elevated in patients with infectious diarrhea of a variety of causes (26); however, the physiological value of this observation remains unresolved. Under a number of proinflammatory conditions, expression of iNOS exacerbates intestinal mucosal injury (37, 46, 48, 53), whereas iNOS may ameliorate mucosal damage during the acute phase of intestinal injury (7, 9, 15, 32, 40, 51). We recently showed, by incubation of C. parvum-infected ileal mucosa with a selective iNOS inhibitor in Ussing chambers, that iNOS was not detrimental to short-term maintenance of barrier function (14). Here we determined the in vivo effect of iNOS expression on barrier function and acute epithelial infection by C. parvum.

iNOS promoted epithelial defense against C. parvum infection in vivo. Specifically, ongoing treatment of C. parvum-infected piglets with a selective iNOS inhibitor, beginning at the time of infection and continuing until the typical onset of peak villous atrophy and diarrhea, resulted in significant increases in villous epithelial parasitism and oocyst excretion. Similarly, iNOS knockout or inhibition in mice resulted in increased susceptibility to Cryptosporidium infection, enhanced oocyst shedding, and delayed parasite clearance (29, 30). In addition, our findings suggest that neither iNOS expression nor the intensified parasitism resulting from iNOS inhibition was detrimental to mucosal barrier function in vivo. Although we observed greater villous atrophy in infected piglets treated with the iNOS inhibitor, this was unlikely to be biologically relevant, because villous atrophy was already profound and diarrhea was not exacerbated by iNOS inhibition. Thus iNOS expression does not appear to contribute to the pathogenic sequelae of C. parvum infection.

A number of physiological approaches were undertaken to determine the means by which iNOS inhibition intensified enterocyte parasitism in C. parvum-infected piglets. Inhibition of iNOS did not diminish epithelial secretion or reduce the severity of diarrhea. Clinically, the use of antidiarrheal drugs for treatment of C. parvum infection is controversial, inasmuch as they are suspected to exacerbate parasitism by delaying transit of autoinfective stages of the parasite. In addition, inhibition of iNOS did not decrease crypt cell proliferation or replacement rate of the infected epithelium as determined by in vivo studies using BrdU. Therefore, intensified parasitism resulting from iNOS inhibition could not be attributed to a longer duration of epithelial exposure to infective organisms or delay in ejection of infected enterocytes from the villi. Accordingly, these findings support a direct effect of iNOS and NO in defense of the enterocytes against infection by C. parvum.

It appears that the epithelium is primarily responsible for iNOS-mediated defense in C. parvum infection. The epithelium was the predominant site of iNOS expression. Also, C. parvum infection was confined to the epithelium without extension to other effector cells such as macrophages, in which iNOS plays a key role in defense against a number of invasive Protozoa, such as Toxoplasma gondii and Entamoeba histolytica (1, 31). In vitro studies performed with noninvasive intestinal pathogens, such as Giardia, have shown that NO produced at the apical surface of enterocytes can exert cytostatic effects on pathogen replication (8). Furthermore, exogenous NO inhibits excystation of C. parvum sporozoites and reduces their viability in vitro (29). Postulated mechanisms of NO antimicrobial action include inactivation of critical metabolic pathways through formation of iron-dinitrosyl-dithiolate complexes and generation of reactive nitrogen intermediates such as peroxynitrite (10, 23). We recently showed that peroxynitrite is generated at sites of iNOS expression in C. parvum infection and that treatment with peroxynitrite scavengers similarly exacerbates C. parvum infection (13).

Whether C. parvum is capable of directly inducing epithelial iNOS expression remains unexplored. A role for subepithelial factors in induction of epithelial iNOS is supported by studies demonstrating that many epithelia fail to maintain constitutive iNOS expression when cultured ex vivo (16, 18) and that soluble mediators and direct lymphocyte-enterocyte interaction are required to sustain epithelial iNOS expression (18). A role for subepithelial induction of epithelial iNOS is also suggested by the inability of cell culture studies to reveal a role for iNOS in epithelial models of the infection.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant K08 DK-02868 (to J. L. Gookin) and Center for Gastrointestinal and Bowel Disease Grant P30 DK-34987.


   ACKNOWLEDGMENTS
 
We thank the Laboratory for Advanced Electron and Light Optical Methods, North Carolina State University, College of Veterinary Medicine, for assistance with scanning electron microscopy and Philip Ruckart for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Gookin, North Carolina State Univ. College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC 27606 (e-mail: Jody_Gookin{at}ncsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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