An Increase in Epithelial Cell Apoptosis Is Associated with Chronic Intestinal Nematode Infection

It is well established that homeostasis of the intestinal epitheliumbecomes dysregulated during gastrointestinal helminth infectionand is under immune control. An increase in both enterocyteproliferation and the subsequent generation of crypt hyperplasiaare hallmarks of chronic infection with Trichuris muris, a largeintestinal dwelling nematode. The effect of this parasitic infectionon apoptosis induction in the large intestine and its regulationhas been neglected. To address this, mice of resistant and susceptiblephenotypes were infected with different doses of T. muris, andthe levels of epithelial cell apoptosis were determined. Itis clear that apoptosis is induced during chronic T. muris infection.This occurs mainly at the base of the cecal crypt, within thestem cell region. The level of apoptosis induced is independentof worm number, suggesting that it is not a consequence of worm-induceddamage but rather a mechanism for controlling cell number withinthe crypt. Neutralization of both gamma interferon and tumornecrosis factor alpha caused a significant reduction in thelevels of apoptosis, showing that proinflammatory cytokinesgenerated in response to chronic infection play an importantrole in apoptosis induction in this system. It is proposed thatthe generation of proinflammatory cytokines during chronic T.muris infection may play a positive role, by promoting intestinalepithelial cell apoptosis, to counter infection-induced epithelialhyperplasia.

INTRODUCTION

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Introduction

Gastrointestinal helminths infect over 1 billion people worldwide.Although rarely causing death, such diseases are associatedwith high levels of morbidity and bear a high economic burdenwithin areas where infection is endemic.

Trichuris muris, a natural intestinal parasite of mice, hasbeen extensively utilized as a laboratory model for the studyof human whipworm Trichuris trichiura. Transmission of T. murisoccurs via a direct feco-oral route through the ingestion ofinfective eggs. The intestinal epithelium forms an intimatehost-parasite interface, as upon ingestion of infective eggslarvae hatch and penetrate the epithelium, forming syncytialtunnels within which they reside throughout infection.

Immune-mediated expulsion of intestinal dwelling nematodes caninvolve the interplay between CD4⁺ T cells and the gut epithelium.Resistance and susceptibility to T. muris infection are tightlyassociated with a polarization in the T helper (TH) cytokineresponse generated. Resistance is characterized by the productionof TH2 cytokines, namely, interleukin-4 (IL-4), IL-5, IL-9,and IL-13 (5, 8, 15, 17). Indeed, IL-13 regulation of intestinalepithelial cell turnover is an important component of the hostprotective (13) response. Resistant animals are capable of parasiteexpulsion before worms reach maturity. Susceptible animals mounta TH1 response, generating high levels of gamma interferon (IFN- ${gamma}$ ),IL-12, and IL-18 (6, 7, 15, 27). Such animals fail to expelthe parasite, and infection progresses to chronicity.

Dysregulation of epithelial homeostasis is apparent during insultto the intestine with a number of bacterial, viral, protozoan,and helminth infections (9, 10, 19, 25, 46, 49). The developmentof a marked crypt cell hyperplasia coupled with villus atrophyare typical of such infections. Proinflammatory cytokine productionplays a key role within the intestinal environment during insultand/or disease. Indeed, both IFN- ${gamma}$ and tumor necrosis factoralpha (TNF- ${alpha}$ ) levels are elevated during Crohn's disease andare thought to be important in disease pathogenesis (33, 36,53).

Animals that are susceptible to T. muris infection mount a TH1immune response, regardless of whether a high- or a low-doseinfection is given. Susceptible animals fail to expel the parasite,and therefore infection becomes chronic. During chronic T. murisinfection, a TH1 immune response can be detected not only inthe draining lymph node but also in the gut tissue. It has beendemonstrated that high levels of IFN- ${gamma}$ within the intestine drivesthe development of pathology during chronic T. muris infection.The neutralization of IFN- ${gamma}$ during infection causes a reductionin both crypt length and epithelial cell proliferation (2),identifying a key role for IFN- ${gamma}$ nematode associated alterationin epithelial architecture.

The pleiotropic cytokine TNF- ${alpha}$ can promote inflammation throughleukocyte recruitment and activation, as well as enhancing thesecretion of other proinflammatory cytokines such as IL-1 andIL-6 (45). Moreover, the levels of TNF- ${alpha}$ are increased afterT. muris infection (1). TNF- ${alpha}$ can induce epithelial cell apoptosisin vivo (23) and has been suggested to play an integral rolein the development of pathology associated with inflammatorybowel disease and graft-versus-host disease (GVHD) (11).

This study defines for the first time that apoptosis is inducedafter chronic T. muris infection and importantly that the productionof the proinflammatory cytokines TNF- ${alpha}$ and IFN- ${gamma}$ play a role ininfection associated programmed cell death. Given that an excessof one billion people harbor chronic helminth infection, worm-inducedperturbation of the epithelium and its resultant impact on gutfunction bears important implications for how and what we consideras normal or "steady state" when regarding intestinal physiologyand concerning immunocompetence at mucosal surfaces.

MATERIALS AND METHODS

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Materials and Methods

Animals. Male AKR, BALB/c, and C57BL/6 were obtained from Harlan-Olac,Ltd., United Kingdom. C.B-17^SCID/SCID SCID, TNF p55^–/–,and p75^–/– mice were from Jackson Laboratories.All mice were 6- to 8-week-old males. All experiments were performedunder the regulations of the Home Office Scientific ProceduresAct (1986).

Parasite. T. muris was maintained as previously described (52). Mice wereinfected by oral gavage to give either 200 to 300 embryonatedeggs (high level) or 10 to 20 eggs (low dose) in double-distilledH₂O. Worm burdens were assessed at various time points postinfectionby methods described previously (16).

Purification and administration of anti-IFN- ${gamma}$ MAb. Rat immunoglobulin G1 monoclonal antibody (MAb) XMG1.6 (anti-IFN- ${gamma}$ )and GL113 (isotype control MAb) were purified from supernatantsby passage over a protein G-Sepharose column and concentrationby using Centricon Centriprep tubes.

The MAbs were administered at 1 mg per 200 µl of phosphate-bufferedsaline by intraperitoneal injection. Injections were given every4 days starting at day –2 to day 42 postinfection (p.i.).

Tissue preparation. Cecum samples were removed and flushed out by using saline.Samples were fixed intact in Carnoy fixative for 30 min priorto storage in 70% ethanol. Tissues were prepared by using thegut bundle technique (40). Tissues were then paraffin embeddedby using standard histological techniques. Two nonserial 3-µmsections were mounted per slide and stained with hematoxylinand eosin.

Detection of apoptotic cells. Sections were stained with hematoxylin and eosin to allow thevisualization of apoptotic cells. Such cells are detected onthe basis of their morphology by using light microscopy, a methodthat has been used extensively (28, 29, 35, 41). Typically,apoptotic cells appear pink and circular, with a crescent-shapednucleus, and are bubbled up out of the plain of focus. Tunnellabeling is another method that has can be used to detect apoptosisin the intestinal epithelium. However, this technique is proneto false-positive and false-negative results compared to morphologicalassessment, as well as failing to distinguish between DNA cleavedby apoptosis and DNA fragments cleaved by other processes (39).For the purpose of this investigation, therefore, morphologicalanalysis was deemed the most reliable method to use.

Levels of epithelial cell proliferation. Groups of four mice were injected intraperitoneally with 10mg of bromodeoxyuridine (BrdU; Sigma, Poole, United Kingdom)40 min prior to sacrifice. All animals were killed at the sametime within and between experiments to minimize any differencesin proliferation attributable to variation in circadian rhythm.Detection of nuclei that had incorporated BrdU was performedby immunohistochemistry using a monoclonal anti-BrdU antibody(Mas 250b; Harlan Serum Laboratories, Loughborough, United Kingdom)as described previously (13). Sections were analyzed by scoring50 cecal crypts per mouse with four mice per group.

Scoring. Full-length longitudinal sections of crypts were selected foranalysis. All histological sections were scored blindly. Thescoring commenced with the cell at the midpoint at the baseof the crypt, which was designated as position 1, and continueduntil the crypt-crypt table was reached. For each mouse 50 cryptswere scored, with four mice in each group. This method of scoringallows the generation of statistically valid results (41) andwas used to determine the levels of apoptotic and proliferatingcells. In this way both the position and the overall numbersof apoptosing cells in the cecum can be determined.

Determination of the levels of IFN- ${gamma}$ and IL-12p40 secretion. Mesenteric lymph node cells (MLNC) were removed, cultured, andrestimulated for 24 h under conditions previously described(8). Sandwich enzyme-linked immunosorbent assay was performedto analyze cytokine levels, using pairs of MAbs (IFN- ${gamma}$ R46A2and XMG1.2 and IL-12 C15.6 and C17.8; BD Pharmingen, San Diego,CA). The amount of each cytokine was determined by referenceto commercially available recombinant murine standards. Thesensitivity of the assay was determined by taking the mean plusthree standard deviations of 16 control wells containing mediumonly.

Detection of TNF- ${alpha}$ and IFN- ${gamma}$ in the cecum. The presence of IFN- ${gamma}$ and TNF- ${alpha}$ in whole gut was determined byreverse transcription-PCR. Briefly, tissue samples were snap-frozenin TRIzol (Gibco, United Kingdom), and the RNA isolated wasreverse transcribed by using IMPROMII (Promega). The primersused were the IFN- ${gamma}$ sense (5'- AGCTCTTCCTCATGGCTGTTTC-3') andantisense (5'-ATGTTGTTGCTGATGGCCTGA-3') primers and the TNF- ${alpha}$ sense (5'-TCTTCTCATTCCTGCTTGTGG-3') and antisense (5'- GACAACCTGGGAGTAGACAAGGT-3')primers. For each reaction, cDNA (0.5 µg) was used ina 25-µl PCR.

Measurement of crypt length and epithelial area. The area of the epithelium was assessed by using a computer-assistedZeiss Axiohome microscope system to mark around the area ofinterest. Analysis was performed on hematoxylin-and-eosin-stainedsections, at four mice per group, with three to four circumferencesper mouse. The circumference of the lumen was subtracted fromthe circumference of the muscularis (which runs under the baseof the crypts) to give the area of the epithelium. Individualcrypt lengths were determined by using the same system, selectingwell orientated crypts, and measuring from the base of the cryptto the lumen. Fifty crypts per mouse were measured, with fourmice per group.

Statistical analysis. Statistical analysis was performed by using a one-way analysisof variance (ANOVA) with a Tukey test to determine the significancefor multiple comparisons. Analysis was performed by using theAabel statistic program. The Student t test was also used foranalysis. For both ANOVA and Student t tests, P values of <0.05were considered statistically significant.

RESULTS

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Results

Both epithelial cell apoptosis and proliferation are elevated during chronic nematode infection. Worm expulsion kinetics following a high-dose infection of susceptibleAKR and resistant BALB/c mice displayed the expected trend,with BALB/c mice expelling all parasites by day 21 p.i., whereasin AKR mice infection developed to patency, with worms stillpresent at day 42 p.i. (Fig. 1A).

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FIG. 1. Levels of apoptosis during T. muris infection. (A) AKR ( ${blacksquare}$ ) and BALB/c ( ${square}$ ) mice were infected with 200 T. muris eggs by oral gavage. Worm burdens were assessed at various time points postinfection. ND, not detected. (B) The levels of apoptosis were assessed at days 14, 21, and 42 p.i. in AKR ( ${blacksquare}$ ) and BALB/c ( ${square}$ ) mice. Apoptotic cells were identified by their morphology on sections stained with hematoxylin and eosin. Four mice per group were scored, with 50 crypts per mouse. (C) Location of epithelial cells within the crypt. The percentage of cells apoptosing at each position was assessed. Symbols: •, AKR mice; ${circ}$ , BALB/c mice. A "0" value is the base of the crypt, and "30" is the lumen. (Di) Absence of apoptosing cells in naive AKR cecum. Scale bar, 20 µm. (Dii) Apoptosing cells in AKR ceca at day 42 p.i. (black arrows). Scale bar, 15 µm. (E) Epithelial cell proliferation was assessed by BrdU incorporation in AKR mice ( ${blacksquare}$ ) and BALB/c mice ( ${square}$ ). (F) Histology of BrdU staining in the cecal epithelium. Scale bar, 30 µm. BrdU-positive cells are stained brown. (G) Crypt length in AKR ( ${blacksquare}$ ) and BALB/c ( ${square}$ ) mice. Each datum point represents the mean of four animals ± the standard error of the mean (SEM). *, the Student t test showed a statistically significant increase in AKR mice versus BALB/c mice (P < 0.001).

Apoptosis was assessed during a time course of infection, withanimals sacrificed at days 14, 21, and 42 p.i. Apoptosis levelsin naive animals were also assessed for the purpose of comparison(Fig. 1B). It is clear that the percentage of intestinal epithelialcells undergoing apoptosis in the BALB/c mouse never exceedsthe baseline levels seen in naive animals. However, in susceptibleAKR mice, a significant elevation in apoptosis is detected atday 42 p.i., once infection has reached chronicity (P < 0.0007)(Fig. 1B, C, and D). The levels of epithelial cell proliferationwere determined by using BrdU incorporation to identify cellsin S phase of the cell cycle. In accordance with previous studies(2, 13), the levels of proliferation were elevated in chronicallyinfected animals (Fig. 1E and F). This temporal associationof proliferation and apoptosis highlights that chronic nematodeinfection can have profound effects on epithelial homeostasis,the dysregulation of which results in the generation of crypthyperplasia (Fig. 1G).

The induction of epithelial cell apoptosis is not due to physical damage elicited by the parasite. In order to investigate whether the elevation in the numbersof cells apoptosing in the intestine is simply a consequenceof the physical damage elicited by the parasites, the levelsof apoptosis observed after a low-dose infection were investigated.Mice were infected with 10 to 20 infective T. muris eggs. Wormburdens and apoptosis levels were assessed at day 42 p.i. Low-doseinfection established in AKR mice, with the mean worm burdenof 13 ± 2.16 compared to 89.6 ± 19.87 during ahigh-dose infection (Fig. 2A). If the apoptosis seen was a simpleresponse to damage caused by the physical presence of the parasite,we would expect to see much higher levels of apoptosis in animalswhere worm numbers were greater. However, there was no significantdifference between the percentage of cells undergoing apoptosisin the ceca of animals given low- or high-dose infections (Fig.2B), despite the highly significant difference in worm burdens(Fig. 2A) (P < 0.0001). It is also interesting that the cellsundergoing apoptosis were located near the base of the crypt,away from the worms, which are embedded within tunnels nearthe epithelial surface at this late stage of infection (Fig.1C and D and Fig. 2C). Indeed, no apoptosis could be seen inthe epithelial tunnels surrounding the worms (Fig. 2C). Again,this suggests that worm-induced damage is not directly responsiblefor the high levels of apoptosis seen with chronic infection.This finding is consistent with studies by Li et al. (30), whodemonstrate that Trichinella spiralis fails to induce epithelialcell apoptosis in vitro.

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FIG. 2. Apoptosis during low-dose T. muris infection. AKR mice were given either a low-dose ( ${square}$ ) T. muris infection (25 eggs) or a high-dose ( ${blacksquare}$ ) infection (200 eggs). (A) Worm burdens were assessed at day 42 p.i. (B) Apoptosis levels were assessed at day 42 p.i. Each bar represents the mean of four animals ± the S.E.M. *, the Student t test reveals a statistically significant difference between the two groups (P < 0.001). (Ci) Mature worms in epithelial tunnel at day 42 p.i. The arrow indicates the anterior end. Scale bar, 50 µm. (Cii) Image of worm showing epithelial tunnel surrounding the parasite, along with an absence of apoptosing cells in close proximity. Scale bar, 25 µm.

TNF- ${alpha}$ can stimulate epithelial cell apoptosis both in vivo (11)and in vitro (11, 20, 43). Furthermore, IFN- ${gamma}$ can induce apoptosis(36, 53). In an attempt to determine what factors might be contributingto the increase in apoptosis levels seen in the large intestineduring chronic helminth infection, the role of TNF- ${alpha}$ and IFN- ${gamma}$ were investigated. Both are produced by permissive hosts (1,15), which develop a TH1-like intestinal colitis (independentof dose) and are therefore feasible candidates for the inductionof apoptosis in this system. Indeed, the immune response toT. muris is well characterized. Elevated levels of IFN- ${gamma}$ , IL-12,IL-18, and TNF- ${alpha}$ are detected in both the MLNC and the intestinalmucosa of susceptible animals (2, 13, 15, 27). Moreover, chronicinfection is associated with enhanced leukocyte infiltrationinto the gut (31).

TNF- ${alpha}$ levels are increased in both the MLNC and the gut of animals susceptible to T. muris. The expression of TNF- ${alpha}$ in both susceptible (AKR) and resistant(BALB/c) mice was assessed in the cecum through a time courseof infection. AKR mice demonstrated enhanced mRNA levels forTNF- ${alpha}$ at days 21 and 42 p.i. compared to BALB/c mice. (Fig. 3Ai).Moreover, during the peak of cytokine production (day 21 p.i.)the message for TNF- ${alpha}$ was significantly upregulated in the mesentericlymph node of susceptible AKR mice compared to resistant mice(Fig. 3Aii). Furthermore, low-dose infection of C57BL/6 micealso results in an elevation in the levels of TNF- ${alpha}$ in the cecum(Fig. 3Aiii).

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FIG. 3. Apoptosis in TNFR-deficient animals during chronic T. muris infection. (A) The expression of TNF- ${alpha}$ in the gut (i) and MLNC (ii) of AKR (susceptible) and BALB/c (resistant) mice was assessed by PCR at various times p.i. (iii) TNF- ${alpha}$ expression in the gut tissue of C57BL/6 mice following low-dose infection was assessed by PCR. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) levels were assessed as a control transcript. N, naive animals; 21, 21 days p.i.; 42, 42 days p.i. (B) p55^–/–, p75^–/–, and WT mice were infected with T. muris. The levels of apoptosis were assessed at day 42 p.i. and are expressed as the total percentage of cells apoptosing (i) and as the percentage of cells apoptosing in the base of the crypt (positions 1 to 4) (ii) and higher up the crypt axis (positions 11 to 30) (iii). Bars: ${square}$ , naive animals; ${blacksquare}$ , infected animals. (C) The area of the epithelium was assessed at day 42 p.i. Bars: ${square}$ , naive animals; ${blacksquare}$ , infected animals. Each datum point represents the mean of four animals ± the SEM. *, ANOVA and Tukey tests showed a statistically significant increase in infected versus naive animals (P < 0.001); ${Psi}$ , ANOVA and Tukey tests showed a statistically significant reduction in gene-deficient mice compared to WT mice (P < 0.001).

p55^–/– and p75^–/– mice display significant differences in levels of apoptosis during infection. To test the hypothesis that TNF- ${alpha}$ may contribute to the elevationin apoptosis during chronic infection, we infected mice deficientin either the p55 or the p75 TNF receptor (TNFR). The p55 receptoris associated with a death domain (38) and is thought to bethe main TNFR associated with apoptosis induction (3). However,it is clear that the induction of apoptosis through the p75receptor can also occur, although the pathways are thought tooccur independently of each other (21, 22, 50). Gene-deficientanimals received a high-dose T. muris infection that progressedto chronicity. Since wild-type (WT) animals are resistant tosuch a high level of infection, they were given a low-dose levelinfection. This renders animals susceptible to infection, allowingchronic infection to develop. Therefore, both gene-deficientand WT animals were susceptible to infection with T. muris.Despite infection reaching chronicity in all three mouse strains,there were significant differences in the levels of apoptosisdetected between each strain (Fig. 3B). Infected WT mice displayeda significant increase in the levels of apoptosis versus naivemice (F, 22.95; P < 0.001) (Fig. 3Bi), but both p55 and p75^–/–animals show no significant increase. Moreover, the levels ofapoptosis in infected p55^–/– and p75^–/–animals are significantly reduced compared to WT (P > 0.001).Interestingly, p75^–/– display reduced levels ofapoptosis in comparison with p55^–/–, despite p55being the classical receptor associated with apoptosis. Thedistribution of apoptosing cells within the crypt unit alsodiffers between p55 and p75 mice. It would appear that signalingthrough p75 induces apoptosis in the base in the base of thecrypt (Fig. 3Bii ), whereas p55-induced apoptosis occurs furtherup the crypt, in cells that are more differentiated (Fig. 3Biii).These data suggest a distributional separation of both receptorswithin the crypt unit. The area of epithelium was determinedto indicate the level of hyperplasia and therefore dysregulationof intestinal homeostasis. It is apparent from Fig. 3C thatthe area of epithelium is increased in p55^–/– andp75^–/– animals over WT mice. Moreover, the elevationin crypt hyperplasia seen in p75^–/– is slightlygreater than that in p55^–/– mice, correlating witha larger reduction in epithelial cell apoptosis in the stemcell region. Ultimately, the failure of these mice to upregulateepithelial cell apoptosis to levels displayed by WT animals,despite the persistence of T. muris indicates that TNF- ${alpha}$ enhancesepithelial cell apoptosis during chronic helminth infection.Whether this is through the direct ligation of an epithelialcell specific receptor, or indirectly through the recruitmentof inflammatory cells/enhanced inflammatory mediator productionremains to be determined.

Neutralization of IFN- ${gamma}$ in SCID mice inhibits T. muris-associated epithelial cell apoptosis without altering the outcome of infection. The data presented thus far indicate that the elevated levelsof TNF- ${alpha}$ in the intestine may play an important role in the inductionof epithelial cell apoptosis during chronic T. muris infection.Coincident with TNF- ${alpha}$ expression, susceptible animals also demonstrateelevated levels of IFN- ${gamma}$ in the cecal mucosa, as has been documentedfor a high-dose infection in AKR mice (13, 2) and during low-doseinfection in C57BL/6 mice (Fig. 4A). SCID mice are deficientin B cells and T cells and therefore have no adaptive immunity.It has previously been shown that the neutralization of IFN- ${gamma}$ in SCID mice can reduce the levels of proliferating cells inthe intestine during T. muris infection (2), suggesting a rolefor IFN- ${gamma}$ in the maintenance of epithelial homeostasis. Althoughthe source of this IFN- ${gamma}$ remains unknown, it has been suggestedto be derived from NK cells, macrophages, and myeloid cells(42, 54). Moreover, it has been shown that IFN- ${gamma}$ can induce epithelialcell apoptosis (11, 43, 55). To determine whether IFN- ${gamma}$ playeda role in epithelial cell apoptosis during T. muris infection,the cytokine was neutralized in SCID mice for the duration ofinfection (42 days). SCID mice were utilized for the presentstudy since neutralization of IFN- ${gamma}$ in immunocompetent animalscauses a switch to a TH2 immune response and subsequent parasiteexpulsion. Therefore, the use of SCID mice allows the effectof IFN- ${gamma}$ neutralization on apoptosis to be investigated duringchronic infection. Indeed, all treatment groups remained susceptibleto infection (Fig. 4B). Despite the high worm numbers in allof these animals, the levels of apoptosis were, as with theTNFR^–/– animals, much lower than those detectedin immunocompetent C57BL/6 and AKR mice at day 42 p.i. (Fig.1B and 3B). Mice in which IFN- ${gamma}$ was neutralized show a significantreduction in apoptosis levels compared to control treated animals(F, 5.92; P < 0.01) (Fig. 4C and D). This suggests that IFN- ${gamma}$ plays a contributory role in the development of cecal epithelialcell apoptosis during infection. Interestingly, the neutralizationof IFN- ${gamma}$ appeared to cause the greatest reduction in the apoptosisof cells positioned at the base of the crypt (Fig. 4Di). Thesecells also show the greatest reduction in proliferation afterIFN- ${gamma}$ neutralization (2).

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FIG. 4. Apoptosis levels in SCID mice after neutralization of IFN- ${gamma}$ . SCID mice received either anti-IFN- ${gamma}$ (XMG1.6) or a control MAb GL113 at 1 mg/injection every 4 days for the duration of infection. (A) IFN- ${gamma}$ is expressed in the cecum during low-dose infection in C57BL/6 mice (B) Worm recovery from SCID mice treated with either anti-IFN- ${gamma}$ or control MAb. Each value represents a mean of four animals ± the SEM. (C) The levels of apoptosis were assessed at day 42 p.i. ( ${square}$ , naive mice; ${blacksquare}$ , infected mice). (D) The position of apoptosing cells in the ceca of infected animals was assessed in the base of the crypt (cell positions 1 to 4) (i) and higher up the crypt (cell positions 11 to 15) (ii). Each value represents the mean of four animals ± the SEM. *, ANOVA and Tukey tests revealed a statistically significant difference between naive and infected animals and between control and anti-IFN- ${gamma}$ -treated mice (P < 0.01).

The levels of IFN- ${gamma}$ produced by MLNC from TNFR^–/–animals shows WT, p55, and p75^–/– mice all makethe characteristic TH1 response with elevated levels of IFN- ${gamma}$ and IL-12 (Fig. 5A and B) and low levels of TH2 cytokines (datanot shown). However, despite the persistence of infection inall three strains, the production of parasite specific IFN- ${gamma}$ was significantly reduced in p75^–/– animals comparedto both WT and p55^–/– mice. This correlates withthe lower levels of apoptosis seen in the TNF p75^–/–animals.

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FIG. 5. The levels of IFN- ${gamma}$ and IL-12 secreted by MLNC isolated at day 42 p.i. were determined in WT, p55^–/–, and p75^–/– animals by ELISA. Each value represents the mean of four animals ± the SEM. *, ANOVA and the Tukey test showed a statistically significant increase in infected versus naive animals; ${Psi}$ , statistically significant difference between WT and gene-deficient mice (P < 0.05). Bars: ${square}$ , naive animals; ${blacksquare}$ , infected animals. ND, not detected. All cytokine levels shown are over the limit of detection.

DISCUSSION

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Discussion

We show here for the first time that chronic T. muris infectionis associated with an elevation in the level of epithelial cellapoptosis in the large intestine. Moreover, the demonstrationthat both TNF- ${alpha}$ and IFN- ${gamma}$ play a role in the development of apoptosisin a naturally occurring model of epithelial cell apoptosisis novel.

The basal levels of apoptosis in the colon are low comparedto the small intestine, and this is thought to be influencedin part by the high levels of the antiapoptotic molecule BCL-2in the bases of the crypts. Certainly, the finding that infectioninduces apoptosis within the epithelial cells positioned atthe base of the crypt is surprising.

It is evident that the upregulation of apoptosis is not merelya consequence of physical damage to the intestine induced byworm burrowing, since both low- and high-dose infections resultin equivalent levels of apoptosis. This elevation in apoptosisis only detected during chronic infection, despite the activeburrowing of larvae through the intestinal epithelium duringearly infection. Moreover, there exists spatial separation betweenthe worms and the apoptosis. During chronic infection the majorityof apoptosing cells are located at the base of the crypt, distantfrom the parasite, the anterior end of which is embedded within epithelial tunnels near the gut lumen.

The data presented here suggest that the increase in apoptosisis a product of the host response to chronic infection ratherthan infection per se. Apoptosis is detected only when infectionreaches chronicity, at which point high levels of proinflammatorycytokines are detected in the intestinal mucosa of infectedanimals (Fig. 3 and 4). Infection of TNF- ${alpha}$ receptor-deficientanimals revealed TNF- ${alpha}$ signaling to play a role in the inductionof intestinal apoptosis associated with chronic infection, withboth p55^–/– and p75^–/– animals demonstratinga significant reduction in apoptosis levels compared to theWT strain. Signaling through either the p55 or the p75 receptoralone shows a reduction in apoptosis levels. This suggests thatsignaling through both receptors is important for optimal apoptosisinduction. This has been demonstrated in in vitro experimentswith IEC-6 cells, where stimulation through both p55 and 75resulted in enhanced apoptosis (32).

Although SCID animals lack B and T cells, it is assumed thatother sources of both IFN- ${gamma}$ and TNF- ${alpha}$ do exist. After in vivodepletion of IFN- ${gamma}$ , we see a complete loss of apoptosis. TNF- ${alpha}$ fails to act in a compensatory manner in these animals (althoughthe levels may be subthreshold). It is interesting that thelevels of apoptosis in SCID animals with or without treatmentare much lower than those seen during chronic infection in animmunocompetent animal. This suggests that during chronic T.muris infection T-cell-derived cytokines may be important inmediating the apoptosis seen during infection of immunocompetentmice. Activated T cells have been identified as key playersin driving the mucosal pathology associated with a number ofinflammatory diseases of the intestine, including GVHD, celiacdisease, and the inflammation seen during anti-CD3 treatment(4, 32, 34, 51). Interestingly, the pathology associated withGVHD bears striking similarities with that seen during gut nematodeinfection. As with T. muris infection, the development of bothcrypt hyperplasia and epithelial cell apoptosis has been reportedin GVHD (24, 37, 47, 48). Moreover, a role for both IFN- ${gamma}$ andTNF- ${alpha}$ in the development GVHD-related pathology is clear (12,14, 48). Indeed, the neutralization of TNF- ${alpha}$ inhibits the developmentof GVHD-associated epithelial cell apoptosis (12, 47).

Many questions still exist regarding the mechanism of actionfor both TNF- ${alpha}$ and IFN- ${gamma}$ in this system. Whether the cytokinesact through direct ligation of receptors on epithelial cellsremains to be determined. It is certainly possible that TNF- ${alpha}$ may promote apoptosis in an indirect manner by enhancing theproduction of IFN- ${gamma}$ and by the recruitment of inflammatory cellsinto the intestinal microenvironment. The release of free radicalsand inflammatory mediators by infiltrating cells can induceoxidative stress, DNA damage, and epithelial cell apoptosis.The synergistic nature of these cytokines should not be ignored;a number of studies have demonstrated that IFN- ${gamma}$ and TNF- ${alpha}$ canact together to inhibit cell growth (18). Moreover, it has beenshown that that IFN- ${gamma}$ can upregulate TNFR expression (44).

The elevated level of apoptosis found in the gut during chronicT. muris infection is coincident with a profound crypt cellhyperplasia and epithelial cell proliferation (Fig. 1, 3, and4) (2, 13). It is reasonable to suggest that the induction ofapoptosis is acting in favor of the host to regulate crypt size.Certainly, there must exist a crypt length, which when exceededwill be deleterious for the host. Apoptosis would, therefore,be an effective regulatory mechanism to bring about homeostasisat the epithelial level. Indeed, apoptosis occurs predominantlynear the base of the crypts, in the stem cell compartment. Ultimately,death of a stem cell would have much more profound effect onlimiting crypt size than would death of a differentiated enterocyte.Whether the induction of apoptosis in this compartment occursduring chemical insult or chronic inflammation is difficultto determine from the literature, since positional analysisof apoptosing cells rarely performed. However, it is feasibleto suggest that this mechanism of crypt cell regulation mayoperate in a number of inflammatory conditions to curb the developmentof excessive hyperplasic change.

Taken together, these data support a "positive" role for proinflammatorycytokines in the induction of apoptosis at the epithelial levelby inducing intestinal homeostasis in an attempt to controlthe dysregulation of epithelial architecture seen during T.muris infection. Although the induction of both proinflammatorycytokines and apoptosis are typically thought to be detrimentalduring intestinal colitis, they may in fact act in a host-protectivemanner during chronic T. muris infection by controlling thelevel of homeostatic dysregulation in the gut. Ultimately, thisis beneficial for both host and parasite.

ACKNOWLEDGMENTS

This study was supported by BBSRC, The Wellcome Trust, and Epistem,Ltd.

FOOTNOTES

* Corresponding author. Mailing address: Michael Smith Building, University of Manchester, Oxford Road, Manchester M139PT, United Kingdom. Phone: 44 161 2755240. Fax: 44 161 2751498. E-mail: richard.k.grencis{at}manchester.ac.uk.

Published ahead of print on 22 January 2007.

Editor: J. F. Urban, Jr.

Present address: Department of Biochemistry and Molecular Biology,Life Sciences, University of Georgia, Athens, GA 30602.

REFERENCES

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