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The Journal of Immunology, 2005, 175: 3207-3213.
Copyright © 2005 by The American Association of Immunologists

Intraepithelial NK Cell-Derived IL-13 Induces Intestinal Pathology Associated with Nematode Infection1

Jacqueline R. McDermott*, Neil E. Humphreys*, Simon P. Forman*, Debra D. Donaldson{dagger} and Richard K. Grencis2,*

* Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom; and {dagger} Department of Respiratory Disease, Wyeth Research, Cambridge, MA 02140


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-13 is a Th2-derived cytokine associated with pathological changes in asthma and ulcerative colitis. Moreover, it plays a major role in the control of gut nematode infection and associated immunopathology. The current paradigm is that these effects are due to T cell-derived IL-13. We show in this study that an innate source of IL-13, the intraepithelial NK cell, is responsible for the disruption of intestinal tissue architecture and induction of goblet cell hyperplasia that characterizes infection with the intestinal helminth Trichinella spiralis. IL-13 or IL-4R{alpha} (but not IL-4) null mice failed to induce intestinal pathology. Unexpectedly, SCID and athymic mice developed the same pathology found in immunocompetent mice following infection. Moreover, immunodeficient mice expressed IL-13 in the intestine, and abnormal mucosal pathology was reduced by in vivo administration of a soluble IL-13 antagonist. IL-13 expression was induced in non-T intraepithelial CD3– NK cells. Epithelial cells expressed the IL-13 signaling receptor, IL-13R{alpha}1, and after infection, IL-4R{alpha}. Furthermore, the soluble IL-13 decoy receptor IL-13R{alpha}2, which regulates IL-13 responses, was also induced upon infection. These data provide the first evidence that intestinal tissue restructuring during helminth infection is an innate event dependent on IL-13 production by NK cells resident in the epithelium of the intestine.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Many mucosal diseases are characterized by abnormal pathology, in which the tissue architecture is disrupted by the actions of a variety of cytokines. Inflammatory bowel disorders induce intestinal pathology that is associated with elevated proinflammatory cytokines (1, 2). Levels of TNF-{alpha} and IFN-{gamma} are high in Crohn’s disease (1), whereas IL-13, a Th2-type cytokine, drives the pathophysiology of oxazalone colitis, an experimental model with pathological features similar to ulcerative colitis (1, 3). Further evidence points to IL-13 as the factor responsible for the pathogenesis of asthma (4, 5, 6, 7). For example, administration of IL-13 to the airways of mice results in distinctive asthmatic pathology of inflammatory cell infiltration, airway hyperresponsiveness, and mucus hypersecretion (4, 5), while mice genetically deficient for IL-13 fail to develop this pathology when challenged with model airway allergens (6). IL-13-secreting NKT cells have been implicated in the pathogenesis of both experimental asthma and ulcerative colitis models (3, 8), and NK cells have been implicated in the development of allergen-induced airway inflammation in mice (9), although it is not known whether they contribute to the responses following Th2-inducing infection. Many pathological features associated with both asthma and inflammatory bowel disorders are identical with those induced by intestinal nematode infection of both the small and large bowel (3, 7, 10). These parasites induce in the host a strong Th2 response (10), but the etiology, in particular the role of cytokines, in intestinal tissue remodeling during these infections is poorly defined. In this study, we have sought to determine the role of IL-13 on enteric pathology during infection with the intestinal nematode, Trichinella spiralis, in a mouse model.

T. spiralis occupies a niche within enterocytes of the jejunum and elicits a well-characterized type 2 cytokine response orchestrated by CD4+ T cells (11). Following infection, CD4+ T cells express IL-4, IL-5, IL-9, and IL-10 in the mesenteric lymph nodes (11) and are subsequently recruited to the gut mucosa (12). In addition, there is a marked eosinophilia in response to IL-5 (13) and a pronounced mastocytosis (14). Expulsion of the parasites from the intestine is dependent on mast cells recruited to the mucosa in response to IL-4 and IL-9 (15, 16, 17). Furthermore, there are goblet cell hyperplasia and mucus hypersecretion that may provide mucosal protection for the host or create an inhospitable environment for the parasite (18). Moreover, distinctive changes in villus and crypt architecture occur that may also be an attempt by the host to drive out the parasite by diminishing the area available for habitation (18). T cells are required for worm expulsion from the gut, but this is not IL-4 dependent, because mice genetically deficient for IL-4 can expel normally (19). However, expulsion is inhibited in mice deficient in the IL-4/IL-13 signaling molecules IL-4R{alpha} or STAT6, suggesting a role for IL-13 (19, 20). IL-13 is certainly critical for expulsion of another gastrointestinal nematode, Nippostrongylus brasiliensis, which inhabits the lumen of the small intestine (21), and resolution of Trichuris muris infection of the large bowel is also dependent on IL-13 via the production of TNF-{alpha} (22). In addition, both T. muris (23) and N. brasiliensis (24) infection generate changes in mucosal tissue architecture, including villus atrophy, crypt hyperplasia, and thickening of the muscularis externa. The causative factor or factors for these changes are unknown, although IFN-{gamma} has been implicated in enterocyte proliferation during T. muris infection (23). IL-13 is also known to induce liver fibrosis in schistosomiasis (25), and is a major contributor to pulmonary granuloma in a mouse model of the same disease (26). Although T cells have been assumed to be the source of IL-13 during helminth infection, this has not been confirmed, and the possibility of an innate source of the cytokine has not been addressed. Likewise, the cellular source and target for IL-13 in asthma have yet to be definitively identified. A fundamental question relates to a possible role of IL-13 in tissue remodeling during intestinal helminth infections. In this study, using T. spiralis infection as a model of Th2-induced inflammation in the small intestine, we have investigated the effect of IL-13 on enteric tissue restructuring and attempted to identify its cellular source. We show that IL-13 is responsible for villus atrophy and crypt and goblet cell hyperplasia, and that it is not driven by the adaptive immune response, but rather by early innate responses to infection. SCID and athymic mice that cannot mount adaptive immune responses exhibit the same enteropathy as immunocompetent mice. Following infection, IL-13 is up-regulated in the jejunum of these mice and is localized to the epithelial layer. Moreover, the potent source of the IL-13 is a population of intraepithelial dwelling CD3– NK cells. Finally, blockade of IL-13 activity by IL-13 decoy receptor significantly reduces infection-associated intestinal pathological changes. These data show for the first time that disruption of mucosal tissue architecture induced by T. spiralis infection is generated by nonadaptive immune events and is IL-13 dependent, but T cell independent. Also, IL-13 is produced by NK cells in response to infection and is the factor responsible for disease-associated changes in tissue architecture in this model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and infection

BALB/c, C.B-17 SCID, and BALB/c nu/nu mice were purchased from Harlan-Olac. IL-4–/– (27), IL-13–/– (28, 29), and IL-4R{alpha}–/– (30) mice were generated, as described, and bred at University of Manchester under specific pathogen-free conditions. All transgenic mice were on a BALB/c background. Mice were infected with T. spiralis at 6–8 wk of age. Maintenance, infection, and recovery of T. spiralis were as previously described (31). Mice were infected by oral gavage with 300 larvae on day 0. All experiments were performed under the regulations of the Home Office Scientific Procedures Act.

Worm burden

Small intestines were removed on day 14 postinfection (p.i.) and opened longitudinally. Intestines were placed in gauze and incubated in PBS at 37°C. The number of worms suspended in PBS was counted after 4 h.

Histology

Sections of jejunum were taken at 15 cm proximal to the pylorus days 7–8 p.i. They were fixed in neutral buffered formaldehyde (NBF)3 overnight, then processed and embedded in paraffin. Tissue sections (5 µm) were cut, dewaxed, rehydrated, and stained with periodic acid and Schiff’s reagent (PAS) or PAS, followed by Alcian blue to visualize goblet cells. After washing, sections were counterstained with Mayer’s hemalum and mounted. The number of goblet cells per 20 villus crypt units (VCU) was counted on each section. The length of 20 villi and the depth of 20 crypts per section were determined using a graticule eye piece.

Blocking IL-13 in vivo

Soluble IL-13R{alpha}2-Fc (kind gift from D. Donaldson, Wyeth Research, Cambridge, MA) or a control fusion protein was administered (200 µg/mouse) i.p. to SCID and BALB/c mice on days 0, 2, 4, and 6 p.i.

Isolation of epithelium

Sections of jejunum (15 cm proximal to the pylorus) were removed days 7–8 p.i. and flushed with ice-cold, Ca2+-free HBSS (Invitrogen Life Technologies). Sections were trimmed of fat, mesentery, and Peyer’s patches, and cut into 2-mm2 pieces that were washed in HBSS containing 2% FCS (Invitrogen Life Technologies). Tissues were incubated in HBSS supplemented with 10% FCS, EDTA (Sigma-Aldrich), and DTT (Sigma-Aldrich). After 15 min, tissues were shaken vigorously and supernatant containing epithelium was removed. This was repeated once. Epithelia were washed in HBSS twice and passed through a 40-µm cell strainer. Cells were resuspended in either RPMI 1640 (Invitrogen Life Technologies) for flow cytometry or TRIzol (Invitrogen Life Technologies) for RNA extraction.

RNA extraction and RT-PCR

Epithelia or sections of jejunum were homogenized in TRIzol, and total RNA was extracted, according to the manufacturers’ protocol. Reverse-transcriptase reaction was performed using ImProm-II (Promega). The following primers were used to determine the level of specific mRNA: IL-13, sense, 5'-ctccctctgacccttaaggag-3', and antisense, 5'-gaaggggccgtggcgaaacag-3'; IL-13R{alpha}1, sense, 5'-gcacgataatatggacgtgg-3', and antisense, 5'-ttgacgacttttctccaggc-3'; IL-13R{alpha}2, sense, 5'-atggcttttgtgcatatcagatgct-3', and antisense, 5'-gacaaatgcgtatctt-3'; IL-4R{alpha}, sense, 5'-gagtgagtggagtcctagcatc-3', and antisense, 5'-gctgaagtaacagaacaggc-3'; TNF-{alpha}, sense, 5'-tcttctcattcctgcttgtgg-3', and antisense, 5'-gacaacctgggagtagacaaggt-3'.

DNA was amplified by a Dyad DNA engine (MJ Research) under the following conditions: IL-13 and TNF-{alpha}, 35 cycles of 1 min at 95°C, 30 s at 60°C, and 1 min at 72°C; IL-13R{alpha}1, 35 cycles of 1 min at 95°C, 30 s at 62°C, and 1 min at 72°C; IL-13R{alpha}2, 35 cycles of 1 min at 95°C, 30 s at 54°C, and 1 min at 72°C.

Flow cytometry and cell sorting

Cells were blocked with Fc block (anti-CD16, CD32) (BD Pharmingen) for 10 min. The following primary Abs were applied to cells on ice for 30 min: anti-CD45 PE and IgG2b PE (Serotec); anti-{gamma}{delta} TCR FITC, anti-CD49b PE, anti-CD4 PE, anti-CD8 PE, hamster IgG FITC, and rat IgM FITC (BD Pharmingen). Cells were washed and fixed with 1% formaldehyde in PBS. Cells were run through a FACSCalibur (BD Biosciences), and data were analyzed using CellQuest Pro. Isolated epithelial cells were further purified, and minority intraepithelial lymphocyte (IEL) populations were identified and sorted using a FACSVantage (BD Biosciences) cell sorter. Abs used to identify subpopulations of IELs were as described above. Epithelial cells were identified using FITC-labeled UEA-1, a lectin that has previously been used to sort purify small intestinal epithelial cells (32). For NK cell (CD49b+) sorting, anti-CD3{epsilon} FITC (BD Pharmingen) was used to ensure that selected NK cells were CD3 negative. Sorted cells were either placed directly in TRIzol (Invitrogen Life Technologies) for RNA extraction, or in RPMI 1640 for further cell culture.

For intracellular cytokine staining of sorted purified intraepithelial NK cells, isolated cells were stimulated for 4 h at 37°C with PMA (50 ng/ml) and ionomycin (500 ng/ml). Brefeldin A (1 µg/ml) (all Sigma-Aldrich) was added for the final 2 h of culture. After washing, cells were fixed in 2% formaldehyde for 20 min and washed in 0.1% saponin. Biotinylated anti-IL-13 (PeproTech) Ab was then added, or isotype control; these cells were further incubated with streptavidin-allophycocyanin (Caltag Laboratories). Cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest Pro software.

Statistics

Statistical significance was determined using Student t test. Values of p <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Effect of IL-4 and IL-13 on intestinal pathology induced by T. spiralis

IL-4 and IL-13 are key components of Th2 responses; therefore, we wanted to investigate their respective effects on T. spiralis expulsion and intestinal tissue pathology. On day 8 p.i., the small intestines of wild-type BALB/c mice showed a significant increase in intestinal goblet cell numbers, increased crypt depth, and decreased villus length compared with uninfected mice (Fig. 1, A, B, F, and G). Goblet cells appeared larger in infected animals, indicating hypersecretion of mucus. In addition, the muscularis externa was thickened and villi were truncated and had formed clubbed branches. Tissue architecture of infected IL-4-deficient mice was similarly disrupted, although there was no significant increase in numbers of goblet cells (Fig. 1, C, F, and G). However, villus atrophy was absent from both IL-4R{alpha} (Fig. 1, D and F)- and IL-13-deficient mice (Fig. 1, E and F), and although infected crypts were deeper than those of naive IL-4R{alpha}- or IL-13-deficient mice, they showed significantly less hyperplasia than infected wild-type animals. Numbers of goblet cells were not elevated in IL-4-, IL-4R{alpha}-, or IL-13-deficient mice following infection (Fig. 1G). On day 14 p.i., both BALB/c wild-type mice and IL-4-deficient mice were expelling their worm burdens, but mice deficient for either IL-4R{alpha} or IL-13 still harbored significant numbers of worms (Fig. 1H).



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  		FIGURE 1. Effect of IL-13 and IL-4 on T. spiralis-induced enteropathy and worm expulsion. Representative plates showing jejuna sections fixed in NBF and stained with PAS and Alcian blue/Mayer’s hemalum, from naive (A) and day 8 p.i. BALB/c mice (B) and day 8 p.i. IL-4–/– (C), IL-4R{alpha}–/– (D), and IL-13–/– mice (E). ME, muscularis externa; bar = 100 µm. Jejunum villi length (•) and crypt depth ({circ}) were measured in each case (F); points represent mean of four mice/group ± SE. Changes in villi length and crypt depth response between days 0 and 8 were assessed for significance within each mouse strain: *, p < 0.05; **, p < 0.01. In addition, the villi and crypt measurements at day 8 p.i. from BALB/c mice were compared with those from knockout mice for significant difference: IL-4R{alpha}–/– vs BALB/c, {dagger}{dagger}, p < 0.005; and IL-13–/– vs BALB/c, {dagger}, p < 0.05. Goblet cell count within 20 VCU (G); *, p < 0.05, significantly different from naives. Intestinal worm burdens at day 14 p.i. (H); *, p < 0.05, significantly different from BALB/c; four mice/group ± SE.

 
Role of adaptive immune response in nematode-induced intestinal pathology

Given that disruption to intestinal architecture appears early on in infection, we were interested in whether this was a T cell effect. We infected athymic mice with T. spiralis, and although they were unable to efficiently expel their worm burden as expected, their intestinal pathology was identical with infected wild-type BALB/c mice (Figs. 1F and 2, A and B). This indicated that normal T cells of thymic origin were not responsible for abnormal intestinal pathology associated with infection. To further explore the possibility that pathology was the result of innate immune events, we infected SCID mice with T. spiralis. Similar to athymic mice, infected SCID mice developed a significant crypt hyperplasia, goblet cell hyperplasia, and villus atrophy compared with uninfected animals, indicating that neither T nor B cells are required to induce this effect (Fig. 2).



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  		FIGURE 2. Enteropathy of T. spiralis-infected immunodeficient mice. Villi length and crypt depth (A) and goblet cells per 20 VCU (B) measured in jejunum in naive and day 8 p.i. athymic mice and SCID mice. Representative plates showing jejuna sections fixed in NBF and stained with PAS/Mayer’s hemalum from naive (C) and day 8 p.i. SCID mice (D). Bar = 100 µm. *, p < 0.05; **, p < 0.01; ***, p < 0.001, significantly different from naive animals. Four mice/group ± SE.

 
Expression of IL-13 in the absence of adaptive immunity

Results from infected cytokine-deficient mice showed that IL-13 and IL-4R{alpha} were required for the maximal development of anomalous tissue architecture. Because immunodeficient mice also developed this pathology, we investigated whether IL-13 was up-regulated in the absence of an adaptive immune response. We found that SCID mice expressed IL-13 mRNA in the total jejunum, but only when infected (Fig. 3A). In addition, we observed that mRNA for IL-13R{alpha}1, which combines with IL-4R{alpha} to allow IL-13 signaling, was expressed in the total jejunum of both naive and infected SCID mice (Fig. 3B). Furthermore, we found that mRNA for IL-13R{alpha}2 is also up-regulated in the jejunum following infection, indicating that the gut is attempting to regulate the response (Fig. 3C). Previous work has shown that IL-13 is required for resistance to T. muris within the large bowel and that its action is due to the induction of TNF-{alpha} (22). Moreover, others have shown that the effect of IL-4 during T. spiralis infection is due to its induction of TNF-{alpha} (33). Given that TNF-{alpha} is an important regulator of intestinal pathology in Crohn’s disease (1), we chose to investigate whether the action of IL-13 was due to it up-regulating TNF-{alpha}. Using mesenteric lymph nodes and jejunum of immunocompetent infected BALB/c mice as positive controls for TNF-{alpha} expression, we were unable to detect significant amplification of TNF-{alpha} mRNA in the intestine of infected SCID mice at a time point when aberrant pathology was established and IL-13 is abundant (Fig. 3D), thus suggesting that IL-13 alone mediates pathology in SCID mice.



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  		FIGURE 3. Jejunal IL-13, IL-13R{alpha}1, IL-13R{alpha}2, and TNF-{alpha} expression in absence of adaptive immunity. RNA was extracted from jejuna of naive SCID or day 8 p.i. (three animals per group). PCR products were run on agarose gel containing ethidium bromide. IL-13 (A), IL-13R{alpha}1 (B), IL-13R{alpha}2 (C), TNF-{alpha} (D). Positive control for TNF-{alpha}: BALB/c jejunum (++) and mesenteric lymph node (+) day 8 p.i.

 
Abrogation of IL-13 in the absence of adaptive immunity

To determine whether IL-13 was indeed responsible for nematode-induced intestinal pathology in immunodeficient mice, infected SCID mice were infected with T. spiralis and IL-13 function blocked by administration of soluble IL-13R{alpha}2 Ig fusion protein. Infected mice that received the control fusion protein displayed crypt hyperplasia and villus atrophy, whereas those that received the soluble (s)IL-13R{alpha}2 fusion protein had significantly longer villi and reduced crypt depth (Fig. 4, A, B, and D). sIL-13R{alpha}2 treatment did not completely block goblet cell hyperplasia, but it was significantly reduced compared with control infected animals (Fig. 4, A–C).When immunocompetent BALB/c mice were treated with sIL-13R{alpha}2, we found a small reduction in goblet cell numbers following infection, although not significant (Fig. 4C); however, as found with SCID mice (Fig. 4, A and B), treatment did affect goblet cell size, with cells appearing smaller, suggesting a reduction of mucus secretion. It is possible that greater amounts of fusion protein are required in immunocompetent mice to completely block goblet cell hyperplasia. sIL-13R{alpha}2 treatment of BALB/c mice also blocked infection-induced villus atrophy and crypt hyperplasia (Fig. 4B and D).



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  		FIGURE 4. Effect of neutralizing IL-13 on T. spiralis-induced enteropathy. Representative plates showing PAS/Mayer’s hemalum staining within SCID mice treated with a control fusion protein (A) or sIL-13R{alpha}2 (B), day 7 p.i. Bar = 100 µm. Net changes ({Delta}) in goblet cell number per 20 VCU (C) and villus length and crypt depth (D) plotted for BALB/c ({blacksquare}) and SCID ({cjs2108}) mice, with or without sIL-13R{alpha}2. Histograms represent mean of four mice/group. **, p < 0.01.

 
Innate source of intestinal IL-13 during nematode infection

Immunodeficient SCID mice expressed IL-13R{alpha}1 in the jejunum constitutively and IL-13 following infection (Fig. 3, A and B). We were interested in investigating the source of IL-13 in the intestine and the cells that express its receptor. Epithelium was isolated from jejuna of naive or infected SCID mice and analyzed by RT-PCR for IL-13 and IL-13R expression. We found that IL-13 mRNA was up-regulated in the epithelium following infection, and that these cells also expressed IL-13R{alpha}1, suggesting that they can directly respond to IL-13 (Fig. 5, A and B). This suggests that both the cellular source and the responding population of cells reside within the epithelial fraction of the jejunum. It is known that SCID mice have a low number of immune cells, such as NK cells in the spleen, and that athymic mice have small numbers of nonthymic generated intraepithelial cells in the gut. Small intestinal epithelium of SCID mice does not contain CD3+ cells (34), but the total cellular composition of the intestinal epithelium in SCID mice is not fully known. To determine the cellular source of IL-13, we first analyzed the cellular composition of jejunal epithelium from both BALB/c and SCID mice by flow cytometry (Fig. 5, C and D). Just over 10% of cells within the jejunal epithelium of naive BALB/c mice expressed the leukocyte surface molecule CD45. Following infection, the percentage of CD45+ intraepithelial cells in BALB/c mice decreases, probably reflecting the diluting effect of increased epithelial cell proliferation. NK cells (expressing CD49b), a possible candidate population for IL-13 secretion (3, 35), make up ~2% of BALB/c jejunal epithelium with a similar percentage in SCID mice. The remaining 7% of CD45+ cells in BALB/c mice that were negative for {gamma}{delta} TCR or CD49b were not identified, but were likely to be B cells or {alpha}{beta} T cells.



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  		FIGURE 5. Epithelial expression of IL-13 and IL-13R{alpha}1 following T. spiralis infection. Jejunal epithelium was isolated from naive SCID mice or day 8 p.i. RNA was extracted and RT-PCR performed. PCR products were run on ethidium bromide containing agarose gel. Epithelial IL-13 (A) and IL-13R{alpha}1 (B). Three animals per group. Percentage of leukocytes as assessed by FACS in the total epithelial cell compartment of naive and infected BALB/c (C) and SCID mice (D). Four to five animals per group ± SE.

 
To precisely identify the source of IL-13 from isolated epithelium, cells from day 7 infected wild-type and SCID mice were sorted by flow cytometry into columnar epithelial UEA-1+ CD45– cells and UEA-1– CD45+ leukocytes (Fig. 6, A and D). In naive BALB/c mice, RT-PCR analysis clearly showed that IL-13 mRNA is not present in either population. Upon infection, IL-13 mRNA is up-regulated only in the sorted UEA-1– CD49b+ population (Fig. 6B). It was not present in either intraepithelial sorted CD4+ or CD8+ cells. Correspondingly, IL-13R{alpha}1 mRNA was only expressed by UEA-1+ CD45– cells. We have previously shown that IL-13mRNA was not expressed in jejunal tissue from naive SCID mice (Fig. 3A); we now show that the CD49b+ population from the epithelium of infected SCID mice expresses IL-13mRNA. Furthermore, mRNA for IL-13R{alpha}1, IL-4R{alpha}, and IL-13{alpha}2 were all expressed by the UEA-1+ CD45– population of cells (Fig. 6C). FACS analysis confirmed that the majority of CD45+ SCID IELs are also CD49b+, and, using intracellular cytokine staining, it was also confirmed that IL-13 protein was produced by the CD45+ population from the epithelium of infected SCID mice (Fig. 6D).



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  		FIGURE 6. Determination of cellular source of IL-13 within infected epithelium. Epithelium was stripped from naive or day 7 p.i. jejuna of BALB/c or SCID mice, and double stained using either pan-leukocyte marker CD45, CD49b, CD4, or CD8 and UEA-1. Representative data from BALB/c CD45, UEA-1 staining, and resulting FACS-sorted populations are shown in A. mRNA expression for IL-13 and IL-13R{alpha}1 for naive and infected BALB/c mice (B) and for IL-13, IL-13R{alpha}1, IL-13R{alpha}2, and IL-4R{alpha} for naive and infected SCID mice (C) was assessed in sorted populations, five animals per group. Representative CD45 and UEA-1 FACS data from the total jejunal epithelium of SCID mice (Di); CD49b staining on total IEL population isolated from infected SCID jejunal epithelium (Dii) (open histogram isotype control; closed CD49b staining). Intracellular fluorescent staining for IL-13 within jejunal epithelial cells isolated and sorted for CD45 from infected SCID mice day 7 p.i. (Diii), five animals per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The data presented in this work demonstrate for the first time that enteropathy associated with gastrointestinal nematode infection does not require T lymphocytes or indeed the presence of acquired immune responses. Moreover, by the use of mice lacking IL-4, IL-13, or IL-4R{alpha}, we have shown that IL-4 is dispensable, but IL-13 is essential for the enteropathy observed in both wild-type and immunodeficient animals. In vivo blocking of IL-13-induced crypt hyperplasia and villus atrophy in infected immunodeficient animals confirmed the functional presence of the protein and its role in enteropathy. Finally, we show IL-13 produced by NK cells is responsible for the intestinal tissue restructuring and goblet cell hyperplasia associated with T. spiralis infection. This could also be the factor that generates the same pathology during infection by other intestinal nematodes such as N. brasiliensis and T. muris. Moreover, NK cells may also be the source of IL-13 that induces at least some of the lung pathophysiology of allergic inflammation in the lungs (9). We have shown that IL-13 expression can be induced in the absence of B and T cells, and as such there raises the intriguing question of what factors or cell type may be stimulating IL-13 production from NK cells. In the context of infection, the parasite itself may induce secretion of the cytokine during mechanical disruption of the epithelium, or in the case of asthma it may be an allergen. It is unclear whether IL-13 acts directly or indirectly. In this study, we have ruled out TNF-{alpha}, but others have shown that lung epithelial proliferation is due to TGF-{alpha} induced by IL-13 (36) and that lung fibrosis is induced by TGF-{beta} under the control of IL-13-secreting macrophages (37). A number of growth factors have also been implicated in intestinal epithelial proliferation, including epidermal growth factor (38) and keratinocyte growth factor (39). However, any relationship with IL-13 has yet to be defined. We have shown that intestinal epithelial cells express IL-13R{alpha}1 and IL-4R{alpha}, which would allow binding of IL-13 with high affinity. This indicates that intestinal epithelial cells are ideally placed to directly respond to the NK cell-derived IL-13. Equally, Kuperman et al. (7) showed that IL-13-regulated features of asthma could only be induced if STAT-6 was expressed in lung epithelial cells. Furthermore, either rIL-13 or rIL-4 applied to the airways can elicit their effects on the lung in the absence of both T and B cells. However, neither cytokine has any effect in IL-4R{alpha}-deficient mice, thus indicating that the responder cell is an IL-4R{alpha}-expressing nonhemopoietic cell (5). Other cell types of the lung and intestine can also respond to IL-13. Both IL-4 and IL-13 can be detected in intestinal smooth muscle during nematode infection (40) and are required, with STAT-6, to induce muscle contractility, a putative host mechanism for parasite expulsion (40, 41). Likewise, airway smooth muscle can produce IL-13 and has been linked to an autocrine pathway inducing smooth muscle hypertrophy, a feature of asthma pathophysiology (42). Moreover, a gene array study by Lee et al. (43) has highlighted vast numbers of distinctive genes that are switched on following IL-13 treatment of lung epithelial cells, fibroblasts, and smooth muscle cells.

Previous studies have shown that IL-13 is important for the development of intestinal goblet cell hyperplasia in N. brasiliensis infection (28) and that hyperplasia is induced only in the presence of STAT-6 during T. spiralis infection (44). In addition to villus/crypt architectural disruption, we have shown that IL-13 controls the development of mucus-secreting goblet cells during T. spiralis infection. Accordingly, Shekels et al. (45) showed that during T. spiralis infection, levels of mucus secretion and mucin gene expression were not affected in the absence of IL-4, IFN-{gamma}, or TNF-{alpha}. Observations in mouse models of asthma have shown that IL-13 induces mucus hypersecretion in the airways (4, 5, 6, 7). Intestinal goblet cells are derived from epithelial stem cells in the crypts of Lieberkühn, although the IL-13-driven mechanism of differentiation of these cells or those in the airway is not known. Expression of IL-13Rs or IL-4Rs has yet to be identified on goblet cells. However, there is evidence that stimulation of epidermal growth factor receptors up-regulates mucin genes and goblet cell numbers in airway epithelial cells (46) and that this is under the control of IL-13 (47). Additionally, Atherton et al. (48) showed that IL-13 can act directly on bronchial epithelial cells to induce goblet cell hyperplasia via MAPK and PI3K pathways.

Recent data have clearly shown that a second IL-13R acts as a regulator of IL-13-induced inflammation (49, 50). Soluble IL-13R{alpha}2 binds IL-13 with high affinity, but does not provide a signal (51, 52). Instead it mops up excess IL-13, dampening down the response. Schistosome-infected mice deficient for IL-13R{alpha}2 displayed exacerbated liver fibrosis, but this pathology was ameliorated when mice were treated with exogenous sIL-13R{alpha}2-Fc fusion protein (49). We report that this second IL-13R is up-regulated in the jejunum in response to T. spiralis infection, suggesting that it is a control mechanism for IL-13-induced intestinal inflammation, at least in the absence of T and B cells. Furthermore, IL-13R{alpha}2 is expressed by epithelial cells themselves, suggesting a homeostatic control mechanism is in place.

This is the first study that identifies IL-13 as the critical cytokine in the generation of the aberrant intestinal pathology characteristic of nematode infection. A previous study has shown that TNF-{alpha}, under the control of IL-4, is important for villus atrophy and crypt hyperplasia during T. spiralis infection (33). Our study has found that the absence of IL-4 had no effect on tissue architecture or parasite resistance, but suggests that it may be required for optimal goblet cell hyperplasia in concert with IL-13. Furthermore, TNF-{alpha} was absent in the intestine of infected immunodeficient mice that developed abnormal enteropathy. These different findings may be attributable, at least in part, to the different mouse strains used in these studies. STAT-6 is required for both IL-4 and IL-13 signaling, and is essential for the expulsion of both T. spiralis and N. brasiliensis from the intestine (20, 21). We have shown that IL-13 (and IL-4R{alpha}), but not IL-4, is required for T. spiralis expulsion. Urban et al. (20) found that worm expulsion was enhanced when mice were treated with exogenous IL-4, but, like us, found that mice deficient for IL-4 expel their worm burden at the same rate as wild-type mice. They also showed that IL-4-deficient mice failed to expel if treated with sIL-13R{alpha}2, thus indicating that both IL-13 and IL-4 are important in resistance to this nematode. Interestingly, IL-4R{alpha} expression by nonhemopoietic cells is enough to expel N. brasiliensis from the intestine, whereas expulsion of T. spiralis requires receptor expression by both bone marrow- and nonbone marrow-derived cells (19). This indicates that IL-13 and/or IL-4 can directly stimulate intestinal epithelial cells. However, the relative effects of compartmentalized IL-4R{alpha} expression on nematode-induced changes in tissue architecture remain to be defined.

The present study clearly shows that aberrant tissue pathology associated with intestinal nematode infection is a nonadaptive immune event orchestrated by NK cell production of IL-13 acting directly upon the adjoining epithelium. It also raises the distinct possibility that this is an integral component of host-protective immunity generated against mucosal invading pathogens, and that changes previously believed to be solely under T cell control may in fact be mediated by cellular components of the innate immune system. Moreover, this finding may shed further light on the pathogenesis of both asthma and IL-13-controlled intestinal colitis.


   Acknowledgments
 
We are grateful to Dr. Andrew McKenzie for providing breeding pairs of IL-13–/– mice, Frank Brombacher for breeding pairs of IL-4R{alpha} mice, and Drs. E. B. Bell and A. J. Bancroft for helpful comments on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by the British Medical Research Council and Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Richard K. Grencis, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, U.K. E-mail address: Richard.K.Grencis{at}manchester.ac.uk Back

3 Abbreviations used in this paper: NBF, neutral buffered formaldehyde; IEL, intraepithelial lymphocyte; PAS, periodic acid and Schiff’s reagent; p.i., postinfection; sIL, soluble interleukin; VCU, villus crypt unit. Back

Received for publication March 8, 2005. Accepted for publication June 9, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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