Nematode Infection Induces Intestinal Pathology Associated With

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

This information is current as Jacqueline R. McDermott, Neil E. Humphreys, Simon P. of September 29, 2021. Forman, Debra D. Donaldson and Richard K. Grencis J Immunol 2005; 175:3207-3213; ; doi: 10.4049/jimmunol.175.5.3207 http://www.jimmunol.org/content/175/5/3207 Downloaded from

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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,† and Richard K. Grencis2*

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␣ (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 Downloaded from antagonist. IL-13 expression was induced in non-T intraepithelial CD3؊ NK cells. Epithelial cells expressed the IL-13 signaling receptor, IL-13R␣1, and after infection, IL-4R␣. Furthermore, the soluble IL-13 decoy receptor IL-13R␣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. The Journal of Immunology, 2005, 175: 3207–3213. http://www.jimmunol.org/ any mucosal diseases are characterized by abnormal tinal tissue remodeling during these infections is poorly defined. In pathology, in which the tissue architecture is disrupted this study, we have sought to determine the role of IL-13 on enteric M by the actions of a variety of cytokines. Inflammatory pathology during infection with the intestinal nematode, bowel disorders induce intestinal pathology that is associated with Trichinella spiralis, in a mouse model. elevated proinflammatory cytokines (1, 2). Levels of TNF-␣ and T. spiralis occupies a niche within enterocytes of the jejunum IFN-␥ are high in Crohn’s disease (1), whereas IL-13, a Th2-type and elicits a well-characterized type 2 cytokine response orches- cytokine, drives the pathophysiology of oxazalone colitis, an ex- trated by CD4ϩ T cells (11). Following infection, CD4ϩ T cells perimental model with pathological features similar to ulcerative express IL-4, IL-5, IL-9, and IL-10 in the mesenteric lymph nodes by guest on September 29, 2021 colitis (1, 3). Further evidence points to IL-13 as the factor re- (11) and are subsequently recruited to the gut mucosa (12). In sponsible for the pathogenesis of asthma (4–7). For example, ad- addition, there is a marked eosinophilia in response to IL-5 (13) ministration of IL-13 to the airways of mice results in distinctive and a pronounced mastocytosis (14). Expulsion of the parasites asthmatic pathology of inflammatory cell infiltration, airway hy- from the intestine is dependent on mast cells recruited to the mu- perresponsiveness, and mucus hypersecretion (4, 5), while mice cosa in response to IL-4 and IL-9 (15–17). Furthermore, there are genetically deficient for IL-13 fail to develop this pathology when goblet cell hyperplasia and mucus hypersecretion that may provide challenged with model airway allergens (6). IL-13-secreting NKT mucosal protection for the host or create an inhospitable environ- cells have been implicated in the pathogenesis of both experimen- ment for the parasite (18). Moreover, distinctive changes in villus tal asthma and ulcerative colitis models (3, 8), and NK cells have and crypt architecture occur that may also be an attempt by the been implicated in the development of allergen-induced airway host to drive out the parasite by diminishing the area available for inflammation in mice (9), although it is not known whether they habitation (18). T cells are required for worm expulsion from the contribute to the responses following Th2-inducing infection. gut, but this is not IL-4 dependent, because mice genetically de- Many pathological features associated with both asthma and in- ficient for IL-4 can expel normally (19). However, expulsion is flammatory bowel disorders are identical with those induced by inhibited in mice deficient in the IL-4/IL-13 signaling molecules intestinal nematode infection of both the small and large bowel (3, IL-4R␣ or STAT6, suggesting a role for IL-13 (19, 20). IL-13 is 7, 10). These parasites induce in the host a strong Th2 response certainly critical for expulsion of another gastrointestinal nema- (10), but the etiology, in particular the role of cytokines, in intes- tode, 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 *Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom; ␣ and †Department of Respiratory Disease, Wyeth Research, Cambridge, MA 02140 TNF- (22). In addition, both T. muris (23) and N. brasiliensis Received for publication March 8, 2005. Accepted for publication June 9, 2005. (24) infection generate changes in mucosal tissue architecture, in- The costs of publication of this article were defrayed in part by the payment of page cluding villus atrophy, crypt hyperplasia, and thickening of the charges. This article must therefore be hereby marked advertisement in accordance muscularis externa. The causative factor or factors for these with 18 U.S.C. Section 1734 solely to indicate this fact. changes are unknown, although IFN-␥ has been implicated in en- 1 This work was supported by the British Medical Research Council and Wellcome terocyte proliferation during T. muris infection (23). IL-13 is also Trust. known to induce liver fibrosis in schistosomiasis (25), and is a 2 Address correspondence and reprint requests to Dr. Richard K. Grencis, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Oxford Road, major contributor to pulmonary granuloma in a mouse model of Manchester M13 9PT, U.K. E-mail address: [email protected] the same disease (26). Although T cells have been assumed to be

the source of IL-13 during helminth infection, this has not been mented with 10% FCS, EDTA (Sigma-Aldrich), and DTT (Sigma-Al- confirmed, and the possibility of an innate source of the cytokine drich). After 15 min, tissues were shaken vigorously and supernatant con- has not been addressed. Likewise, the cellular source and target for taining epithelium was removed. This was repeated once. Epithelia were washed in HBSS twice and passed through a 40-␮m cell strainer. Cells IL-13 in asthma have yet to be definitively identified. A funda- were resuspended in either RPMI 1640 (Invitrogen Life Technologies) for mental question relates to a possible role of IL-13 in tissue remod- flow cytometry or TRIzol (Invitrogen Life Technologies) for RNA eling during intestinal helminth infections. In this study, using T. extraction. spiralis infection as a model of Th2-induced inflammation in the RNA extraction and RT-PCR small intestine, we have investigated the effect of IL-13 on enteric tissue restructuring and attempted to identify its cellular source. Epithelia or sections of jejunum were homogenized in TRIzol, and total RNA was extracted, according to the manufacturers’ protocol. Reverse- We show that IL-13 is responsible for villus atrophy and crypt and transcriptase reaction was performed using ImProm-II (Promega). The fol- goblet cell hyperplasia, and that it is not driven by the adaptive lowing primers were used to determine the level of specific mRNA: IL-13, immune response, but rather by early innate responses to infection. sense, 5Ј-ctccctctgacccttaaggag-3Ј, and antisense, 5Ј-gaaggggccgtggcgaaa Ј ␣ Ј Ј Ј SCID and athymic mice that cannot mount adaptive immune re- cag-3 ; IL-13R 1, sense, 5 -gcacgataatatggacgtgg-3 , and antisense, 5 - ttgacgacttttctccaggc-3Ј; IL-13R␣2, sense, 5Ј-atggcttttgtgcatatcagatgct-3Ј, sponses exhibit the same enteropathy as immunocompetent mice. and antisense, 5Ј-gacaaatgcgtatctt-3Ј; IL-4R␣, sense, 5Ј-gagtgagtggagtc Following infection, IL-13 is up-regulated in the jejunum of these ctagcatc-3Ј, and antisense, 5Ј-gctgaagtaacagaacaggc-3Ј; TNF-␣, sense, 5Ј- mice and is localized to the epithelial layer. Moreover, the potent tcttctcattcctgcttgtgg-3Ј, and antisense, 5Ј-gacaacctgggagtagacaaggt-3Ј. source of the IL-13 is a population of intraepithelial dwelling DNA was amplified by a Dyad DNA engine (MJ Research) under ␣ CD3Ϫ NK cells. Finally, blockade of IL-13 activity by IL-13 de- the following conditions: IL-13 and TNF- , 35 cycles of 1 min at 95°C, 30 s at 60°C, and 1 min at 72°C; IL-13R␣1, 35 cycles of 1 min at 95°C, coy receptor significantly reduces infection-associated intestinal 30 s at 62°C, and 1 min at 72°C; IL-13R␣2, 35 cycles of 1 min at Downloaded from pathological changes. These data show for the first time that dis- 95°C, 30 s at 54°C, and 1 min at 72°C. ruption of mucosal tissue architecture induced by T. spiralis in- Flow cytometry and cell sorting fection is generated by nonadaptive immune events and is IL-13 dependent, but T cell independent. Also, IL-13 is produced by NK Cells were blocked with Fc block (anti-CD16, CD32) (BD Pharmingen) for cells in response to infection and is the factor responsible for dis- 10 min. The following primary Abs were applied to cells on ice for 30 min: anti-CD45 PE and IgG2b PE (Serotec); anti-␥␦ TCR FITC, anti-CD49b ease-associated changes in tissue architecture in this model. PE, anti-CD4 PE, anti-CD8 PE, hamster IgG FITC, and rat IgM FITC (BD http://www.jimmunol.org/ Pharmingen). Cells were washed and fixed with 1% formaldehyde in PBS. Materials and Methods Cells were run through a FACSCalibur (BD Biosciences), and data were Animals and infection analyzed using CellQuest Pro. Isolated epithelial cells were further purified, and minority intraepithelial lymphocyte (IEL) populations were iden- BALB/c, C.B-17 SCID, and BALB/c nu/nu mice were purchased from tified and sorted using a FACSVantage (BD Biosciences) cell sorter. Abs Harlan-Olac. IL-4Ϫ/Ϫ (27), IL-13Ϫ/Ϫ (28, 29), and IL-4R␣Ϫ/Ϫ (30) mice used to identify subpopulations of IELs were as described above. Epithelial were generated, as described, and bred at University of Manchester under cells were identified using FITC-labeled UEA-1, a lectin that has previ- specific pathogen-free conditions. All transgenic mice were on a BALB/c ously been used to sort purify small intestinal epithelial cells (32). For NK background. Mice were infected with T. spiralis at 6–8 wk of age. Main- cell (CD49bϩ) sorting, anti-CD3⑀ FITC (BD Pharmingen) was used to

tenance, infection, and recovery of T. spiralis were as previously described ensure that selected NK cells were CD3 negative. Sorted cells were either by guest on September 29, 2021 (31). Mice were infected by oral gavage with 300 larvae on day 0. All placed directly in TRIzol (Invitrogen Life Technologies) for RNA extrac- experiments were performed under the regulations of the Home Office tion, or in RPMI 1640 for further cell culture. Scientific Procedures Act. For intracellular cytokine staining of sorted purified intraepithelial NK cells, isolated cells were stimulated for4hat37°C with PMA (50 ng/ml) Worm burden and ionomycin (500 ng/ml). Brefeldin A (1 ␮g/ml) (all Sigma-Aldrich) was added for the final2hofculture. After washing, cells were fixed in 2% Small intestines were removed on day 14 postinfection (p.i.) and opened formaldehyde for 20 min and washed in 0.1% saponin. Biotinylated anti- longitudinally. Intestines were placed in gauze and incubated in PBS at IL-13 (PeproTech) Ab was then added, or isotype control; these cells were 37°C. The number of worms suspended in PBS was counted after 4 h. further incubated with streptavidin-allophycocyanin (Caltag Laboratories). Histology Cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest Pro software. 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, Statistics ␮ then processed and embedded in paraffin. Tissue sections (5 m) were cut, Statistical significance was determined using Student t test. Values of p dewaxed, rehydrated, and stained with periodic acid and Schiff’s reagent Ͻ0.05 were considered significant. (PAS) or PAS, followed by Alcian blue to visualize goblet cells. After washing, sections were counterstained with Mayer’s hemalum and Results 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 Effect of IL-4 and IL-13 on intestinal pathology induced by T. per section were determined using a graticule eye piece. spiralis Blocking IL-13 in vivo IL-4 and IL-13 are key components of Th2 responses; therefore, we wanted to investigate their respective effects on T. spiralis ex- Soluble IL-13R␣2-Fc (kind gift from D. Donaldson, Wyeth Research, Cambridge, MA) or a control fusion protein was administered (200 ␮g/ pulsion and intestinal tissue pathology. On day 8 p.i., the small mouse) i.p. to SCID and BALB/c mice on days 0, 2, 4, and 6 p.i. intestines of wild-type BALB/c mice showed a significant increase in intestinal goblet cell numbers, increased crypt depth, and de- Isolation of epithelium creased villus length compared with uninfected mice (Fig. 1, A, B, Sections of jejunum (15 cm proximal to the pylorus) were removed days F, and G). Goblet cells appeared larger in infected animals, indi- ϩ 7–8 p.i. and flushed with ice-cold, Ca2 -free HBSS (Invitrogen Life Tech- cating hypersecretion of mucus. In addition, the muscularis externa nologies). Sections were trimmed of fat, mesentery, and Peyer’s patches, was thickened and villi were truncated and had formed clubbed and cut into 2-mm2 pieces that were washed in HBSS containing 2% FCS (Invitrogen Life Technologies). Tissues were incubated in HBSS supple- 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 3 Abbreviations used in this paper: NBF, neutral buffered formaldehyde; IEL, intra- ␣ epithelial lymphocyte; PAS, periodic acid and Schiff’s reagent; p.i., postinfection; atrophy was absent from both IL-4R (Fig. 1, D and F)- and IL- sIL, soluble interleukin; VCU, villus crypt unit. 13-deficient mice (Fig. 1, E and F), and although infected crypts The Journal of Immunology 3209

IL-4R␣ or IL-13 still harbored signiﬁcant numbers of worms (Fig. 1H).

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). Downloaded from

Expression of IL-13 in the absence of adaptive immunity Results from infected cytokine-deﬁcient mice showed that IL-13 and IL-4R␣ were required for the maximal development of anom- alous tissue architecture. Because immunodeﬁcient mice also de-

veloped this pathology, we investigated whether IL-13 was up- http://www.jimmunol.org/ 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␣1, which combines with IL-4R␣ 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␣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 by guest on September 29, 2021

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-4Ϫ/Ϫ (C), IL-4R␣Ϫ/Ϫ (D), and IL-13Ϫ/Ϫ mice (E). ME, muscularis externa; bar ϭ 100 ␮m. Jejunum villi length (F) and crypt depth (E) were measured in each case (F); points represent mean of four mice/group Ϯ SE. Changes in villi length and crypt depth response between days 0 and 8 were assessed for -p Ͻ 0.01. In ad ,ءء ;p Ͻ 0.05 ,ء :significance within each mouse strain dition, 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␣Ϫ/Ϫ vs BALB/c, ††, p Ͻ 0.005; and IL-13Ϫ/Ϫ vs BALB/c, †, p Ͻ p Ͻ 0.05, significantly ,ء ;(Goblet cell count within 20 VCU (G .0.05 p Ͻ ,ء ;(different from naives. Intestinal worm burdens at day 14 p.i. (H 0.05, significantly different from BALB/c; four mice/group Ϯ SE.

FIGURE 2. Enteropathy of T. spiralis-infected immunodeficient mice. were deeper than those of naive IL-4R␣- or IL-13-deficient mice, Villi length and crypt depth (A) and goblet cells per 20 VCU (B) measured they showed significantly less hyperplasia than infected wild-type in jejunum in naive and day 8 p.i. athymic mice and SCID mice. Repre- animals. Numbers of goblet cells were not elevated in IL-4-, IL- sentative plates showing jejuna sections fixed in NBF and stained with 4R␣-, or IL-13-deficient mice following infection (Fig. 1G). On PAS/Mayer’s hemalum from naive (C) and day 8 p.i. SCID mice (D). p Ͻ 0.001, significantly ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء .day 14 p.i., both BALB/c wild-type mice and IL-4-deficient mice Bar ϭ 100 ␮m were expelling their worm burdens, but mice deficient for either different from naive animals. Four mice/group Ϯ SE. 3210 NK-DERIVED IL-13 INDUCES INTESTINAL PATHOLOGY

Infected mice that received the control fusion protein displayed crypt hyperplasia and villus atrophy, whereas those that received the soluble (s)IL-13R␣2 fusion protein had significantly longer villi and reduced crypt depth (Fig. 4, A, B, and D). sIL-13R␣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␣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 com- FIGURE 3. Jejunal IL-13, IL-13R␣1, IL-13R␣2, and TNF-␣ expression pletely block goblet cell hyperplasia. sIL-13R␣2 treatment of 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 BALB/c mice also blocked infection-induced villus atrophy and agarose gel containing ethidium bromide. IL-13 (A), IL-13R␣1(B), IL- crypt hyperplasia (Fig. 4B and D). 13R␣2(C), TNF-␣ (D). Positive control for TNF-␣: BALB/c jejunum (ϩϩ) and mesenteric lymph node (ϩ) day 8 p.i. Innate source of intestinal IL-13 during nematode infection Downloaded from Immunodeficient SCID mice expressed IL-13R␣1 in the jejunum for resistance to T. muris within the large bowel and that its action constitutively and IL-13 following infection (Fig. 3, A and B). We is due to the induction of TNF-␣ (22). Moreover, others have were interested in investigating the source of IL-13 in the intestine shown that the effect of IL-4 during T. spiralis infection is due to and the cells that express its receptor. Epithelium was isolated its induction of TNF-␣ (33). Given that TNF-␣ is an important from jejuna of naive or infected SCID mice and analyzed by RT- regulator of intestinal pathology in Crohn’s disease (1), we chose PCR for IL-13 and IL-13R expression. We found that IL-13 http://www.jimmunol.org/ to investigate whether the action of IL-13 was due to it up-regu- mRNA was up-regulated in the epithelium following infection, and lating TNF-␣. Using mesenteric lymph nodes and jejunum of im- that these cells also expressed IL-13R␣1, suggesting that they can munocompetent infected BALB/c mice as positive controls for directly respond to IL-13 (Fig. 5, A and B). This suggests that both TNF-␣ expression, we were unable to detect significant amplifi- the cellular source and the responding population of cells reside cation of TNF-␣ mRNA in the intestine of infected SCID mice at within the epithelial fraction of the jejunum. It is known that SCID a time point when aberrant pathology was established and IL-13 is mice have a low number of immune cells, such as NK cells in the abundant (Fig. 3D), thus suggesting that IL-13 alone mediates pa- spleen, and that athymic mice have small numbers of nonthymic thology in SCID mice. generated intraepithelial cells in the gut. Small intestinal epithe- ϩ lium of SCID mice does not contain CD3 cells (34), but the total by guest on September 29, 2021 Abrogation of IL-13 in the absence of adaptive immunity cellular composition of the intestinal epithelium in SCID mice is To determine whether IL-13 was indeed responsible for nematode- not fully known. To determine the cellular source of IL-13, we first induced intestinal pathology in immunodeficient mice, infected analyzed the cellular composition of jejunal epithelium from both SCID mice were infected with T. spiralis and IL-13 function BALB/c and SCID mice by flow cytometry (Fig. 5, C and D). Just blocked by administration of soluble IL-13R␣2 Ig fusion protein. over 10% of cells within the jejunal epithelium of naive BALB/c

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␣2 (B), day 7 p.i. Bar ϭ 100 ␮m. Net changes (⌬) in goblet cell number per 20 VCU (C) and villus length and crypt depth (D) plotted for BALB/c (f) and SCID (u) mice, with or without sIL-13R␣2. Histograms represent mean .p Ͻ 0.01 ,ءء .of four mice/group The Journal of Immunology 3211 Downloaded from http://www.jimmunol.org/ FIGURE 6. Determination of cellular source of IL-13 within infected epithelium. Epithelium was stripped from naive or day 7 p.i. jejuna of FIGURE 5. Epithelial expression of IL-13 and IL-13R␣1 following T. BALB/c or SCID mice, and double stained using either pan-leukocyte spiralis infection. Jejunal epithelium was isolated from naive SCID mice marker CD45, CD49b, CD4, or CD8 and UEA-1. Representative data from or day 8 p.i. RNA was extracted and RT-PCR performed. PCR products BALB/c CD45, UEA-1 staining, and resulting FACS-sorted populations were run on ethidium bromide containing agarose gel. Epithelial IL-13 (A) are shown in A. mRNA expression for IL-13 and IL-13R␣1 for naive and and IL-13R␣1(B). Three animals per group. Percentage of leukocytes infected BALB/c mice (B) and for IL-13, IL-13R␣1, IL-13R␣2, and IL- as assessed by FACS in the total epithelial cell compartment of naive 4R␣ for naive and infected SCID mice (C) was assessed in sorted popu- and infected BALB/c (C) and SCID mice (D). Four to five animals per lations, five animals per group. Representative CD45 and UEA-1 FACS group Ϯ SE. data from the total jejunal epithelium of SCID mice (Di); CD49b staining by guest on September 29, 2021 on total IEL population isolated from infected SCID jejunal epithelium (Dii) (open histogram isotype control; closed CD49b staining). Intracellu- mice expressed the leukocyte surface molecule CD45. Following lar fluorescent staining for IL-13 within jejunal epithelial cells isolated and ϩ sorted for CD45 from infected SCID mice day 7 p.i. (Diii), five animals per infection, the percentage of CD45 intraepithelial cells in BALB/c group. 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 ϳ2% of BALB/c jejunal epithelium with a similar percentage in Discussion SCID mice. The remaining 7% of CD45ϩ cells in BALB/c mice The data presented in this work demonstrate for the first time that that were negative for ␥␦ TCR or CD49b were not identified, but enteropathy associated with gastrointestinal nematode infection were likely to be B cells or ␣␤ T cells. does not require T lymphocytes or indeed the presence of acquired To precisely identify the source of IL-13 from isolated epithe- immune responses. Moreover, by the use of mice lacking IL-4, lium, cells from day 7 infected wild-type and SCID mice were IL-13, or IL-4R␣, we have shown that IL-4 is dispensable, but sorted by flow cytometry into columnar epithelial UEA-1ϩ CD45Ϫ IL-13 is essential for the enteropathy observed in both wild-type cells and UEA-1Ϫ CD45ϩ leukocytes (Fig. 6, A and D). In naive and immunodeficient animals. In vivo blocking of IL-13-induced BALB/c mice, RT-PCR analysis clearly showed that IL-13 mRNA crypt hyperplasia and villus atrophy in infected immunodeficient is not present in either population. Upon infection, IL-13 mRNA is animals confirmed the functional presence of the protein and its up-regulated only in the sorted UEA-1Ϫ CD49bϩ population (Fig. role in enteropathy. Finally, we show IL-13 produced by NK cells 6B). It was not present in either intraepithelial sorted CD4ϩ or is responsible for the intestinal tissue restructuring and goblet cell CD8ϩ cells. Correspondingly, IL-13R␣1 mRNA was only ex- hyperplasia associated with T. spiralis infection. This could also be pressed by UEA-1ϩ CD45Ϫ cells. We have previously shown that the factor that generates the same pathology during infection by IL-13mRNA was not expressed in jejunal tissue from naive SCID other intestinal nematodes such as N. brasiliensis and T. muris. mice (Fig. 3A); we now show that the CD49bϩ population from Moreover, NK cells may also be the source of IL-13 that induces the epithelium of infected SCID mice expresses IL-13mRNA. Fur- at least some of the lung pathophysiology of allergic inflammation thermore, mRNA for IL-13R␣1, IL-4R␣, and IL-13␣2 were all in the lungs (9). We have shown that IL-13 expression can be expressed by the UEA-1ϩ CD45Ϫ population of cells (Fig. 6C). induced in the absence of B and T cells, and as such there raises FACS analysis confirmed that the majority of CD45ϩ SCID IELs the intriguing question of what factors or cell type may be stim- are also CD49bϩ, and, using intracellular cytokine staining, it was ulating IL-13 production from NK cells. In the context of infec- also confirmed that IL-13 protein was produced by the CD45ϩ tion, the parasite itself may induce secretion of the cytokine during population from the epithelium of infected SCID mice (Fig. 6D). mechanical disruption of the epithelium, or in the case of asthma 3212 NK-DERIVED IL-13 INDUCES INTESTINAL PATHOLOGY it may be an allergen. It is unclear whether IL-13 acts directly or This is the first study that identifies IL-13 as the critical cytokine indirectly. In this study, we have ruled out TNF-␣, but others have in the generation of the aberrant intestinal pathology characteristic shown that lung epithelial proliferation is due to TGF-␣ induced of nematode infection. A previous study has shown that TNF-␣, by IL-13 (36) and that lung fibrosis is induced by TGF-␤ under the under the control of IL-4, is important for villus atrophy and crypt control of IL-13-secreting macrophages (37). A number of growth hyperplasia during T. spiralis infection (33). Our study has found factors have also been implicated in intestinal epithelial prolifer- that the absence of IL-4 had no effect on tissue architecture or ation, including epidermal growth factor (38) and keratinocyte parasite resistance, but suggests that it may be required for optimal growth factor (39). However, any relationship with IL-13 has yet goblet cell hyperplasia in concert with IL-13. Furthermore, TNF-␣ to be defined. We have shown that intestinal epithelial cells ex- was absent in the intestine of infected immunodeficient mice that press IL-13R␣1 and IL-4R␣, which would allow binding of IL-13 developed abnormal enteropathy. These different findings may be with high affinity. This indicates that intestinal epithelial cells are attributable, at least in part, to the different mouse strains used in ideally placed to directly respond to the NK cell-derived IL-13. these studies. STAT-6 is required for both IL-4 and IL-13 signal- Equally, Kuperman et al. (7) showed that IL-13-regulated features ing, and is essential for the expulsion of both T. spiralis and N. of asthma could only be induced if STAT-6 was expressed in lung brasiliensis from the intestine (20, 21). We have shown that IL-13 epithelial cells. Furthermore, either rIL-13 or rIL-4 applied to the (and IL-4R␣), but not IL-4, is required for T. spiralis expulsion. airways can elicit their effects on the lung in the absence of both Urban et al. (20) found that worm expulsion was enhanced when T and B cells. However, neither cytokine has any effect in IL- mice were treated with exogenous IL-4, but, like us, found that 4R␣-deficient mice, thus indicating that the responder cell is an mice deficient for IL-4 expel their worm burden at the same rate as IL-4R␣-expressing nonhemopoietic cell (5). Other cell types of the wild-type mice. They also showed that IL-4-deficient mice failed Downloaded from ␣ lung and intestine can also respond to IL-13. Both IL-4 and IL-13 to expel if treated with sIL-13R 2, thus indicating that both IL-13 can be detected in intestinal smooth muscle during nematode in- and IL-4 are important in resistance to this nematode. Interest- ␣ fection (40) and are required, with STAT-6, to induce muscle con- ingly, IL-4R expression by nonhemopoietic cells is enough to tractility, a putative host mechanism for parasite expulsion (40, expel N. brasiliensis from the intestine, whereas expulsion of T. 41). Likewise, airway smooth muscle can produce IL-13 and has spiralis requires receptor expression by both bone marrow- and been linked to an autocrine pathway inducing smooth muscle hy- nonbone marrow-derived cells (19). This indicates that IL-13 http://www.jimmunol.org/ and/or IL-4 can directly stimulate intestinal epithelial cells. How- pertrophy, a feature of asthma pathophysiology (42). Moreover, a ever, the relative effects of compartmentalized IL-4R␣ expression gene array study by Lee et al. (43) has highlighted vast numbers of on nematode-induced changes in tissue architecture remain to be distinctive genes that are switched on following IL-13 treatment of defined. lung epithelial cells, fibroblasts, and smooth muscle cells. The present study clearly shows that aberrant tissue pathology Previous studies have shown that IL-13 is important for the associated with intestinal nematode infection is a nonadaptive im- development of intestinal goblet cell hyperplasia in N. brasiliensis mune event orchestrated by NK cell production of IL-13 acting infection (28) and that hyperplasia is induced only in the presence directly upon the adjoining epithelium. It also raises the distinct of STAT-6 during T. spiralis infection (44). In addition to villus/ by guest on September 29, 2021 possibility that this is an integral component of host-protective crypt architectural disruption, we have shown that IL-13 controls immunity generated against mucosal invading pathogens, and that the development of mucus-secreting goblet cells during T. spiralis changes previously believed to be solely under T cell control may infection. Accordingly, Shekels et al. (45) showed that during T. in fact be mediated by cellular components of the innate immune spiralis infection, levels of mucus secretion and mucin gene ex- system. Moreover, this finding may shed further light on the patho- ␥ pression were not affected in the absence of IL-4, IFN- ,or genesis of both asthma and IL-13-controlled intestinal colitis. TNF-␣. Observations in mouse models of asthma have shown that IL-13 induces mucus hypersecretion in the airways (4–7). Intes- Acknowledgments tinal goblet cells are derived from epithelial stem cells in the crypts We are grateful to Dr. Andrew McKenzie for providing breeding pairs of of Lieberku¨hn, although the IL-13-driven mechanism of differen- IL-13Ϫ/Ϫ mice, Frank Brombacher for breeding pairs of IL-4R␣ mice, and tiation of these cells or those in the airway is not known. Expres- Drs. E. B. Bell and A. J. Bancroft for helpful comments on the manuscript. sion of IL-13Rs or IL-4Rs has yet to be identified on goblet cells. However, there is evidence that stimulation of epidermal growth Disclosures factor receptors up-regulates mucin genes and goblet cell numbers The authors have no financial conflict of interest. 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 References can act directly on bronchial epithelial cells to induce goblet cell 1. Bouma, G., and W. Strober. 2003. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 3: 521–533. hyperplasia via MAPK and PI3K pathways. 2. Strober, W., I. J. Fuss, and R. S. Blumberg. 2002. The immunology of mucosal Recent data have clearly shown that a second IL-13R acts as a models of inflammation. Annu. Rev. Immunol. 20: 495–549. 3. Heller, F., I. J. Fuss, E. E. Nieuwenhuis, R. S. Blumberg, and W. Strober. 2002. regulator of IL-13-induced inflammation (49, 50). 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