Transmembrane Tumor Necrosis Factor Alpha Is Required for ...

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Dec 1, 2008 - SIPBS, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, .... immunosorbent assay (ELISA) kit was a kind gift from Hugh Miller,.
INFECTION AND IMMUNITY, Sept. 2009, p. 3879–3885 0019-9567/09/$08.00⫹0 doi:10.1128/IAI.01461-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 9

Transmembrane Tumor Necrosis Factor Alpha Is Required for Enteropathy and Is Sufficient To Promote Parasite Expulsion in Gastrointestinal Helminth Infection䌤 M. X. Ierna,1 H. E. Scales,1 C. Mueller,2 and C. E. Lawrence1* SIPBS, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, United Kingdom,1 and Institute of Pathology, Division of Experimental Pathology, University of Bern, CH-3010 Bern, Switzerland2 Received 1 December 2008/Returned for modification 1 January 2009/Accepted 22 June 2009

To study the specific role of transmembrane tumor necrosis factor (tmTNF) in protective and pathological responses against the gastrointestinal helminth Trichinella spiralis, we compared the immune responses of TNF-␣/lymphotoxin alpha (LT␣)ⴚ/ⴚ mice expressing noncleavable transgenic tmTNF to those of TNF-␣/ LT␣ⴚ/ⴚ and wild-type mice. The susceptibility of TNF-␣/LT␣ⴚ/ⴚ mice to T. spiralis infection was associated with impaired induction of a protective Th2 response and the lack of mucosal mastocytosis. Although tmTNFexpressing transgenic (tmTNF-tg) mice also had a reduced Th2 response, the mast cell response was greater than that observed in TNF-␣/LT␣ⴚ/ⴚ mice and was sufficient to induce the expulsion of the parasite. T. spiralis infection of tmTNF-tg mice resulted in significant intestinal pathology characterized by villus atrophy and crypt hyperplasia comparable to that induced following the infection of wild-type mice, while pathology in TNF-␣/LT␣ⴚ/ⴚ mice was significantly reduced. Our data thus indicate a role for tmTNF in host defense against gastrointestinal helminths and in the accompanying enteropathy. Furthermore, they also demonstrate that TNF-␣ is required for the induction of Th2 immune responses related to infection with gastrointestinal helminth parasites.

forms of TNF-␣ have been shown to exert different effects both in vitro and in vivo (37). The expression of tmTNF on CD4⫹ T cells provides a costimulatory signal to human B cells (5). Transgenic mice expressing a noncleavable mutant of tmTNF-␣ (tmTNF-tg mice) are resistant to Mycobacterium bovis bacillus Calmette-Gue´rin, while TNF-␣/lymphotoxin alpha (LT␣)⫺/⫺ mice succumb to infection (41). Mice overexpressing tmTNF are prone to developing arthritis (1), and mice expressing tmTNF but not soluble TNF-␣ are susceptible to concanavalin A-induced liver disease (30). Furthermore, tmTNF-tg mice treated with lipopolysaccharide and D-galactosamine have reduced mortality compared to wild-type mice. A number of studies also implicate TNF-␣ in Th2-mediated immune responses. TNF-␣ has been shown to be necessary for the development of allergic airway inflammation (38) and mast cell-mediated gastric allergic inflammation (14). The expulsion of gastrointestinal (GI) helminths has been shown to be dependent on Th2-mediated cytokine responses, and following the blockade of TNF-␣ in interleukin-4 (IL-4)⫺/⫺ mice, parasite expulsion is delayed, suggesting that TNF-␣ plays an important role in the IL-13-mediated expulsion of Trichuris muris (3). Results from previous studies using p55 TNFR⫺/⫺ mice suggest that TNF-␣ plays a role in the development of enteropathy but not in the expulsion of Trichinella spiralis from the small intestine (32) and that p75 TNFR⫺/⫺ mice develop increased pathology (C. E. Lawrence, unpublished data). The aim of this study was to evaluate the roles played by soluble TNF-␣ and tmTNF-␣ in the development of protective Th2 immune responses and pathological responses following T. spiralis infection of wild-type, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice. Our results showed that soluble TNF-␣ but not tmTNF-␣ is required for the induction of protective Th2 responses and

Tumor necrosis factor alpha (TNF-␣) is a proinflammatory cytokine considered to play important roles in Th1-mediated protective immune responses. TNF-␣ is of particular importance in the control of intracellular pathogens such as Mycobacterium tuberculosis and Leishmania species (6). TNF-␣ is also implicated in a variety of immunopathological conditions; anti-TNF-␣ treatment inhibits collagen-induced arthritis in mice (46) and the development of intestinal and skin lesions in murine graft-versus-host disease (43). TNF-␣ also plays an important role in the development of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn’s disease, with increased TNF-␣ secretion from mononuclear cells isolated from the lamina propriae of IBD patients (40). Furthermore, treatment of wild-type mice with exogenous TNF-␣ leads to rapid development of severe intestinal lesions characterized by villus atrophy, with apoptotic epithelial cells and edema (15, 20, 44). TNF-␣ is synthesized as a 26-kDa precursor, which may be proteolytically cleaved by the transmembrane matrix metalloprotease TNF-␣-converting enzyme into a 17-kDa secreted monomer, while noncleaved TNF-␣ is present as a type II transmembrane protein, transmembrane TNF-␣ (tmTNF-␣) (16, 36). Membrane-bound TNF has been shown to be the major activating ligand of the p75 TNF receptor (TNFR) and to suppress the proinflammatory activity of TNF-␣ signaling via the p55 TNFR (17, 42). Membrane-bound and soluble

* Corresponding author. Mailing address: SIPBS, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, United Kingdom. Phone: 0141 548 2104. Fax: 0141 552 2562. E-mail: catherine.lawrence @strath.ac.uk. 䌤 Published ahead of print on 29 June 2009. 3879

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subsequent parasite loss and that tmTNF-␣ is required for the development of pathological responses.

MATERIALS AND METHODS Animals and infection. The maintenance and recovery of T. spiralis and the infection of animals were essentially as described previously (32). Female 8- to 12-week-old mice were infected with 400 T. spiralis larvae on day 0 and killed at various times postinfection (p.i.) as detailed below. Worm burdens were assessed as described previously (32). TNF-␣/LT␣⫺/⫺ mice and tmTNF-tg mice with a C57BL/6 background were described previously (37). The transgene is under the control of the TNF promoter. The AU-rich regulatory elements at the 3⬘ end of the TNF gene have been maintained to ensure appropriate gene regulation. To generate a noncleavable mutant of murine TNF, two deletions (Leu⫺12 to Leu⫺10 and Leu⫺2 to Leu1) and an amino acid substitution (Lys113Glu11) were introduced into a genomic TNF clone. C57BL/6 mice were purchased from Harlan Olac. All mice were bred, maintained, and housed in the University of Strathclyde animal facility under standard conditions with free access to food and water, and all procedures were performed in accordance with the Animals (Scientific Procedures) Act of 1986. Quantitation of intestinal pathology. Mucosal architecture and epithelial cell mitotic activity in jejunum samples taken 10 cm from the pylorus were assessed (32). The entire small intestine was removed and weighed, and 1-cm samples were fixed in 25% acetic acid–75% ethanol and stained with Schiff reagent (Sigma, Poole, United Kingdom). Specimens were microdissected, and villus and crypt lengths were measured using an eyepiece micrometer. Ten villi and 10 crypts from each sample were measured. MPO assay. Myeloperoxidase (MPO) was measured in snap-frozen samples of small intestine tissue (33). Briefly, samples were homogenized in cold 1% (wt/ vol) hexadecyltrimethylammonium bromide (Sigma, Poole, United Kingdom) in phosphate buffer, pH 6.0, and sonicated for 15 s. After being snap-frozen in liquid nitrogen and thawed three times, the homogenate was centrifuged for 15 min at 12,000 ⫻ g at 4°C, and the supernatant was removed for the MPO assay. To 10 ␮l of supernatant in a flat-bottom 96-well microtiter plate, 200 ␮l of 50 mM phosphate buffer, pH 6, containing 0.4 mg/ml of substrate o-phenylenediamine (Sigma) and 0.05% H2O2 (Sigma) was added. After 20 min, the reaction was stopped by the addition of 50 ␮l of 0.4 M H2SO4 and the absorbance at 490 nm was determined using a plate reader (Titertek Multiscan, Eflab, Finland). Sample enzyme activity was measured using a standard curve of horseradish peroxidase activity (Boehringer Mannheim, Lewes, United Kingdom). Mast cell quantitation. Consecutive samples of jejunum tissue were obtained and fixed in Carnoy’s fixative and then processed using standard histological techniques. Sections were stained with 0.5% toluidine blue (Sigma) in 0.5 M HCl for mast cell visualization and counterstained with 0.5% Safranine O (Sigma) for 2 min. The mucosal mast cells (MMC) in 10 villus-crypt units (VCU) per sample were counted, and the data were expressed as the mean number of MMC per VCU. mMCP-1 analysis. The mouse mast cell protease 1 (mMCP-1) sandwich enzyme-linked immunosorbent assay (ELISA) kit was a kind gift from Hugh Miller, University of Edinburgh, Edinburgh (33). Lymphocyte culture and cytokine responses. Single-cell suspensions of splenocytes were prepared and incubated in the presence of T. spiralis larval homogenate (50 ␮g/ml protein). Culture supernatants were harvested after 24 h, and IL-4, IL-9, IL-18, IL-13, and gamma interferon (IFN-␥) levels were measured by ELISAs using paired antibodies (32). TNF-␣ levels in sera were determined by an ELISA according to the instructions of the assay kit manufacturer (PharMingen, Oxford, United Kingdom). Measurement of antibody responses. Parasite-specific immunoglobulin G1 (IgG1) and IgG2a and total IgE levels were determined as described previously (32). T. spiralis larval homogenate at 10 ␮g/ml was used as a target antigen in the ELISA mixture, and sera were diluted 1/100. IgG1 and IgG2a were detected using biotinylated anti-mouse IgG1 or IgG2a (PharMingen) at 2 ␮g/ml each, followed by streptavidin-peroxidase (SAPU, Carluke, United Kingdom). Total serum IgE levels were measured by an ELISA, anti-mouse IgE was used as the capture antibody, and IgE was detected using biotinylated anti-mouse IgE (PharMingen). An IgE monoclonal antibody specific for trinitrophenyl (PharMingen) was used as the standard. Statistics. Results are expressed as means ⫾ standard errors of the means (SEM). Statistical differences (P ⱕ 0.05) between experimental groups were determined using the Mann-Whitney U test for nonparametric data.

FIG. 1. Roles of soluble TNF-␣ and tmTNF-␣ in the expulsion of T. spiralis from the intestine and the accumulation of muscle larvae. (A) The establishment of T. spiralis in and its expulsion from wild-type (WT), TNF-␣/LT␣⫺/⫺ (TNF/LT KO), and tmTNF-tg mice at days 6 and 14 p.i. were evaluated. The small intestines were excised, and the worms present were counted. (B) The accumulation of muscle larvae in wild-type, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice at day 30 p.i. was measured. Data are expressed as the mean number of worms per mouse ⫹ SEM, and five mice per group were used. The experiments were repeated twice, with similar results. ⴱ, significantly different from the value for mice at day 6 p.i.; §, significantly different from the result for wild-type mice (P ⬍ 0.05).

RESULTS Expulsion of adult T. spiralis is delayed in the absence of soluble TNF-␣. To assess the effect of the absence of soluble TNF-␣ and tmTNF-␣ on the immune expulsion of T. spiralis, wild-type, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice were infected with 400 T. spiralis larvae. The levels of T. spiralis establishment in the intestines at day 6 p.i. did not differ significantly among the mouse strains. While wild-type and tmTNF-tg mice had significantly reduced intestinal worm burdens at day 14 p.i., TNF-␣/LT␣⫺/⫺ mice failed to expel the adult worms, having significantly higher intestinal worm burdens than wildtype mice. No significant differences between tmTNF-tg and wild-type mice at day 14 p.i. were observed (Fig. 1A). While a small number of adult worms remained in tmTNF-tg mice 30 days p.i., this outcome was not significantly different from that for wild-type or TNF-␣/LT␣⫺/⫺ mice. Burdens in muscles from tmTNF-tg and TNF-␣/LT␣⫺/⫺ mice were not significantly different from those in muscles obtained from wild-type mice (Fig. 1B). Enteropathy is ameliorated in the absence of soluble TNF-␣ and tmTNF-␣. In order to assess the effects of soluble TNF-␣ and tmTNF-␣ on the induction of the intestinal inflammation accompanying helminth infection, gut weights, villus heights, and crypt lengths were measured. As seen previously, the infection of wild-type mice with T. spiralis resulted in significant crypt hyperplasia and villus atrophy at both days 6 and 14 p.i., and these developments were accompanied by a significant increase in the mean wet weight of the guts (Fig. 2A and B). Similarly, the infection of tmTNF-tg mice resulted in significant enteropathy, accompanied by a significant increase in gut weight at both days 6 and 14 p.i., while TNF-␣/LT␣⫺/⫺ mice exhibited significantly reduced enteropathy in comparison to both wild-type mice and tmTNF-tg mice. The MPO level is commonly used as an index of neutrophil infiltration and in-

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FIG. 2. Roles of soluble TNF-␣ and tmTNF-␣ in the development of enteropathy following infection with T. spiralis. Crypt depths (bottom) and villus lengths (top) (A), wet weights of the entire small intestine (B), and MPO levels (C) in uninfected and infected wild-type (WT), TNF-␣/LT␣⫺/⫺ (TNF/LTKO), and tmTNF-tg mice at days 6 and 14 p.i. were determined. Data are expressed as means ⫹ SEM, and five mice per group were used. The experiments were repeated twice, with similar results. ⴱ, significantly different from the value for uninfected mice; §, significantly different from the value for wild-type mice; †, significantly different from the value for TNF-␣/LT␣⫺/⫺ mice (P ⬍ 0.05).

flammation. From the data in Fig. 2C, it can be seen that the development of enteropathy in wild-type mice and tmTNF-tg mice was associated with significant increases in levels of MPO; in contrast, infection of TNF-␣/LT␣⫺/⫺ mice did not result in any significant alterations in levels of MPO. No significant evidence of gross intestinal abnormality was seen in uninfected mice of any strain. Taken together, these results suggest that the intestinal inflammation in TNF-␣/LT␣⫺/⫺ mice following infection with T. spiralis was significantly reduced in comparison to that in wild-type mice and tmTNF-tg mice. Mucosal mastocytosis is significantly reduced in the absence of soluble TNF-␣. Mastocytosis and the production of mMCP-1 are important features of infection with T. spiralis, and mast cells have been implicated in both the development of enteropathy and expulsion following infection (28, 33). To assess the role of soluble TNF-␣ and tmTNF-␣ in the development of mastocytosis following infection with T. spiralis, the number of mast cells was determined. At day 6 p.i., comparable, significant levels of mastocytosis were present in wild-type and tmTNF-tg mice; while profound mastocytosis had developed in wild-type and tmTNF-tg mice on day 14 p.i., the level in tmTNF-tg mice was significantly lower than that in wild-type

mice. The numbers of mast cells observed in TNF-␣/LT␣⫺/⫺ mice were significantly lower than those in both wild-type and tmTNF-tg mice at both time points. Mast cells were barely detectable in uninfected wild-type, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice (Fig. 3, left). Detected levels of mMCP-1 correlated with the numbers of mast cells observed in the three different mouse lines following infection with T. spiralis. While mMCP-1 levels in tmTNF-tg mice were significantly elevated in comparison to those in infected TNF␣/LT␣⫺/⫺ mice, they were still lower than those in wild-type mice (Fig. 3, right). In the absence of soluble TNF-␣, infection with T. spiralis results in attenuated secretion of IL-4 while the secretion of IL-18 is increased. The effects of soluble TNF-␣ and tmTNF-␣ on the Th1-Th2 balance during T. spiralis infection were examined. Spleen cells were used since TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice lack mesenteric lymph nodes (29). IL-4 production by antigen-stimulated spleen cells from TNF-␣/ LT␣⫺/⫺ and tmTNF-tg mice at day 14 p.i. was significantly reduced in comparison to that in cells from wild-type mice (Fig. 4A). Levels of IL-9 were determined since IL-9 is involved in mast cell generation and function and is required for

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FIG. 3. TNF-␣ is required for the development of mucosal mastocytosis following infection with T. spiralis. (A) Jejunum samples from infected wild-type (WT), TNF-␣/LT␣⫺/⫺ (TNF/LT KO), and tmTNF-tg mice at days 6 and 14 p.i. and from uninfected counterparts were fixed in Carnoy’s fixative, processed, and stained with 0.5% toluidine blue, and the MMC in 10 VCU from each sample were counted. (B) Serum titers of mMCP-1 from uninfected and infected mice were determined by ELISA. Data are expressed as means ⫹ SEM. Five mice per group were used. The experiments were repeated twice, with similar results. ⴱ, significantly different from the value for uninfected mice; §, significantly different from the value for wild-type mice; †, significantly different from the value for TNF-␣/LT␣⫺/⫺ mice (P ⬍ 0.05).

parasite loss (13). Figure 4C demonstrates that although IL-9 levels in both wild-type and tmTNF-tg mice were significantly elevated in comparison to those in TNF-␣/LT␣⫺/⫺ mice, levels in tmTNF-tg mice were lower than those in wild-type mice. Neither IL-13 production nor IFN-␥ production in either mutant mouse strain was significantly altered compared to that in wild-type mice (Fig. 4B and D). In contrast, IL-18 production in both TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice was significantly increased compared to that in wild-type mice following infection (Fig. 4E). Levels of TNF-␣ secreted by splenocytes from

infected wild-type mice were similar to those reported previously (32), while no TNF secreted by splenocytes from either TNF-␣/LT␣⫺/⫺ or tmTNF-tg mice was detected (data not shown). The total amount of IgE in serum is significantly reduced in the absence of soluble TNF-␣. In order to further evaluate the in vivo Th1-Th2 balance, total serum IgE and antigen-specific IgG1 and IgG2a levels in uninfected and T. spiralis-infected wild-type, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice were determined. While total IgE titers in the sera of wild-type, TNF-␣/

FIG. 4. Roles of soluble TNF-␣ and tmTNF-␣ in cytokine secretion following infection with T. spiralis. Spleens were removed from wild-type (WT), TNF-␣/LT␣⫺/⫺ (TNF/LT KO), and tmTNF-tg mice at day 14 p.i. Splenocytes (106 cells) were cultured in single-cell suspensions with 50 ␮g/ml TAg. The secretion of IL-4, IL-13, IL-9, IFN-␥, and IL-18 was measured by ELISAs. Data are expressed as mean cytokine concentrations in picograms per milliliter ⫹ SEM, and five mice per group were used. The experiments were repeated twice, with similar results. ⴱ, significantly different from the value for uninfected mice; §, significantly different from the value for wild-type mice; †, significantly different from the value for TNF-␣/LT␣⫺/⫺ mice (P ⬍ 0.05).

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FIG. 5. Roles of soluble TNF-␣ and tmTNF-␣ in antibody responses following infection with T. spiralis. Total IgE (A) and TAgspecific IgG1 (B) and IgG2a (C) titers in sera taken from uninfected and infected wild-type (WT), TNF-␣/LT␣⫺/⫺ (TNF/LT KO), and tmTNF-tg mice on day 14 p.i. were measured. IgE titers were measured by a sandwich ELISA using a purified IgE standard and were expressed as mean levels in micrograms per milliliter ⫹ SEM. Antigenspecific IgG1 and IgG2a titers were assessed by an ELISA using TAg at 50 ␮g/ml as the target antigen; sera were serially diluted, and optical density readings at 1:640 dilutions were used. Data were expressed as the mean optical density (OD) ⫹ SEM; five mice per group were used. The experiments were repeated twice, with similar results. ⴱ, significantly different from the value for uninfected mice; §, significantly different from the value for wild-type mice; †, significantly different from the value for TNF-␣/LT␣⫺/⫺ mice (P ⬍ 0.05).

LT␣⫺/⫺, and tmTNF-tg mice increased following infection, on day 14 p.i., IgE levels in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice were reduced compared to those in wild-type mice (Fig. 5, top). Similarly, T-antigen (TAg)-specific IgG1 titers in wildtype, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice following infection were elevated compared to those in uninfected mice, and TAgspecific IgG1 levels in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice were significantly lower than those in wild-type mice on day 14 p.i. (Fig. 5, middle). TAg-specific IgG2a titers in all three experimental groups of mice were not significantly elevated following infection, and no significant differences among wildtype, TNF-␣/LT␣⫺/⫺, and tmTNF-tg mice were observed (Fig. 5, bottom).

In this study, we have demonstrated that in the absence of soluble TNF-␣, the expulsion of T. spiralis from mice is significantly delayed and enteropathy and mastocytosis are reduced. The expulsion of the parasite from tmTNF-tg mice, which lack soluble TNF-␣, and intestinal pathology and mastocytosis in these mice were not significantly altered in comparison to expulsion from and intestinal pathology and mastocytosis in wild-type mice. The IL-4 responses elicited by ex vivo restimulation of spleen cells from both TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice were significantly reduced compared to those from wildtype mice, while IL-13 production was unaffected. Levels of IL-9 in infected TNF-␣/LT␣⫺/⫺ mice were not increased and those in infected wild-type mice were elevated compared to those in uninfected counterparts. IL-9 levels in tmTNF-tg mice increased after infection but were significantly lower than those in wild-type mice. IL-18 secretion was significantly increased following infection; however, IFN-␥ production in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice was not significantly different from that in wild-type mice. Consistent with the reduced IL-4 secretion, IgE production in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice was significantly reduced compared to that in wild-type mice. The expulsion of T. spiralis has been shown previously to be mediated by a Th2 response (18, 32), with expulsion from IL-4⫺/⫺ mice (32) and IL-13⫺/⫺ mice (35) being delayed. However, the role of TNF in GI helminth infection is less clear. The administration of TNF to wild-type or IL-4-deficient mice had no effect on parasite expulsion or on Th2 responses, despite severe TNF-induced enteropathy (Lawrence, unpublished). The expulsion of T. spiralis from female p55 TNFR⫺/⫺ mice (32) and female p75 TNFR⫺/⫺ mice (C. E. Lawrence et al., unpublished data) had the same kinetics as that from wild-type mice. While female p55 TNFR⫺/⫺ and p75 TNFR⫺/⫺ mice expelled T. muris, expulsion from male mice of the same genotypes was delayed (21, 22). In contrast, the results of the present study showed that T. spiralis expulsion from female TNF-␣/LT␣⫺/⫺ mice but not tmTNF-tg mice was significantly delayed compared to that from wild-type mice. TNF-␣, LT␣, and particularly LT␤ are crucial for lymphoid organogenesis (9). Mice which do not secrete TNF but have normal tmTNF support many features of lymphoid organ structure except for the generation of primary B-cell follicles (45). tmTNF is required for lymphoid tissue structure but secreted TNF is required for inflammatory lesion development in experimental autoimmune encephalomyelitis induced in tmTNF knock-in mice, which in contrast to the tmTNF-tg mice used in the present study, still contain a functional LT␣ gene (45). Although both TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice lacked lymph nodes, only TNF-␣/LT␣⫺/⫺ mice were unable to rapidly expel the parasite, suggesting that the lack of lymph nodes was not a contributory factor in the delay of parasite expulsion. TNF-␣ is an important mediator of intestinal pathology. Anti-TNF-␣ treatment of patients with IBD has been shown to be efficacious in ameliorating pathology. Furthermore, the induction of colitis following the adoptive transfer of CD4⫹ CD45RBhi T cells into RAG2⫺/⫺ mice is dependent on nonT-cell-derived TNF-␣ (12). Increased TNF-␣-converting en-

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zyme activity in the colonic mucosa of ulcerative colitis patients has been demonstrated, suggesting that the conversion of TNF-␣ from the transmembrane to the soluble form may be important (7). Interestingly, the transfer of CD4⫹ CD45RBhi T cells expressing only tmTNF induces colitis in tmTNF-tg RAG2⫺/⫺ mice (11), suggesting that the presence of soluble TNF-␣ is not required for the development of colitis. Similarly, in the present study, enteropathy was ameliorated in the absence of TNF-␣ and LT␣ while the presence of tmTNF was sufficient to induce enteropathy following infection with T. spiralis. The development of enteropathy in p55 TNFR⫺/⫺ mice during infection with T. spiralis has been shown previously to be significantly reduced (32), while p75 TNFR⫺/⫺ mice developed enhanced enteropathy (Lawrence et al., unpublished). tmTNF preferentially stimulates p75 TNFR (17), and levels of p75 TNFR in tmTNF-tg mice are lower than those in wild-type mice (8). Shedding of p75 TNFR has been suggested to play a role in limiting inflammatory responses (10, 34). However, tmTNF also binds p55 TNFR (31) and is sufficient to induce inflammatory responses in other infection models (2). Mucosal mastocytosis is a significant histological feature of T. spiralis infection. Peak mastocytosis coincides with the expulsion of the adult worms from the intestine (32), and deficiency in mMCP-1 delays the expulsion of T. spiralis (28) and reduces enteropathy (33) compared to expulsion from and enteropathy in wild-type mice. In the absence of soluble TNF-␣, MMC responses following infection with T. spiralis were significantly delayed. The reduced mastocytosis observed may be due to diminished Th2 responses in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice since IL-4 and IL-9 are necessary for the development of mast cells and the expulsion of some GI helminths (13). Mast cell survival and activation are dependent on IgE through FcεR1 (4, 26). The expulsion of T. spiralis from IgE⫺/⫺ mice is slower than that from wild-type mice and is correlated with reduced levels of mMCP-1 and splenic but not jejunal mastocytosis (19). Thus, the reduction in IgE levels in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice may also contribute to the reduced mastocytosis observed. The roles of LT␣ and TNF-␣ in the development of Th2 responses are unclear. Previous studies have suggested that TNF acts to potentiate ongoing immune responses, since blocking TNF in mice resistant to T. muris has no effect on parasite expulsion whereas susceptible mice treated with TNF have greater Th1 responses (21, 22). We have recently shown that mast cell-derived TNF is required for the generation of both Th2 responses and mastocytosis, necessary for the expulsion of T. spiralis (25). Additionally, mast cell-derived TNF-␣ has been shown to play a crucial role in the induction of Th2 responses by promoting the migration of dendritic cells and enhancing T-cell activation in a murine model of allergy (39). LT␣ is essential for the development of Th2 responses following pulmonary challenge with schistosome egg antigen. While pulmonary inflammation in wild-type mice is associated with elevated IgE and Th2 cytokine responses, LT␣⫺/⫺ mice fail to develop significant IgE responses to schistosome egg antigen and develop a Th1 cytokine profile. In LT␣⫺/⫺ mice, this inflammation is ameliorated by the introduction of endogenous IgE, suggesting that LT␣ and IgE are important in the modulation of the Th1-Th2 balance and pathology (27). This observation is consistent with the reduced IgE, IgG1, and IL-4

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levels and decreased enteropathy in TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice compared to those in wild-type mice following infection with T. spiralis, suggesting that LT␣ may play a role in balancing Th1- and Th2-mediated pathologies. TNF-␣ has been shown previously to have anti-inflammatory properties, inhibiting the production of IL-12 by macrophages stimulated with IFN-␥ in vitro, and protects mice against IL-12-mediated inflammation following infection with “Corynebacterium parvum” (24). Mice deficient in TNF-␣ or its receptors exhibit impaired allergic inflammation (38). Further, it was demonstrated that soluble TNF-␣ but not tmTNF-␣ was required for the recruitment of inflammatory cells and the production of Th2 cytokines. IL-18 levels in both TNF-␣/LT␣⫺/⫺ and tmTNF-tg mice were elevated following infection with T. spiralis. IL-18 appears to function as a mediator of both Th1 and Th2 responses, depending on the cytokine environment (47). Exogenous IL-18 has also been shown previously to inhibit the development of mastocytosis following infection with T. spiralis, and IL-18⫺/⫺ mice expel T. spiralis more rapidly than wild-type mice (23). Thus, the delay in parasite expulsion from the small intestine in TNF-␣/LT␣⫺/⫺ mice may relate to elevated IL-18 secretion, along with decreased IL-9 levels and ensuing mastocytosis. In conclusion, the results of this study corroborate the findings of other studies that both soluble TNF and tmTNF critically contribute to the development of intestinal inflammation. Intriguingly, while TNF-␣/LT␣⫺/⫺ mice failed to rapidly expel T. spiralis, tmTNF-tg mice were able to clear the helminth infection in the absence of secreted TNF-␣. Importantly, TNF is required for the generation of the Th2 response necessary for the expulsion of T. spiralis. This may have important implications for the development of therapies for inflammatory diseases associated with Th2 responses, such as allergic asthma. ACKNOWLEDGMENTS We thank Hugh Miller, University of Edinburgh, Edinburgh, for the provision of mMCP-1 ELISA reagents. This study was supported by Wellcome Trust grants 055503 and 62264 to C.E.L. REFERENCES 1. Alexopoulou, L., M. Pasparakis, and G. Kollias. 1997. A murine transmembrane tumor necrosis factor (TNF) transgene induces arthritis by cooperative p55/p75 TNF receptor signaling. Eur. J. Immunol. 27:2588–2592. 2. Allenbach, C., P. Launois, C. Mueller, and F. Tacchini-Cottier. 2008. An essential role for transmembrane TNF in the resolution of the inflammatory lesion induced by Leishmania major infection. Eur. J. Immunol. 38:720–731. 3. Artis, D., C. S. Potten, K. J. Else, F. D. Finkelman, and R. K. Grencis. 1999. Trichuris muris: host intestinal epithelial cell hyperproliferation during chronic infection is regulated by interferon-gamma. Exp. Parasitol. 92:144–153. 4. Asai, K., J. Kitaura, Y. Kawakami, N. Yamagata, M. Tsai, D. P. Carbone, F. T. Liu, S. J. Galli, and T. Kawakami. 2001. Regulation of mast cell survival by IgE. Immunity 14:791–800. 5. Aversa, G., J. Punnonen, and J. E. de Vries. 1993. The 26-kD transmembrane form of tumor necrosis factor alpha on activated CD4⫹ T cell clones provides a costimulatory signal for human B cell activation. J. Exp. Med. 177: 1575–1585. 6. Botha, T., and B. Ryffel. 2003. Reactivation of latent tuberculosis infection in TNF-deficient mice. J. Immunol. 171:3110–3118. 7. Brynskov, J., P. Foegh, G. Pedersen, C. Ellervik, T. Kirkegaard, A. Bingham, and T. Saermark. 2002. Tumour necrosis factor alpha converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease. Gut 51:37–43. 8. Canault, M., F. Peiretti, C. Mueller, F. Kopp, P. Morange, S. Rihs, H. Portugal, I. Juhan-Vague, and G. Nalbone. 2004. Exclusive expression of transmembrane TNF-alpha in mice reduces the inflammatory response in early lipid lesions of aortic sinus. Atherosclerosis 172:211–218.

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