1. Thymic Stromal Lymphopoietin Is Required for Gastrointestinal Allergy but Not Oral Tolerance 
Ana Belén Blázquez, Lloyd Mayer, M. Cecilia Berin 
Gastroenterology 
Volume 139, Issue 4, Pages e4 (October 2010)
DOI: /j.gastro
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2. Figure 1
Allergic diarrhea is TSLP-dependent. TSLPR+/+ and TSLPR−/− mice (n = 20/group) were sensitized and fed with OVA to induce diarrhea (OVA/OVA). Controls included mice that were sensitized but unfed with OVA or unsensitized and fed with OVA. (A) Percent of TSLPR+/+ (solid circle) or TSLPR−/− (open circle) mice in the OVA/OVA groups developing diarrhea symptoms after each oral antigen challenge. (B) Jejunal mast cells were quantified and expressed as number per high-power field (HPF) in TSLPR+/+ (open square) and TSLPR−/− (solid square) mice. (C) Real-time RT-PCR for mMCP-1 (mouse mast cell protease-1) in the jejunum of TSLPR+/+ (open square) and TSLPR−/− (solid square) mice. Data are expressed as fold increase in gene expression in OVA/OVA vs control mice. (D) OVA-specific IgE levels in serum obtained prior to (open bars) and after (grey bars) repeated oral challenge with OVA. Shown are optical density values for serum diluted 1:256. Results are expressed as mean + standard error of mean of 4 independent experiments. **P < .01, and ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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3. Figure 2
Th2 cytokine production in the gastrointestinal tract is TSLP-dependent. TSLPR+/+ (open square) and TSLPR−/− (solid square) mice were sensitized and repeatedly fed with OVA to induce allergic diarrhea. (A) Cytokine secretion from MLN cells restimulated in vitro with OVA. (B and C) Real-time PCR for IL-4 and IL-13 in the jejunum of mice with allergic diarrhea. Data are expressed as fold change compared with control mice. N = 20 mice/group. **P < .01, ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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4. Figure 3
OVA-induced anaphylaxis is independent of TSLP. TSLPR+/+ and TSLPR−/− mice were intragastrically sensitized with OVA plus CT and challenged with 100 μg of OVA (intraperitoneally). (A) Body temperature was measured 30 minutes after OVA challenge. (B) Serum OVA-specific IgE levels. (C) Cytokine secretion from spleen cells restimulated in vitro with OVA, from naïve (open square) or OVA+CT (solid square) sensitized mice. Data are expressed as a mean + standard error of mean of 5 mice/group. ***P < .01.
Gastroenterology  , e4DOI: ( /j.gastro )
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5. Figure 4
Oral tolerance induction is independent of TSLP. TSLPR+/+ and TSLPR−/− mice were fed for 5 days with 1 mg OVA (solid square) or remained unfed as control (open square). Mice were then immunized by intradermal injection of OVA or phosphate-buffered saline as a control. Change in ear thickness comparing OVA- and phosphate-buffered saline-injected ears was measured at (A) 24 hours and 48 hours after injection, n = 10 mice/group. (B) Serum OVA-specific IgG1 antibody levels in non-fed and OVA-fed mice were measured by enzyme-linked immunosorbent assay. Data are expressed as a mean + standard error of mean of 5 mice/group. **P < .01 and ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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6. Figure 5
Primed TSLPR+/+ MLN cells restore disease in TSLPR−/− mice. MLN cells from TSLPR+/+ mice with allergic diarrhea were isolated and transferred to naïve TSLPR+/+ (open square) and TSLPR−/− (solid square) mice (n = 5/group). Recipient mice were orally challenged with OVA. (A) Cytokine secretion from MLN cells of recipient mice. (B) IL-4 and IL-13 messenger RNA expression in the small intestine. (C) Serum OVA-specific IgE measured after the final OVA feed. (D) Jejunal mast cells expressed as number per high-power field (HPF). (E) Percent of recipient mice experiencing diarrhea after each OVA feed. Data are expressed as mean + standard error of mean of 5 mice per group.
Gastroenterology  , e4DOI: ( /j.gastro )
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7. Figure 6
Lamina propria-derived DCs from TSLPR−/− mice are functional in their ability to induce Th2 responses. (A) OVA-TCR transgenic CD4+ T cells were cultured with DCs isolated from the lamina propria of TSLPR+/+ (open square) and TSLPR−/− (solid square), in the presence of OVA323–339 for 72 hours. (B) DO11.10 CD4+ T cells were transferred to TSLPR+/+ or TSLPR−/− mice and then fed with OVA (open squares) or OVA + CT (solid squares). After 72 hours, cells were isolated from the MLN and cultured with OVA323–339 for 72 hours. IL-4, IL-13, and IFN-γ levels were measured by enzyme-linked immunosorbent assay. Results are expressed as mean + standard error of mean of 3 (A) or 4 (B) experiments.
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8. Figure 7
TSLPR on T cells is necessary for Th2 skewing. TSLPR+/+ and TSLPR−/− DO11.10 CD4+ T cells were cocultured with DCs from the MLN of TSLPR+/+ and TSLPR−/− mice. Cells were cultured ± 50 ng/mL of TSLP. IL-4 and IL-13 secretion were measured by enzyme-linked immunosorbent assay. Data are shown as the difference in cytokine secretion ± exogenous TSLP. Results are expressed as mean + standard of mean of 5 experiments. **P < .01.
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9. Supplementary Figure 1
Cytokine secretion in ovalbumin (OVA) sensitized mice before and after repeated OVA feeding. Cells were isolated from the spleen (before repeated OVA feeding) or from the mesenteric lymph node (after repeated OVA feeding) of thymic stromal lymphopoietin receptor (TSLPR)+/+ (open squares) and TSLPR−/− (solid squares) mice and restimulated in vitro with OVA. Interleukin-13, interleukin-10, and interferon-γ production were measured by enzyme-linked immunosorbent assay. Results are expressed as a mean + standard error of mean of 5 mice/group. *P < .05, ***P < .001.
Gastroenterology  , e4DOI: ( /j.gastro )
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10. Supplementary Figure 2
Thymic stromal lymphopoietin (TSLP) receptor (TSLPR) expression on CD4+ T-cell subsets. Mesenteric lymph node (MLN) cells were isolated and stained for expression of the TSLPR and the interleukin (IL)-7 receptor α subunit, which together comprise the functional TSLP receptor. CD4+ T-cell subsets were identified as CD62L+ CD44− (naïve), CD62L− CD44+ (effector memory), and CD62L+ CD44high (central memory). To assess the impact of activation, 106 MLN cells were cultured overnight in the presence or absence of αCD3/CD28. The red line indicates staining with the minus antibody control, and the blue line indicates antibody staining.
Gastroenterology  , e4DOI: ( /j.gastro )
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11. Supplementary Figure 3
Effect of thymic stromal lymphopoietin (TSLP) on T helper cell (Th) 2 priming in vivo. TSLP receptor (TSLPR)+/+ and TSLPR−/− DO11.10 cells were adoptively transferred to TSLPR+/+ or TSLPR−/− mice. The next day, 100 μg of ovalbumin (OVA) (open squares) or OVA+0.5 μg of recombinant TSLP (solid squares) were injected in the footpad. After 72 hours, cells were isolated from the draining lymph nodes, cultured, and restimulated in vitro with OVA323–339. Interleukin-4 and interleukin-13 secretion were measured by enzyme-linked immunosorbent assay. Data are expressed as mean + standard error of mean of 4 mice per group and are representative of 3 experiments.
Gastroenterology  , e4DOI: ( /j.gastro )
Copyright © 2010 AGA Institute Terms and Conditions