1. Volume 146, Issue 7, Pages 1752-1762.e4 (June 2014)
The Tumor Necrosis Factor Family Member TNFSF14 (LIGHT) Is Required for Resolution of Intestinal Inflammation in Mice 
Petra Krause, Sonja P. Zahner, Gisen Kim, Raziyah B. Shaikh, Marcos W. Steinberg, Mitchell Kronenberg 
Gastroenterology 
Volume 146, Issue 7, Pages e4 (June 2014)
DOI: /j.gastro
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2. Figure 1
LIGHT-deficiency accelerates T-cell transfer–induced colitis. Wild-type CD4+CD45RBhigh T cells were transferred into Tnfsf14−/−Rag1−/− (gray) or Rag1−/− (black) recipients. (A) Body weight was calculated as percentage of individual weight at the time of transfer. Mean and SEM are shown from 6 independent experiments with 2–5 mice in each group in each experiment. (B) Frequency of CD4+ T cells in colon lamina propria 3–4 weeks after T-cell transfer. (C, D, E) Cytokine expression in proximal colon tissue 3–4 weeks after transfer was assessed by real-time polymerase chain reaction and mRNA expression of (C) TNF, (D) IL-17A, and (E) IL-6 mRNA is presented relative to L32 expression. Each symbol represents measurements from an individual mouse. ∗∗P < .01; ∗∗∗P < .001
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3. Figure 2
LIGHT-deficiency aggravates DSS-induced chronic colitis. Colitis was induced in wild-type (black) and LIGHT-deficient (Tnfsf14−/−, gray) mice by administration of 2 or 4 cycles of 3% DSS in drinking water; control mice received normal drinking water without DSS. (A) Body weight and (B) survival were monitored (log-rank test, ∗∗∗P < .0001). Body weight is presented as percentage of starting weight. (C, D) After 29 days large intestines were taken and compared for length. Means and SEM of 5 independent experiments with 3–5 mice per group per experiment are presented. (E) Histology scores of colon and cecum were compared after 2 or 4 cycles of DSS treatment. Means and SEM of 6 independent experiments with 3–5 mice per group per experiment are shown. Mann-Whitney test, ∗∗P < .01; ∗∗∗P < (F) H&E staining of sections from distal colon (upper panel) and cecum (lower panel). e, epithelial disruption, i, inflammatory infiltrate, m, lamina muscularis mucosae, s, submucosal swelling.
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4. Figure 3
Cell infiltration in DSS-induced colitis. Colitis was induced with 2 or 4 cycles of DSS in Tnfsf14−/− (gray) and wild-type (black) mice. Control mice were kept on regular drinking water without DSS. Frequencies of (A) CD4+ T cells, (B) neutrophils, and (C) monocytes were analyzed in large intestine lamina propria after (A, B) 2 and 4 cycles, or (C) 2 cycles of DSS. Blood was taken after 2 cycles of DSS treatment. Mean frequencies and SEM from (A, C) 4 or (B) 5 independent experiments are shown with 3–5 mice per group per experiment. Populations are presented as percentage of live cells expressing (A) CD4+TCRβ+, (B) CD11b+Ly6Ghigh, and (C) CD11b+Ly6GlowLy6Chigh. Each symbol represents measurements from an individual mouse. ∗P < .05; ∗∗P < .01; ∗∗∗P < (D) Colitis was induced with 4 cycles of DSS and 7-μm sections from distal colon or cecum were stained for total collagen (red) and noncollagen protein (green).
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5. Figure 4
Expression of inflammatory mediators in the colon in DSS-induced colitis. Colitis was induced with 2 cycles DSS in wild-type (black) and Tnfsf14−/− mice (gray). At day 12, distal colon tissue was assessed for mRNA expression of (A) cytokines and (B) chemokines by real-time polymerase chain reaction (PCR) using a RT2 Profiler PCR array. Expression is presented relative to the average expression of Gusb, Hprt1, Hsp90ab1, and Gapdh. Each symbol represents an individual mouse; combined data from 2 independent experiments are shown. ∗P < .05; ∗∗P < .01.
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6. Figure 5
LTβR transduces the protective LIGHT-mediated signal. (A) Colitis was induced with 3 cycles of 3% DSS in wild-type (black squares) or HVEM-deficient (Tnfrsf14−/−, gray triangles) mice. Control mice were kept on regular drinking water without DSS (black dotted line). Means and SEM are presented from 3 independent experiments with wild-type no DSS (n = 7), wild-type + DSS (n = 12), and Tnfrsf14−/− + DSS (n = 14). (C) Colitis was induced with 3 cycles of 3% DSS in wild-type (black lines) or Tnfsf14−/− (gray circles) mice. Wild-type mice were injected intraperitoneally twice a week with anti-LTβR monoclonal antibody (black triangles, dotted line) or rat IgG (black squares, solid line) starting at day 1. Means and SEM are presented from 2 independent experiments with Tnfsf14−/− (n = 6), wild-type + IgG (n = 7), wild-type + anti-LTβR (n = 7). (B, D) Wild-type CD4+CD45RBhigh T cells were transferred into Tnfsf14−/−Rag1−/− (gray) or Rag1−/− (black) recipients. Rag1−/− recipients received (B) anti-HVEM monoclonal antibody (dotted line) or hamster IgG (solid line) or (D) anti-LTβR monoclonal antibody (dashed line) or rat IgG (solid line) intraperitoneally twice a week starting at the time of T-cell transfer. (E, F) Anti-LTβR antibody was injected into C57BL/6 mice every 2–3 days starting at day 7 after the first cycle of DSS. Means and SEM for (B, E) 2 independent experiments with total 7–10 mice per group or (D) 6 independent experiments with total 19–21 mice per group. Body weight is calculated as percentage of individual starting weight and statistical analysis was performed between the antibody treated compared with control IgG-treated group; ∗P < .05; ∗∗P < .01; ∗∗∗P < (F) Symbols represent mean histologic scores of cecum and distal colon of individual mice; Mann Whitney test, ∗P < .05.
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7. Figure 6
LIGHT and LTβR expression in lamina propria populations. C57BL/6 mice were treated with 2 cycles of DSS or kept on regular water without DSS, and cell populations were sorted from large intestine lamina propria and assessed for expression of (A, B) LIGHT or (C, D) LTβR mRNA by real-time polymerase chain reaction relative to L32 expression. Cells were sorted into (A) CD45+ and CD45− populations, or (B, D) CD45+ cells were further divided into neutrophils (CD11b+Ly6Ghigh), the remaining CD11b+ and CD11b− cells and compared with CD45− cells. (C) From CD45− cells, podoplanin+ (Pdpn+) fibroblasts were sorted (CD31− and EpCAM− cells) and compared with CD45+ cells. Each symbol represents cells sorted from an individual mouse, except for neutrophils from mice without DSS, which were pooled from 3–4 mice.
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8. Figure 7
Cellular source of cytokines. C57BL/6 mice were treated with 2 cycles of DSS, and the indicated cell populations were sorted from large intestine lamina propria and assessed for expression of (A) IL-6, (B) IL-1β, or (C) Osm by real-time polymerase chain reaction (PCR) relative to L32 expression. (D) NIH3T3 cells were cultured with or without 1 ng/mL IL-1β or 10 ng/mL Osm or a combination of both and IL-6 mRNA expression was measured after 8 hours by real-time PCR relative to L32 expression. One experiment is shown out of 2 performed with similar results.
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9. Supplementary Figure 1
Histologic scores of distal colon and cecum after chronic DSS challenge. Chronic colitis was induced with 2 or 4 cycles of 3% DSS administration in Tnfsf14−/− (gray symbols) or wild-type (black symbols) mice. Control mice received regular drinking water without DSS (no DSS). Tissue sections from (A) distal colon and (B) cecum were stained with H&E and histologic scores were assigned. Means and SEM of 6 independent experiments with 3–5 mice per group per experiment are shown. Mann-Whitney test, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
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10. Supplementary Figure 2
Acute DSS-induced colitis. LIGHT-deficient (gray circles) or wild-type (black squares) mice were given (A) 1.5% or (B) 3% DSS in drinking water for 7 days. Control mice received regular drinking water with no DSS (dashed black line). Body weight was monitored daily and is presented as mean percentage of individual starting weight with SEM. Histologic scores and colon length were analyzed at day 8.
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11. Supplementary Figure 3
Chronic DSS-induced colitis in Rag1−/− and Tnfsf14−/−Rag1−/−. Chronic colitis was induced in Rag1−/− and Tnfsf14−/−Rag1−/− mice with 1 cycle of 2% DSS followed by 7 weeks on regular drinking water without DSS. Samples from distal colon and cecum tissue were embedded in paraffin and 5-μM sections were stained with H&E.
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12. Supplementary Figure 4
Neutrophils in distal colon. Colitis was induced with 2 cycles of 3% DSS in wild-type and Tnfsf14−/− mice. Control mice were kept on regular drinking water with no DSS. Neutrophils were stained with Ly6G-specific monoclonal antibody in 5-μm cryosections of distal colon. The sections from wild-type mice show autofluorescence from epithelial cells.
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13. Supplementary Figure 5
Intestinal barrier function. Wild-type (black) or Tnfsf14−/− mice (gray) were gavaged with 600 g/kg fluorescein isothiocyanate (FITC)–dextran (Sigma, St Louis, MO) und fluorescence intensity was measured 4 hours later in serum. Mice were analyzed without treatment or after 1 cycle of 3% DSS treatment at day 8. FITC-dextran concentration in serum was calculated compared with an FITC-dextran standard curve after subtraction of the fluorescence intensity value of serum from a control mouse that did not receive FITC-dextran.
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14. Supplementary Figure 6
Anti-LTβR antibody does not deplete LTβR-expressing cell populations. (A) CD45+ and CD45−Podoplanin+ (EpCAM−CD31−) cells were sorted from lamina propria of wild-type mice with chronic DSS-induced colitis. LTβR expression was quantified relative to L32 by real-time PCR. (B) Chronic colitis was induced with 2 cycles of DSS and 100 μg anti-LTβR antibody (LLTB2) was injected intraperitoneally every third day starting at day 7 of DSS treatment. Frequency of CD45−podoplanin+ cells was assessed in lamina propria of large intestine (excluding EpCAM+ and CD31+ cells). Symbols represent measurements from individual mice.
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15. Supplementary Figure 7
Chronic DSS-induced colitis in littermates. Colitis was induced with 2 cycles of 3% DSS in LIGHT-deficient or LIGHT-sufficient co-housed littermates. (A) Body weight was monitored and is presented as average percentage of individual starting weight with SEM. (B) Survival curve. Two independent experiments with Tnfsf14−/− (n = 12) and Tnfsf14+/+ (n = 8).
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