Death Receptor 3 Signaling Controls the Balance between Regulatory and Effector Lymphocytes in SAMP1/YitFc Mice with Crohn's Disease-Like Ileitis

Abstract

Death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, has been implicated in regulating T-helper type-1 (T_H1), type-2 (T_H2), and type-17 (T_H17) responses as well as regulatory T cell (T_reg) and innate lymphoid cell (ILC) functions during immune-mediated diseases. However, the role of DR3 in controlling lymphocyte functions in inflammatory bowel disease (IBD) is not fully understood. Recent studies have shown that activation of DR3 signaling modulates T_reg expansion suggesting that stimulation of DR3 represents a potential therapeutic target in human inflammatory diseases, including Crohn's disease (CD). In this study, we tested a specific DR3 agonistic antibody (4C12) in SAMP1/YitFc (SAMP) mice with CD-like ileitis. Interestingly, treatment with 4C12 prior to disease manifestation markedly worsened the severity of ileitis in SAMP mice despite an increase in FoxP3⁺ lymphocytes in mesenteric lymph node (MLN) and small-intestinal lamina propria (LP) cells. Disease exacerbation was dominated by overproduction of both T_H1 and T_H2 cytokines and associated with expansion of dysfunctional CD25^-FoxP3⁺ and ILC group 1 (ILC1) cells. These effects were accompanied by a reduction in CD25⁺FoxP3⁺ and ILC group 3 (ILC3) cells. By comparison, genetic deletion of DR3 effectively reversed the inflammatory phenotype in SAMP mice by promoting the expansion of CD25⁺FoxP3⁺ over CD25^-FoxP3⁺ cells and the production of IL-10 protein. Collectively, our data demonstrate that DR3 signaling modulates a multicellular network, encompassing T_regs, T effectors, and ILCs, governing disease development and progression in SAMP mice with CD-like ileitis. Manipulating DR3 signaling toward the restoration of the balance between protective and inflammatory lymphocytes may represent a novel and targeted therapeutic modality for patients with CD.

Keywords: CD25+/− T cells; Crohn’s disease; SAMP1/YitFc; TL1A; death receptor 3; ileitis; inflammatory bowel disease; innate lymphoid cell; regulatory T cells.

Figure 1

DR3 stimulation accelerates ileitis development in SAMP mice. (A) Representative photomicrographs of ileal sections of SAMP mice treated with control IgG isotype (left panel) or with 4C12 (right panel). Scale bar is 50 µm. (B) Total histologic score represents the sum of five indices including (C) active inflammation, (D) chronic inflammation, (E) transmural inflammation, (F) villous distortion, and (G) monocyte inflammation. Data presented as median ± interquartile range and analyzed by Mann–Whitney test, n = 6. (H) Mucosal architecture of fixed postmortem ileal specimens collected from 4C12-treated and IgG-treated SAMP mice was examined by stereomicroscopy. (I) Cobblestone area expressed as percentage of total specimen calculated in the ileum of 4C12-treated and IgG-treated SAMP mice. Data presented as median ± interquartile range and analyzed by two-tailed unpaired t-test, n = 4–6. Data are representative of three independent experiments.

Figure 2

DR3 stimulation increases FoxP3⁺ T_regs without altering T_H17 cell population during chronic ileitis. (A) Flow-cytometric analysis of mesenteric lymph node cells from IgG- and 4C12-treated SAMP or AKR mice after staining with specific anti-CD4 and anti-FoxP3 Abs or (C) anti-CD4 and anti-IL17 Abs. (B) Relative expression of FoxP3 mRNA and (D) of IL-17A mRNA was measured in total tissue RNA extracted from the terminal ilea of SAMP or AKR mice treated with IgG or 4C12 (10-week-old, n = 4). The relative expression of each target gene was normalized to the relative expression of β-actin in the sample. Data presented as median ± interquartile range and analyzed by two-way ANOVA, with Bonferroni’s post hoc test. Data are representative of three independent experiments.

Figure 3

DR3 stimulation correlates to the expansion of CD25⁻FoxP3⁺ cells during chronic ileitis. (A,B) Flow-cytometric analysis of mesenteric lymph node and (C,D) lamina propria cells from IgG- and 4C12-treated SAMP mice (10-week-old, n = 6–5) after staining with specific anti-CD25 and anti-FoxP3 Abs. The frequency of CD25⁺FoxP3⁺ and CD25⁻FoxP3⁺ cells is indicated. All data are presented as median ± interquartile range. Data in graphs A, B, and C were analyzed by two-tailed unpaired t-test. Data in graph D were analyzed by Mann–Whitney test. Data are representative of three independent experiments.

Figure 4

DR3 deletion ameliorates ileitis severity and expands CD25⁺FoxP3⁺ cells in SAMP mice. (A) Representative photomicrographs of ileal sections of SAMP mice wild-type (DR3_WT) and lacking DR3 (DR3_KO). Scale bar is 50 µm. (B) Total histologic score presented as median ± interquartile range and analyzed by two-tailed unpaired t-test, n = 6. (C) Fixed postmortem ileal specimens collected from DR3_WT and DR3_KO mice (10-week-old, n = 6) and analyzed by stereomicroscopy to assess area of abnormal (i.e., cobblestone lesions) and normal mucosa. (D) Cobblestone area expressed as percentage of total specimen calculated in the ileum of DR3_WT and DR3_KO mice (10-week-old, n = 6). Data presented as median ± interquartile range and analyzed by two-tailed unpaired t-test, n = 4. (E) Flow-cytometric analysis of mesenteric lymph node (MLN) and (F) lamina propria (LP) cells from DR3_WT and DR3_KO mice (10-week-old, n = 6) after staining with specific anti-CD4 and anti-FoxP3. Data presented as median ± interquartile range and analyzed by two-tailed unpaired t-test, n = 6. (G,H) Flow-cytometric analysis of MLN and (I,J) LP cells from DR3_WT and DR3_KO mice (10-week-old, n = 6) after staining with specific anti-CD25 and anti-FoxP3 Abs. Data presented as median ± interquartile range and analyzed by two-tailed unpaired t-test. Data are representative of three independent experiments.

Figure 5

DR3 stimulation triggers T_H1 and T_H2 mediators and reduces anti-inflammatory response during chronic ileitis. Mesenteric lymph node cells from IgG- or 4C12-treated SAMP and AKR mice (10-week-old, n = 6) were cultured in RPMI medium supplemented with anti-CD3/CD28 Abs. After 72 h, the secretion of indicated cytokines was quantified in cell supernatants [IL-10 (A), IL-13 (B), IFN-γ (C), IL-17A (D)]. Data presented as median ± interquartile range and analyzed by two-way ANOVA, with Bonferroni’s post hoc test. Data are representative of three independent experiments.

Figure 6

Genetic deletion of DR3 enhances anti-inflammatory response and reduces T_H1, T_H2, and T_H17 cytokines in SAMP mice. Mesenteric lymph node cells from DR3_WT and DR3_KO mice (10-week-old, n = 6) were cultured in RPMI medium supplemented with anti-CD3/CD28 Abs. After 72 h, the secretion of indicated cytokines was quantified in cell supernatants [IL-10 (A), IL-13 (B), IFN-γ (C), IL-17A (D)]. Data presented as median ± interquartile range and analyzed by two-tailed unpaired t-test. Data are representative of three independent experiments.

Figure 7

Upon death receptor 3 stimulation, increased innate lymphoid cell group 1 (ILC1) and decreased ILC3 frequencies are associated with intestinal inflammation. Flow-cytometric analysis of mesenteric lymph node cells from IgG- or 4C12-treated SAMP mice (10-week-old, n = 5) after staining with specific Abs for detection of ILC populations, including (A) T-bet⁺ ILC1s and (B) receptor-related orphan receptor-γt (ROR-γt⁺) ILC3s. Cell frequencies presented as median ± interquartile range and analyzed by Mann–Whitney test. Data are representative of three independent experiments.