Induced Treg Cells Augment the Th17-Mediated Intestinal Inflammatory Response in a CTLA4-Dependent Manner

<div><p>Th17 cells and Foxp3⁺ regulatory T cells (Tregs) are thought to promote and suppress inflammatory responses, respectively. However, whether they counteract each other or synergize in regulating immune reactions remains controversial. To determine their interactions, we describe the results of experiments employing mouse models of intestinal inflammation by transferring antigen-specific Th cells (Th1, Th2, and Th17) differentiated <i>in vitro</i> followed by the administration of the cognate antigen via enema. We show that cotransfer of induced Tregs (iTregs) suppressed Th1- and Th2-mediated colon inflammation. In contrast, colon inflammation induced by transfer of Th17 cells, was augmented by the cotransfer of iTregs. Furthermore, oral delivery of antigen potentiated Th17-mediated colon inflammation. Administration of a blocking antibody against cytotoxic T lymphocyte-associated antigen 4 (CTLA4) abrogated the effects of cotransfer of iTregs, while the injection of a soluble recombinant immunoglobulin (Ig) fusion protein, CTLA4-Ig substituted for the cotransfer of iTregs. These results suggest that antigen-specific activation of iTregs in a local environment stimulates the Th17-mediated inflammatory response in a CTLA4-dependent manner.</p></div

Anti-CTLA4 antibody abrogates the effects of iTregs, and soluble CTLA4 alone mimics the effects of iTregs.

<p>(A) Effector T cells (Th2 or Th17) were intravenously transferred with or without iTregs in the presence or absence of anti-CTLA4 antibody (20 microg) and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. CTI values were calculated and are shown as mean and standard error (SE). Independent experimental sets were designed for histological analysis, and representative images are shown. (B) iTregs or effector T cells (Th2 or Th17) were intravenously transferred in the presence of anti-CTLA4 antibody (20 microg) or control antibody (20 microg, indicated as minus) and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. (C) Effector T cells (Th2 or Th17) were intravenously transferred in the presence or absence of an indicated amount of CTLA4-Ig, and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. CTI values were calculated and are shown as mean and SE.</p

Antigen-specific iTregs exacerbate Th17-mediated colon inflammation in eosinophil-deficient mice.

<p>deltadblGATA mice were adoptively engrafted with effector T cells (2×10⁷ cells /mouse: Th2/Th17) alone or together with iTregs (1 × 10⁷ cells/mouse). (A) Recipient mice were challenged with OVA and CTI values were determined. (B) Cells were prepared from the cLP and subjected to flow cytometric analysis to determine the percentage of neutrophils. Representative flow cytometry data of two independent experiments are shown.</p

Co-transfer of iTregs or oral administration of OVA stimulates colon thickening mediated by Th17.

<p>(A) Each effector cell (2 × 10⁷ cells per mouse: Th1, Th2, Th17) was adoptively transferred or not (none) with or without iTregs (1 × 10⁷ cells per mouse) into wild-type BALB/c mice and each mouse was immunized with OVA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. CTI values are shown as the mean and standard error (SE). Independent experimental sets were designed for histological analysis, and representative images are shown. (B) Before adoptive transfer, BALB/c mice were continuously supplied with OVA (1 mg/mL) in their drinking water for 7 days to induce oral tolerance (indicated as OVA-fed). Each effector cell (2 × 10⁷ cells/mouse: Th1/Th2/Th17) was adoptively transferred, and mice were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. CTI and histological analysis were performed as described above.</p

<i>Il17a</i>-deficient Th17 cells induce a diminished but significant inflammatory responses.

<p>(A) Th17 cells were differentiated in vitro from naïve CD4⁺T cells derived from <i>Il17a</i>-deficient (<i>Il17a</i>-KO:DO11.10⁺:<i>Rag2</i>-KO) or <i>Il17a</i>-sufficient (<i>Il17a</i>^+/−:DO11.10⁺:<i>Rag2</i>-KO) mice. Eosinophil-deficient deltadblGATA mice were engrafted and treated with OVA as described above. Spleen weights were measured and analyzed (<i>n</i> = 4). The weight-to-length ratio of the colon was calculated and expressed as CTI. Mononuclear cells of the cLP were prepared and subjected to flow cytometric analysis to determine the frequencies of CD11b⁺ Gr-1⁺ cells. Representative flow cytometry data of two separately performed and reproducibly repeated experiments are shown. (B) <i>Il17a</i>^+/− Th17 or <i>Il17a</i>-KO Th17 cells were restimulated using the anti-CD3epsilon-/anti-CD28-conjugated beads (Life Technologies) for 48 h. Secreted cytokines were quantified using ELISA, as described in Materials and Methods. (C) Restimulated cells were subjected to flow cytometric analysis to investigate RORgammat expression as a marker of Th17 cells. The shaded histogram shows the control experiment using an isotype-matched antibody. Frequencies of RORgammat⁺ cells are indicated. (D) deltadblGATA mice were engrafted with <i>Il17a</i>^+/− Th17 or <i>Il17a</i>-KO Th17 cells and immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150244#pone.0150244.g001" target="_blank">Fig 1</a>. Mononuclear cells were prepared from the cLP and spleen and stained with mAbs against CD3epsilon, CD4, and DO11.10 TCR. The frequencies of DO11.10 TCR⁺ cells in the total population of CD3⁺CD4⁺ cells are shown.</p

Adoptive transfer of effector T cells induces colon thickening.

<p>Each effector T cell subset (2 × 10⁷ viable cells) was intravenously transferred to wild-type BALB/c mice, and OVA protein was administered via enema once a week for 4 weeks. The day after the last OVA challenge, the colonic weight-to-length ratio (mg/mm) was calculated as the colon thickness index (CTI) to evaluate the inflammatory response. (A) Three types of effector cells (Th1/Th2/Th17) induced CTI using this model mouse. (B) Representative histological images of HE-stained mid-colonic sections are shown. Scale bars indicate 500 microm. (C) Mononuclear cells were isolated from the spleen and cLP of recipient mice. CD11b⁺ CCR3⁺ and CD11b⁺ Gr-1⁺ cells were gated, and their frequencies (%) were determined using flow cytometric analysis. Representative data of three independent experiments are shown. (D) Mononuclear cells were isolated from the cLP of recipient mice and stained with monoclonal antibodies (mAbs) against CD11b, CCR3, Gr-1, and Siglec-F. The frequencies (%) of Siglec-F⁺ Gr-1^middle cells in the total population of CD11b⁺ CCR3⁺ cells are shown</p

CTLA4-Ig augments the ratio of IL-17A-producing cells in the presence of anti-TGF-beta antibody.

<p>(A) CD4⁺ T cells prepared from DO11.10⁺:<i>Rag2</i>-KO mice were stimulated under the condition for Th17 cell lineage, namely medium supplemented with IL-6, IL-23, TGF-beta1, IL-1beta and TNF-alpha, in the absence (control) or presence of CTLA4-Ig (+CTLA4-Ig, 20microg/mL). After 7 days, cells were restimulated with PMA (20 ng /mL) and ionomycin (1 μM) in the presence of monensin for 4 h. Cells were stained with anti-CD4 and treated with FVD and subjected to the analysis for intracellular expression of the indicated cytokines (IL-17A and IL-17F) and FoxP3. (B) Th17 cells were stimulated again using the APCs (irradiated splenocytes derived from <i>Rag2</i>-KO mice) and OVA peptide in the medium without cytokines in the absence (control) or presence of CTLA4-Ig (+CTLA4-Ig, 20microg/mL). After 6 days, cells were examined as described in (A). (C) Th17 cells were differentiated using the medium supplement with IL-6, IL-23 IL-1beta and TNF-alpha, but not with TGF-beta1 (indicated as Th17 recipe without TGF-beta1), in the absence (none) or presence (+anti-TGF-beta1,2,3, 10microg/mL) of anti-TGF-beta antibody in conjunction with CTLA4-Ig (20 microg/mL) or not. After 6 days, cells were examined as described in (A). (D) Th17 cells were re-stimulated as described in (B) in the absence (none) or presence (+anti-TGF-beta1,2,3) of anti-TGF-beta antibody in conjunction with CTLA4-Ig or not as shown in (C). After 6 days, cells were examined as described in (A).</p

Defect in antigen-induced NHR in antigen-specific IgE-Tg mice.

<p>IgE-Tg mice as well as antigen-immunized mice and T cell-transferred mice were challenged 3–4 times with OVA or saline, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g006" target="_blank">6</a>. Six hours after the 3rd challenge, antigen-specific serum IgE, IgG, IgG₁, IgG_2a, IgG_2b, and IgG₃ were determined (A). The number of sneezes evoked by histamine (B) and accumulation of lymphocytes, neutrophils, and eosinophils in NALF (C) of IgE-Tg mice were examined 6 h after the 4th challenge. Data are expressed as mean ± SEM for 4–8 animals. *<i>p</i> < 0.05, **<i>p</i> < 0.01 (Dunn’s test). N.D.: not detectable.</p

Antigen-induced NHR in immunized mice.

<p>Mice were immunized with 4-time i.p. injection of OVA plus alum. Two weeks after the last immunization, mice were challenged once a day with daily i.n. injection of OVA or BSA solution, or of saline on days 35–38 and 41–43. Then, these mice were challenged with OVA, BSA, or saline on day 44 (A). On days 41–44, the number of sneezes was counted for 5 min just after i.n. administration of OVA, BSA, or saline (B). The BSA- and histamine-evoked sneezing response was evaluated at 6 h after 0 (day 34)- to 4 (day 38)-time challenge with OVA or saline (C). Time course of histamine-evoked sneezing response after 4-time challenge on days 35–38 with OVA or saline was evaluated (D). Data are expressed as mean ± SEM for 4–10 animals. *<i>p</i> < 0.05, compared with saline-challenged control mice (Mann-Whitney <i>U</i> test).</p

Effect of anti-CD4 mAb on antigen-induced NHR.

<p>Immunized mice were challenged 4 times with OVA or saline, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146686#pone.0146686.g002" target="_blank">Fig 2</a>. Anti-CD4 mAb or control rat IgG was administered twice, that is, at 9 and 6 days before the last challenge. Six hours after the last challenge, the CD3⁺CD4⁺ population in the spleen was determined by flow cytometry (A). Representative data from 3 independent experiments are shown. The number of sneezes evoked by histamine (B) and the accumulation of lymphocytes, neutrophils, and eosinophils in NALF (C) were also examined. Data are expressed as mean ± SEM for 4–6 animals. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.01, compared with OVA-challenged control mice (Dunn’s test).</p