1. Volume 146, Issue 1, Pages 166-175 (January 2014)
Endogenous Regulation of Visceral Pain via Production of Opioids by Colitogenic CD4+ T Cells in Mice 
Jérôme Boué, Lilian Basso, Nicolas Cenac, Catherine Blanpied, Malvyne Rolli–Derkinderen, Michel Neunlist, Nathalie Vergnolle, Gilles Dietrich 
Gastroenterology 
Volume 146, Issue 1, Pages (January 2014)
DOI: /j.gastro
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2. Figure 1
Th1 and Th17 cells produce enkephalin-containing peptides. (A) CD4+ T cells isolated from naive DO11.10 mice were differentiated into Th1 (white bars) or Th17 (black bars) in vitro. IFN-γ and IL-17 were quantified by CBA in supernatants recovered 24 hours after an additional incubation of the cells with anti-CD3/anti-CD28 mAbs. (B) PENK mRNA in Th1 (white bars) and Th17 (black bars) was quantified by quantitative real-time polymerase chain reaction. mRNA content was normalized to HPRT mRNA and quantified relative to standard mouse brain complementary DNA. In panels A and B, data are expressed as mean ± SEM of 3 independent experiments performed in duplicate. Statistical analysis was performed using Mann–Whitney U test (*P < .05). (C) Assessment of intracytoplasmic Met-enkephalin–containing peptides by cytofluorometry. Th1 (left panel) or Th17 (right panel) lymphocytes were incubated with either control rabbit IgG (white histogram) or rabbit anti–Met-enkephalin polyclonal IgG antibodies (black histogram). (D) Assessment of intracytoplasmic Met-enkephalin–containing peptides by immunofluorescence staining. Th1 (upper panels) or Th17 (lower panels) were incubated with either normal control rabbit IgG (left panels) or anti–Met-enkephalin rabbit IgG (right panels). Fluorescence images were acquired by confocal microscopy. Scale bar = 5 μm. Panels C and D show one representative experiment out of 3 performed.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2014 AGA Institute Terms and Conditions

3. Figure 2
Colitis induced by adoptive transfer of CD4+CD45RBhigh T lymphocytes is associated with PENK mRNA up-regulation in mucosal CD4+ T lymphocytes. (A) The time course of the disease was monitored in SCID mice passively transferred with CD4+CD45RBhigh alone (black symbols, n = 15) or together with CD4+CD45RBlow T lymphocytes (white symbols, n = 13) by weight loss until the mice were killed 6 weeks later. Statistical analysis was performed using repeated-measures 2-way analysis of variance and subsequent Bonferroni post hoc test when appropriate (***P < .001). (B) The intensity of disease was assessed 6 weeks after T-cell transfer by measuring wall thickness (left panel), macroscopic appearance of the colon (middle panel), and histological analysis of tissue damage (right panel). (C) Proinflammatory cytokines (IL-1β and tumor necrosis factor α) and chemokines (CCL2 and KC) were quantified by CBA in colonic samples from SCID mice transferred with CD4+CD45RBhigh T cells (black histogram) or CD4+CD45RBhigh plus CD4+CD45RBlow T cells (white histogram). In panels B and C, data are expressed as mean ± SEM. **P < .01; ***P < .001 (Mann–Whitney U test). (D) Colonic tissue samples recovered 6 weeks after T-cell transfer from CD4+CD45RBhigh plus CD4+CD45RBlow T cell–transferred SCID mice (upper panel) and CD4+CD45RBhigh T cell–transferred SCID mice (lower panel) were stained with anti-CD3 (lymphocytes), anti-F4/80 (macrophages), or anti–Ly-6B.2 (neutrophils) antibodies. The picture depicts a representative immunostaining. Cell nuclei were stained with DAPI. Fluorescence images were acquired by confocal microscopy. Scale bar = 5 μm. (E) CD4+CD45RBhigh T cells isolated from naive BALB/c mice (white bars) and lamina propria mononuclear cells isolated from SCID mice 6 weeks after transfer of CD4+ CD45RBhigh T cells (black bars) were incubated with anti-CD3/anti-CD28 mAbs for 24 hours. IFN-γ (left panel) and IL-17 (middle panel) were quantified by CBA in supernatants. PENK mRNA content (right panel) was quantified by quantitative polymerase chain reaction as described in Figure 1B. Data are expressed as mean ± SEM of at least 3 independent experiments performed in duplicate. *P < .05; **P < .01 (Mann–Whitney U test).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2014 AGA Institute Terms and Conditions

4. Figure 3
Colitogenic Th1 and Th17 lymphocytes isolated from inflamed intestinal mucosa produce endogenous opioids. (A) Intracytoplasmic accumulation of Met-enkephalin–containing peptides was assessed by cytofluorometry in macrophages (F4/80+ CD4− cells), in total CD4+ T lymphocytes (F4/80− CD4+ cells), and in both Th1 (IFNγ+ IL-17A− T cells) and Th17 (IFNγ− IL-17A+ T cells) subsets of CD4+ T lymphocytes isolated from the lamina propria of SCID mice 6 weeks after adoptive transfer of CD4+CD45RBhigh T lymphocytes. (B) Expression of Met-enkephalin–containing peptides was assessed by immunofluorescence staining on lamina propria mononuclear cells isolated from SCID mice 6 weeks after transfer of CD4+CD45RBhigh T lymphocytes. Cells were stained with rabbit anti–Met-enkephalin antibodies (green) together with either anti-CD3 (red, upper panel) or anti-F4/80 (red, lower panel) antibodies. Cell nuclei were stained with DAPI. Fluorescence images were acquired by confocal microscopy. Scale bar = 5 μm. (C) Expression of Met-enkephalin–containing peptides was assessed by cytofluorometry in CK18+ intestinal epithelial cells from SCID mice 6 weeks after adoptive transfer of CD4+CD45RBhigh T lymphocytes.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2014 AGA Institute Terms and Conditions

5. Figure 4
mRNA expression of endogenous opioids in myenteric plexus is not altered in CD4+CD45RBhigh-induced colitis. Longitudinal muscle/myenteric plexus was isolated from nontransferred SCID mice (white histogram, n = 10) or SCID mice 6 weeks after CD4+CD45RBhigh T-cell transfer (black histogram, n = 10). PENK (left panel) and PDYN (right panel) mRNA were quantified by quantitative polymerase chain reaction as described in Figure 1B. Data are expressed as mean ± SEM. Statistical analyses were performed using Mann–Whitney U test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2014 AGA Institute Terms and Conditions

6. Figure 5
Peripheral opioid receptor blockade increases inflammation-induced colonic sensitivity. (A) Colonic sensitivity was measured 30 minutes after intraperitoneal administration of PBS in nontransferred SCID mice (open squares, n = 10) or either PBS (open circles, n = 18) or naloxone methiodide (closed circles, n = 17) in mice 6 weeks after CD4+CD45RBhigh T-cell transfer. Abdominal muscle contraction was recorded in response to distention pressures of 15, 30, 45, and 60 mm Hg. Data are expressed as mean ± SEM. Statistical analysis was performed using repeated-measures 2-way analysis of variance and subsequent Bonferroni post hoc test when appropriate; **P < .01, ***P < .001 (comparison with CD4+CD45RBhigh T cell–transferred SCID mice treated with PBS). (B) Inflamed CD4+CD45RBhigh T cell–transferred SCID mice intraperitoneally injected with PBS (white histogram) or naloxone methiodide (black histogram) were killed after colorectal distention and colonic tissue damage was assessed by measuring wall thickness (left panel) and scoring macroscopic (middle panel) and microscopic (right panel) injury. (C) The fold increase in visceral sensitivity after treatment with naloxone methiodide was compared between normal nontransferred SCID mice (white bars, n = 10), SCID mice transferred with both CD45RBhigh and CD45RBlow T lymphocytes (gray bars, n = 8) and SCID mice transferred with CD45RBhigh T lymphocytes (black bars, n = 17). The fold increase in abdominal muscle activity in response to distention pressures of 45 and 60 mm Hg was calculated as the ratio between treatments with naloxone methiodide and PBS. The fold increase induced by naloxone methiodide was calculated for each mouse relative to the average of abdominal response measured in PBS-treated mice. Results are expressed as mean ± SEM. ***P < .001 (Mann–Whitney U test).
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Accumulation of colitogenic CD4+ T cells in the inflamed intestine from mice with DSS-induced colitis reduces visceral inflammatory pain while enhancing colonic tissue damage. (A) The intensity of disease was assessed in normal mice (D0, n = 22), in mice on day 5 after treatment with DSS (D5, n = 23), and in mice treated with DSS for 5 days and then with water until day 10 (D10, n = 17). The intensity of disease was scored by measuring wall thickness (left panel), macroscopic appearance of the colon (middle panel), and histological analysis of tissue damage (right panel). Data are expressed as mean ± SEM. (B) The density of CD3+ T lymphocytes in inflamed intestine from mice on day 5 after treatment with DSS (D5, n = 10) and mice treated with DSS for 5 days and then with water until day 10 (D10, n = 12) was estimated by quantifying specific anti-CD3 fluorescence intensity relative to the tissue surface delimited manually with DAPI staining.16 Each point, corresponding to one animal, represents the mean of 4 different histological examinations. In panels A and B, statistical analyses were performed using Mann–Whitney U test. (C) Intracytoplasmic accumulation of Met-enkephalin–containing peptides was assessed by cytofluorometry in CD4+ T lymphocytes isolated from the lamina propria of mice treated with DSS for 5 days and then with water until day 10. (D) Colonic sensitivity was measured in normal mice (D0, open circles, n = 11), in mice on day 5 after treatment with DSS (D5, open squares, n = 10), and in mice treated with DSS for 5 days and then with water until day 10 (D10, closed circles, n = 15). Abdominal muscle contraction was recorded in response to distention pressure of 15, 30, and 45 mm Hg. Data are expressed as mean ± SEM. Statistical analysis was performed using repeated-measures 2-way analysis of variance and subsequent Bonferroni post hoc test when appropriate (**P < .01, ***P < .001; DSS-treated mice [D5] vs normal mice [D0]). (E) Colonic sensitivity was measured, as described in the preceding text, 30 minutes after intraperitoneal administration of either PBS (white histogram, n = 15) or naloxone methiodide (black histogram, n = 10) in mice treated with DSS for 5 days and then with water until day 10 (D10 mice). Results are expressed as mean ± SEM. Statistical analyses were performed using Mann–Whitney U test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2014 AGA Institute Terms and Conditions