Volume 137, Issue 5, Pages 1736-1745 (November 2009) Th1/Th17 Immune Response Is Induced by Mesenteric Lymph Node Dendritic Cells in Crohn's Disease  Atsushi Sakuraba, Toshiro Sato, Nobuhiko Kamada, Mina Kitazume, Akira Sugita, Toshifumi Hibi  Gastroenterology  Volume 137, Issue 5, Pages 1736-1745 (November 2009) DOI: 10.1053/j.gastro.2009.07.049 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 The immune status of CD4+ T cells in MLNs of CD. (A) An MLN from the inflamed intestine of a CD patient is shown here, with abundant non-caseating granulomas (left, original magnification, ×10; right, original magnification, ×40 of the solid square in the left). (B and C) Intracellular production of IFN-γ, IL-4, and IL-17A was assessed in isolated CD4+ T cells from MLNs of NC, UC, and CD. Numbers indicate the percentages of cells in each quadrant. (D) Production of IFN-γ, IL-4, and IL-17A was assessed in isolated CD4+ T cells from MLNs of NC, UC, and CD by cytokine bead array or ELISA. ND, not detected. (E) Expression of the transcription factors T-bet, GATA-3, and RORc in CD4+ T lymphocytes from MLNs was assessed by real-time RT-PCR. A total of 4, 10, and 16 independent experiments for NC, UC, and CD, respectively, were performed for C–E. According to the available cell numbers in each experiment, only certain analyses were performed in some experiments. Horizontal bars show the mean value. Gastroenterology 2009 137, 1736-1745DOI: (10.1053/j.gastro.2009.07.049) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Isolation of MLN DCs. (A) Mononuclear cells were isolated from MLNs and analyzed by flow cytometry. Dead cells were excluded by observing the fluorescence intensity of propidium iodide (left; gray square). Lineage negative, MHC II high cells were identified as DCs (middle; gray square). These cells were divided into 3 populations using anti-CD11c, CD123 mAb, and fluorescence intensity of MHC II (right). These were myeloid DC (mDC; CD11c+/CD123−/MHC II+), plasmacytoid DC (pDC; CD11c−/CD123+/MHC II+), and mature DC (CD11c+/CD123+/MHC II+). Representative data of CD are shown. UC and NC showed similar results. (B) MLR between mDCs and allogeneic naïve T cells were performed. The proliferation of T cells was assessed by incorporation of 3H-thymidine. Shown are representative data from 4 individual experiments. Gastroenterology 2009 137, 1736-1745DOI: (10.1053/j.gastro.2009.07.049) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Surface phenotype of MLN DCs. (A) The surface phenotype of MLN DCs was assessed by flow cytometry. The black area shows a histogram plot of each marker and the gray area a histogram plot of respective isotype controls. Representative data from a CD patient are shown. Similar results were obtained from UC and NC. (B) The number of each DC subset was counted and the proportion of each calculated. A total of 3 independent experiments for NC, UC, and CD, respectively, was performed. Data are shown as mean ± SEM. *Indicates P < .05. Gastroenterology 2009 137, 1736-1745DOI: (10.1053/j.gastro.2009.07.049) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 mDCs from CD MLNs induce a Th1 response in allogeneic MLR. (A) MLR was performed between the sorted subgroups of DCs and allogeneic CD4+ CD45RA+ naïve T lymphocytes. Intracellular staining was performed for IFN-γ/IL-4 on the stimulated T cells. Representative data from CD are shown. UC and NC showed similar results. (B) Supernatants of MLR cultures were collected, and the content of IFN-γ, IL-4, and IL-10 were analyzed by CBA. Representative data from CD are shown. UC and NC showed similar results. (C) mDCs and CD4+CD45RA+ naïve T cells were cocultured, and cytokine production of IFN-γ, IL-4, and IL-17 by T cells was analyzed by intracellular cytokine staining as in A. (D) Cytokine concentrations in culture supernatants of MLR were analyzed by CBA. Representative or pooled data from 3 independent experiments for NC, UC, and CD, respectively, are shown in A–D. Data are shown as mean ± SEM in B and D. Gastroenterology 2009 137, 1736-1745DOI: (10.1053/j.gastro.2009.07.049) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Isolated DCs from CD MLNs produce high amounts of IL-23 and low amounts of IL-10 upon stimulation. (A) mDCs and mature DCs were isolated from MLNs of UC and CD, and the production of IL-12p40, IL-12p70, IL-23, and IL-10 in the supernatants was assessed by ELISA. Lipopolysaccharides (LPS) (10 μg/mL) and E faecalis cell extract were used for stimulation. UC, gray bar; CD, black bar. Three independent experiments for UC and CD, respectively, were performed. ND, not detected. (B) The ratio of IL-23p19 to IL-10 was calculated in UC and CD. (C) Mature/mDCs were isolated from MLNs of CD by magnetic sorting. MLR was performed with allogeneic CD4+ CD45RA+ naïve T lymphocytes. MLR was performed with or without the addition of 20 ng/mL of IL-23 or IL-10. (−): medium only. Representative of 3 independent experiments. Data are shown as mean ± SEM, where applicable. Gastroenterology 2009 137, 1736-1745DOI: (10.1053/j.gastro.2009.07.049) Copyright © 2009 AGA Institute Terms and Conditions