Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells
Christoph Becker, Stefan Wirtz, Manfred Blessing, Jaana Pirhonen, Dennis Strand, Oliver Bechthold, Julia Frick, Peter R. Galle, Ingo Autenrieth, Markus F. Neurath
Christoph Becker, Stefan Wirtz, Manfred Blessing, Jaana Pirhonen, Dennis Strand, Oliver Bechthold, Julia Frick, Peter R. Galle, Ingo Autenrieth, Markus F. Neurath
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Article Immunology

Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells

Abstract

IL-12 p40–related cytokines such as IL-12 p35/p40 heterodimer and IL-23 (p19/p40) are potent regulators of adaptive immune responses. Little is known, however, about the transcriptional regulation of the p40 gene in vivo. In an attempt toward this goal, we have generated transgenic mice expressing firefly luciferase under the control of the IL-12 p40 promoter. High constitutive transgene expression was found in the small intestine only, whereas little reporter gene activity was observed in other tissues. Within the small bowel, constitutive promoter activity was restricted to the terminal ileum and associated with high expression of p40 mRNA as well as p40 and IL-23 p19/p40 proteins. The cells constitutively producing IL-12 p40 were identified as CD8α and CD11b double-negative CD11c+ lamina propria dendritic cells (LPDCs) that represent a major cell population in the lamina propria of the small intestine, but not in the colon. FISH directly demonstrated the uptake of bacteria by a subset of LPDCs in the terminal ileum that was associated with p40 expression. Furthermore, little or no p40 protein expression in LPDCs was found in the terminal ileum of germfree mice, indicating a key role of the intestinal flora for constitutive p40 expression. In addition, analysis of transgenic mice with a mutated NF-κB target site in the p40 promoter showed a critical role of NF-κB for constitutive transgene expression. Our data reveal important functional differences between the mucosal immune systems of the small and large bowel in healthy mice and suggest that the high bacterial load in the terminal ileum activates p40 gene transcription in LPDCs through NF-κB. These data suggest a predisposition of the terminal ileum to develop chronic inflammatory responses through IL-23 and thus may provide a molecular explanation for the preferential clinical manifestation of Crohn disease in this part of the gut.

Authors

Christoph Becker, Stefan Wirtz, Manfred Blessing, Jaana Pirhonen, Dennis Strand, Oliver Bechthold, Julia Frick, Peter R. Galle, Ingo Autenrieth, Markus F. Neurath

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IL-12 p40 is produced largely by CD11c+ DCs located below the crypts in ...
IL-12 p40 is produced largely by CD11c+ DCs located below the crypts in the lamina propria. (a) Cryosections of transgenic mice were analyzed by immunohistochemistry for luciferase expression. One representative staining for luciferase in the small intestine (D4) of a transgenic animal out of four is shown (left panel). Luciferase-expressing cells were mainly seen in the lamina propria below the crypts (arrows). No staining was seen in sections from transgenic mice treated with an isotype control Ab right panel), the proximal segments of the small intestine (D1, D2), and in healthy nontransgenic control mice (not shown). (b) Detection of CD11c+ and CD11b+ cells in the lamina propria of transgenic mice. More CD11c+ than CD11b+ cells were detected in the lamina propria, suggesting that many cells in the lamina propria of healthy mice carry surface markers of DCs. No differences in the staining patterns of CD11b+ and CD11c+ cells were noted between the proximal and the distal segments of the small bowel. (c) IL-12 p40 cytokine levels in supernatants from CD11c+ enriched DCs and CD11c–CD11b+ enriched macrophages isolated from the lamina propria of healthy mice. Lamina propria cells were isolated as described in Methods and purified using the MACS system. To measure IL-12 p40 protein production, 500,000 cells/well were seeded out in 1 ml culture medium in triplicate and incubated in the presence or absence of LPS/SAC. After 24 hours, supernatants were removed and assayed for p40 concentration by ELISA. Unstim, unstimulated.