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. 2016 Mar;9(2):336-51.
doi: 10.1038/mi.2015.64. Epub 2015 Jul 15.

Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

A R B M Muzaki  1 , P Tetlak  1 , J Sheng  1 , S C Loh  1 , Y A Setiagani  1 , M Poidinger  2 , F Zolezzi  2 , K Karjalainen  1 , C Ruedl  1
Affiliations

Intestinal CD103(+)CD11b(-) dendritic cells restrain colitis via IFN-γ-induced anti-inflammatory response in epithelial cells

A R B M Muzaki et al. Mucosal Immunol. 2016 Mar.

Abstract

A crosstalk between commensals, gut immune cells, and colonic epithelia is required for a proper function of intestinal mucosal barrier. Here we investigated the importance of two distinct intestinal dendritic cell (DC) subsets in controlling intestinal inflammation. We show that Clec9A-diphtheria toxin receptor (DTR) mice after depletion of CD103(+)CD11b(-) DCs developed severe, low-dose dextran sodium sulfate (DSS)-induced colitis, whereas the lack of CD103(+)CD11b(+) DCs in Clec4a4-DTR mice did not exacerbate intestinal inflammation. The CD103(+)CD11b(-) DC subset has gained a functional specialization that able them to repress inflammation via several epithelial interferon-γ (IFN-γ)-induced proteins. Among others, we identified that epithelial IDO1 and interleukin-18-binding protein (IL-18bp) were strongly modulated by CD103(+)CD11b(-) DCs. Through its preferential property to express IL-12 and IL-15, this particular DC subset can induce lymphocytes in colonic lamina propria and in epithelia to secrete IFN-γ that then can trigger a reversible early anti-inflammatory response in intestinal epithelial cells.

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Figures

Figure 1
Figure 1
Transcriptome of colon dendritic cell (DC) subsets. (a) Sorting strategy for colon DC isolation. Large intestines obtained from 8 mice, either control (steady state (SS)) or dextran sodium sulfate (DSS) treated (day 4 DSS), were pooled and lamina propria (LP) cells were isolated. Procedure was repeated three times independently. Following CD11chighMHCII+ DC subsets were sorted and analyzed in a gene expression microarray: (1) CD103+CD11b−, (2) CD103+CD11b+, and (3) CD103−CD11b+. (b) Transcript heat map of the ∼640 genes that are at least twofold differentially expressed in one comparison (red: upregulated; green: downregulated). Clustering was performed using Pearson's correlation and complete linkage. Heat map was z-score normalized by row. (c) XY plot of the first two components of a principal component analysis (PCA) of all six groups (SS 1–3 and DSS 1–3). (d) Heat map showing differential expression of selected genes involved in DC development and function; heat map was generated as described in b.
Figure 2
Figure 2
Distinct intestinal myeloid cells are ablated in Clec9A- and Clec4a4-–diphtheria toxin receptor (DTR) mice. Colon cells were obtained from DT-treated CX3CR1GFP wild-type (WT) controls, CX3CR1GFP/Clec9A-DTR, and CX3CR1GFP/Clec4a4-DTR mice. (a) Colon lamina propria (LP) cells were analyzed for CD103 and CD11b expression by gating on CD11chighMHC II+ cells (gate 1) and for CX3CR1 and CD64 expression by gating CD11cintMHC II+ cells (gate 2). (b) Mesenteric lymph nodes (MLNs) were obtained from the same mice and analyzed for CD103 and CD11b expression by gating on CD11cintMHCII++ migratory dendritic cells (DCs; gate 3) and classical lymphoid CD11chighMHC II+ DCs (gate 4). Representative dot plots of colons and MLNs isolated from three different mice are shown. Indicated numbers show the percentage of each gated cell subset.
Figure 3
Figure 3
Differential localization of distinct colon lamina propria (LP) dendritic cell (DC) subsets and CX3CR1+ macrophages at steady state and during dextran sodium sulfate (DSS)-mediated colitis. (a) Representative confocal images of Clec9A+ and Clec4a4+ DC subsets in the colonic innate lymphoid follicles (ILFs). Sections were stained with phycoerythrin (PE)-labeled anti-Clec9A antibody (red), allophycocyanin (APC)-labeled anti-Clec4a4 (blue), and fluorescein isothiocyanate (FITC)-labeled anti-CD11c (green). Original magnification × 20. Bars=50 μm. (b) Representative confocal images of Clec9A+ and Clec4a4+ DC subsets in the colonic LP under steady-state conditions and 4 days (4 d) after DSS treatment. Cryosections were stained as above in (a). Original magnification × 20. Bars=50 μm. (c) Representative confocal images of CX3CR1GFP+ cells (green) in the colonic LP under steady-state (SS) conditions and 4 days after DSS treatment. Sections were stained with 4',6-diamidino-2-phenylindole (DAPI; blue, nuclei). Original magnification × 20. Bars=45 μm. (d) Flow cytometry of colon LP cells of CX3CR1GFP mice analyzed at steady state and after 4 days of DSS. CD11c/major histocompatibility complex (MHC class II)-positive cells were analyzed for CD64/CX3CR1GFP. Indicated numbers show the percentage of each gated cell subset. (e) Percentage of differentially expressing CX3CR1 populations in the colon during steady state and upon 4 days of DSS treatment. N=5 mice, Student's t-test ***P>0.001. (f) DC ablation profiles during DSS treatment. LP cells were collected at day 4 from the distal part of colon of DT-treated wild-type (WT), Clec9A–diphtheria toxin receptor (DTR), and Clec4a4-DTR (n=3 each group). Representative fluorescence-activated cell sorting (FACS) dot plots showing the staining profile of CD11c and MHC class II as well as CD103 and CD11b.
Figure 4
Figure 4
Ablation of CD103+CD11b− cells enhances susceptibility to dextran sodium sulfate (DSS)-induced colitis. Wild-type (WT), Clec9A–diphtheria toxin receptor (DTR), and Clec4a4-DTR mice were injected with 20 ng g−1 DT following the schedule described in Methods. Then, 2% DSS was supplied at day 0 ad libidum in the drinking water for 7 consecutive days followed by drinking water at day 8. (a) Body weight was monitored daily over a period of 15 days. Black circles: DT-treated WT control; black triangles: DT-treated Clec9A-DTR; black squares: DT-treated Clec4a4-DTR. Each group: n=8. Values represent the mean±s.d. Three independent experiments were performed with the same numbers of animals. (b) Measurement of colon length at day 8 (cm) of control WT mice (white bar) and DSS-treated DT-injected WT (black bar), Clec4a4-DTR (striped bar), and Clec9A-DTR (gray bar) mice. Each group: n=8. Values represent the mean±s.d. (c) Fecal samples of DT-injected WT controls (black circles), Clec4a4-DTR (black squares), and Clec9A-DTR (black triangles) mice were collected at day 8 upon DSS treatment and scored for blood content. Each group: n>9 mice. Student's t-test significance: ***P>0.001, NS, not significant. (d, e) Increased influx of inflammatory myeloid cells in the absence of Clec9A+CD103+CD11b− dendritic cell (DC) subset at early stages of inflammation (day 4 (d4)). Colon of DT-injected WT, Clec4a4-DTR, and Clec9A-DTR mice were analyzed at day 0 and 4 days upon start of DSS treatment. Representative dot plots of three mice indicating CD11bLy6G+ and CD11bLy6C+ cells (d) and bar chart of 3–4 mice±s.d. Student's t-test significance: **P>0.005. (f) Intestinal permeability as determined by quantifying the amount of fluorescein isothiocyanate (FITC)–dextran levels (μg ml−1) in the serum after its oral gavage. DT-injected WT, Clec9A-DTR, or Clec4a4-DTR mice were tested at days 4 (filled symbols) and 10 (empty symbols) from the beginning of DSS treatment. For each group, 7–9 mice were analyzed. Student t-test significance: *P>0.01; **P>0.005. (g) Clec9A DTR mice do not survive a 5%, high-dose DSS treatment. WT, Clec9A-DTR, and Clec4a4-DTR mice were treated with 5% DSS for 7 consecutive days followed by drinking water at day 8. Body weight was monitored daily over a period of 13 days. Mice were killed when body weight was <75% of their original weight. Black circles: DT-treated WT control; black triangles: DT-treated Clec9A-DTR; white circles: DT-treated Clec4a4-DTR. Each group: n=4. Values represent the mean±s.d.
Figure 5
Figure 5
Depletion of CX3CR1high macrophages leads to severe intestinal inflammation. (a) Flow cytometry analysis of different macrophage and dendritic cell (DC) subsets. CX3CR1-GFP-CD169-DTR and CX3CR1-GFP-WT mice were injected with DT (20 ng per g body weight) and analyzed the following day for the ablation profile of different CD11chighMHC IIhigh DCs and CD11cintMHC II high macrophages, respectively. For DC profiling, anti-CD103 and anti-CD11b were used, whereas for macrophage profiling, cells were stained with anti-CD64 and monitored for CX3CR1 GFP expression. (b–e) Ablation of CX3CR1high macrophages enhances susceptibility to dextran sodium sulfate (DSS)-induced colitis. Wild-type (WT) and CD169–diphtheria toxin receptor (DTR) mice were injected with 20 ng g−1 DT following the schedule described in Methods. (b) Body weight was monitored daily over a period of 15 days. Open circles: DT-treated WT control; filled circles: DT-treated CD169-DTR. Each group: n=5. Values represent the mean±s.d. Two independent experiments were performed with the same numbers of animals. (c) Fecal samples of DT-injected WT controls (open circles) and CD169-DTR (filled circles) mice were collected at day 8 upon DSS treatment and scored for blood content. Each group: n>5 mice. Student's t-test significance: ***P>0.001. (d) Measurement of colon length at day 8 (cm) of control WT mice (gray bar) and DSS-treated DT-injected WT (white bar) or CD169 DTR (black bar) mice. Each group: n=5. Values represent the mean±s.d. (e) Intestinal permeability as determined by quantifying the amount of fluorescein isothiocyanate (FITC)–dextran levels (μg ml−1) in the serum after its oral gavage. DT-injected WT (open circles) and CD169-DTR mice (filled circles) were tested at days 4 and 10 from the beginning of DSS treatment. For each group, 5–9 mice were analyzed.
Figure 6
Figure 6
Epithelial expressed interferon-γ (IFN-γ)-inducible genes are strongly affected by ablation of CD103+CD11b− dendritic cells (DCs). (a) Heat map showing differential expression of selected genes regulated by IFN-γ of colon intestinal epithelial cells (IECs) obtained from wild-type (WT) untreated mice and dextran sodium sulfate (DSS)-treated day 4 WT and Clec9A–diphtheria toxin receptor (DTR) mice (n=3). (b) Gene validation comparing bulk IECs and CD45+ lymphocyte-depleted IECs obtained from DSS-treated animals. IECs were isolated from the colon as described in Methods and loaded on a Percoll gradient to separate the lymphocytes from the epithelial fraction. RNA and subsequently complementary DNA (cDNA) was prepared and validated for Cd3, Ifn-γ, and a series of Ifn-γ-induced genes, including Ido1 and IL-18bp. One representative sample is shown. (c) Quantitative real-time PCR (qPCR) analysis of Ido1 expression in different intestinal DC subsets and IECs at steady state (SS) and 4 days after DSS treatment. N=3±s.e.m. (d) Indoleamine 2,3 dioxygenase (IDO1) is the major tryptophan-degrading enzyme in the colonocytes. IECs obtained from distal part of the colon of DSS-treated WT mice (day 4) were analyzed for Ido1, Ido2, and Tdo expression by semiquantitative real-time PCR (RT-PCR) analysis. Hprt was used as an endogenous mRNA control. Results are representative of 3–4 pooled colons. (e) Ido1 and IL-18bp expression profile during DSS treatment in IECs. WT mice were treated with 1% DSS over 6 days. Colonocytes were isolated from the distal part of three mice every day and monitored by RT-PCR for Ido1 and IL-18bp mRNA expression. (f) qPCR analysis of IL-18bp expression in IECs at steady state and 4 days after DSS treatment. N=3±s.e.m. (g) RT-PCR analysis of Ido1 and IL-18bp in IECs obtained from pooled colons of DT-injected untreated or DSS-treated (day 4) WT, Clec9A-DTR, and Clec4a4-DTR mice. PCR results are representative of three independent IEC isolations. (h) IDO1 protein expression in IECs pooled from three DSS-treated WT or Clec9A DTR mice (day 4). Representative immunoblots for epithelial IDO1 (45 kDa) and β-tubulin control (50 kDa) are shown. (i) Absence of CX3CR1high macrophages does not affect expression of IDO1 and interleukin-18-binding protein (IL-18bp) in IECs during colitis. RT-PCR analysis of Ido1and IL-18bp in IECs obtained from DT-injected untreated or DSS-treated (day 4) WT and CD169-DTR mice. PCR results are representative of three independent IEC isolations.
Figure 7.
Figure 7.
IFN-γ −/− mice show enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. Wild-type (WT) and interferon-γ (IFN-γ)−/− mice were treated as described in Methods. (a) Body weight was monitored daily over a period of 11 days. IFN-γ−/− mice were killed at day 8 because of severe body weight loss (>30%). White circles: CB57/BL6 control; black circles: IFN-γ−/− mice. Each group: n=5. Values represent the mean±s.d. Two independent experiments were performed with the same numbers of animals. (b) Fecal samples of CB57/BL6 control and IFN-γ −/− mice were collected at day 7 upon DSS treatment and scored for blood content. Each group: n>7 mice. Student's t-test significance: ****P>0.0001.
Figure 8
Figure 8
IDO1 and IL-18bp expression is modulated by interferon-γ (IFN-γ). (a) Colonocytes express IFN-γ receptor (IFN-γR). The ex vivo isolated colonocytes and CMT-93 colon epithelial cell line were analyzed by semiquantitative real-time PCR (RT-PCR) analysis for IFN-γ receptor expression. Hprt was used as an endogenous mRNA control. (b) IDO1 and IL-18bp expression is induced by IFN-γ. CMT-93 cells were stimulated overnight with 100 U ml−1 IFN-γ and analyzed for Ido1 and IL-18bp expression by semiquantitative RT-PCR analysis. (c) IFN-γ−/− mice do not upregulate Ido1 and IL-18bp epithelial expression upon dextran sodium sulfate (DSS) treatment. Intestinal epithelial cells (IECs) were collected from untreated or DSS-treated wild-type (WT) and IFN-γ−/− mice and evaluated by semiquantitative RT-PCR. One representative sample from each experimental group of three mice is shown. SS, steady state. (d) Clec9A–diphtheria toxin receptor (DTR) mice have a decreased proportion of IFN-γ-expressing lamina propria (LP) T cells and intraepithelial lymphocytes (IELs). Representative flow cytometry plots of LP and IELs harvested from wild-type (WT) and Clec9A-DTR mice 4 days after DSS treatment and stained for CD4, CD8, and γ/δ T cell receptor (TCR), respectively (representative fluorescence-activated cell sorting (FACS) dot plot, right panel) and stained for intracellular IFN-γ. Quantification of LP CD4+ T cells, LP CD8+ T cells, LP CD4−CD8− T-cell fraction, γ/δ+, and CD8+ IELs expressing IFN-γ. N=6–8 mice pooled from 2 independent experiments±s.e.m. Student's t-test significance: *P>0.01, ***P>0.001, NS, not significant. (e) Quantitative real-time PCR (qPCR) analysis of IL-15, IL-12p40, IL-12p35, and IL-13p19 expression in distinct colon dendritic cell (DC) subsets obtained from control WT mice: CD103+CD11b−, CD103+CD11b+, and CD103−CD11b+. Data are representative of 3 independent experiments with 10 mice pooled in each group.
Figure 9
Figure 9
Immunostimulatory oligonucleotide (ISS-ODN) treatment limits the colitis severity in Clec9A–diphtheria toxin receptor (DTR) mice. DT-injected wild-type (WT) and Clec9A-DTR mice were injected intraperitoneally (i.p.) 10 μg of ISS-ODN at the start of the dextran sodium sulfate (DSS) treatment (2%) and 4 days later. (a) Interferon-γ (IFN-γ) response was measured in the serum collected at day 4. (b) Epithelial Ido1 and IL-18bp expression profile at steady-state or under DSS treatment. Representative samples of three WT and Clec9A-DTR mice are shown. (c) The body weight was monitored daily over a period of 10 days. Black circles: DT-treated WT control; white circles: DT-treated WT control+ISS-ODN; black squares: DT-treated Clec9A-DTR; white squares: DT-treated Clec9A-DTR+ISS-ODN; Each group: n=6 mice from two independent experiments. Values represent the mean±s.d. ND, not detectable.
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References

    1. Brown, E.M., Sadarangani, M. & Finlay, B.B. The role of the immune system in governing host-microbe interactions in the intestine. Nat. Immunol. 14, 660–667 (2013). - PubMed
    1. Macdonald, T.T. & Monteleone, G. Immunity, inflammation, and allergy in the gut. Science 307, 1920–1925 (2005). - PubMed
    1. Ponda, P.P. & Mayer, L. Mucosal epithelium in health and disease. Curr. Mol. Med. 5, 549–556 (2005). - PubMed
    1. Schmitz, H. et al. Altered tight junction structure contributes to the impaired epithelial barrier function in ulcerative colitis. Gastroenterology 116, 301–309 (1999). - PubMed
    1. Peeters, M. et al. Clustering of increased small intestinal permeability in families with Crohn's disease. Gastroenterology 113, 802–807 (1997). - PubMed

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