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. 2016 Mar 23:6:23505.
doi: 10.1038/srep23505.

Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis

Tomokazu Ohta  1   2   3 , Masanaka Sugiyama  1   3   4   5 , Hiroaki Hemmi  1   2   4   5 , Chihiro Yamazaki  5   6 , Soichiro Okura  1   2 , Izumi Sasaki  1   2   5 , Yuri Fukuda  1   2   5 , Takashi Orimo  1   2   3 , Ken J Ishii  7   8 , Katsuaki Hoshino  1   4   5   9 , Florent Ginhoux  10 , Tsuneyasu Kaisho  1   2   4   5
Affiliations

Crucial roles of XCR1-expressing dendritic cells and the XCR1-XCL1 chemokine axis in intestinal immune homeostasis

Tomokazu Ohta et al. Sci Rep. .

Abstract

Intestinal immune homeostasis requires dynamic crosstalk between innate and adaptive immune cells. Dendritic cells (DCs) exist as multiple phenotypically and functionally distinct sub-populations within tissues, where they initiate immune responses and promote homeostasis. In the gut, there exists a minor DC subset defined as CD103(+)CD11b(-) that also expresses the chemokine receptor XCR1. In other tissues, XCR1(+) DCs cross-present antigen and contribute to immunity against viruses and cancer, however the roles of XCR1(+) DCs and XCR1 in the intestine are unknown. We showed that mice lacking XCR1(+) DCs are specifically deficient in intraepithelial and lamina propria (LP) T cell populations, with remaining T cells exhibiting an atypical phenotype and being prone to death, and are also more susceptible to chemically-induced colitis. Mice deficient in either XCR1 or its ligand, XCL1, similarly possess diminished intestinal T cell populations, and an accumulation of XCR1(+) DCs in the gut. Combined with transcriptome and surface marker expression analysis, these observations lead us to hypothesise that T cell-derived XCL1 facilitates intestinal XCR1(+) DC activation and migration, and that XCR1(+) DCs in turn provide support for T cell survival and function. Thus XCR1(+) DCs and the XCR1/XCL1 chemokine axis have previously-unappreciated roles in intestinal immune homeostasis.

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Figures

Figure 1
Figure 1. Intestinal T cell populations are decreased in mice lacking XCR1+ DCs.
(A–D) Percentages (A,C) and total numbers (B,D) of T lineage cells of LP (A,B) and IELs (C,D) from control (Xcr1+/cre) and XCR1-DTA mice. Dead cells and doublets were eliminated by FSC/SSC gating and LIVE/DEAD staining, with live cells subjected to further gating as indicated. (E) Immunofluorescent images of intestinal sections from control and XCR1-DTA mice labelled with anti-TCRβ and anti-Ep-CAM Abs. Scale bars, 500 μm. Means ± s.e.m. of five mice are indicated (B,D). (F) AnnexinV and 7-AAD staining of LP and intraepithelial TCRαβ+ cells, in control and XCR1-DTA mice. Percentage of double-positive cells is indicated. Results are representative of five (A-D) or two (E,F) independent experiments. (*P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test).
Figure 2
Figure 2. Intestinal T cells in mice lacking XCR1+ DCs exhibit an aberrant phenotype.
(A) Intensity of expression of CD62L, CD103, CCR9 and α4β7 integrin on CD4+ and CD8+ T cells in spleen, MLNs and LP from control (Xcr1+/cre, shaded histograms) and XCR1-DTA (open histograms with red lines) mice. Results are representative of four independent experiments. (B,C) Nanostring gene expression analysis of purified LP CD4+ T cells from control and XCR1-DTA mice. Scatter plot (B) shows means of normalized log intensities of individual probes with lines indicating the 2-fold difference threshold. Expression profiles (C) of indicated genes are shown as means ± s.e.m. of three independent experiments. Cells from three or four mice were pooled for each experiment.
Figure 3
Figure 3. XCR1-DTA mice are more susceptible to DSS-induced colitis.
(A,B) Body weight changes (A) and disease activity index (B) after DSS exposure of control (open circles) and XCR1-DTA (filled circles) mice. Mice were given 1.5% DSS in drinking water for 7 days and clinically monitored. Means ± s.e.m. of control (n=8) and XCR1-DTA (n=6) mice are indicated. (C) Colon length after 10 days of DSS treatment. Means are shown as bars. Results are representative of three (A-C) independent experiments. (*P < 0.05; **P < 0.01, Student’s t test).
Figure 4
Figure 4. The XCR1-XCL1 axis is involved in maintenance of intestinal T cell populations.
(A–D) Percentages (A,C) and total numbers (B,D) of T lineage cells of LP and IELs from control (XCL1+/−) and XCL1-deficient (XCL1−/−) mice are shown. (E–H) Percentages (E,G) and total numbers (F,H) of T lineage cells of LP and IELs from control (XCR1+/venus) and XCR1-deficient (XCL1venus/venus) mice are shown. Dead cells and doublets were eliminated by FSC/SSC gating and LIVE/DEAD staining, with live cells subjected to further gating as indicated. Means ± s.e.m. of five mice are shown (B,D,F,H). Results are representative of four independent experiments. (*P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test).
Figure 5
Figure 5. XCR1+ DC distribution is affected in the absence of XCR1 or XCL1.
(A) Nanostring gene expression analysis of sorted LP DC subsets pooled from eight Xcr1+/venus or eight Xcr1venus/venus mice. (B,C) Percentages (B) and total numbers (C) of MLN and LP DCs from control (XCL1+/−) and XCL1-deficient (XCL1−/−) mice are shown. (D,E) Percentages (D) and total numbers (E) of MLN and LP DCs of control (XCR1+/venus) and XCR1-deficient (XCL1venus/venus) mice are shown. Dead cells and doublets were eliminated by FSC/SSC gating and LIVE/DEAD staining, with live cells subjected to further gating as indicated. Results are representative of four (C,E) independent experiments. Means ± s.e.m. of five mice are indicated (C,E). (*P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test).
Figure 6
Figure 6. Intestinal CD4 T cells are closely associated with XCR1+ DCs in vivo.
(A) Xcl1 expression in sorted T cells from spleen and LP, IELs, and splenic B (CD3ε−B220+) and NK (CD3ε−CD49b+) cells pooled from eight wildtype mice, determined by quantitative real-time PCR. (B) Immunofluorescence imaging of intestinal sections from Xcr1+/venus mice labelled with anti-CD4 Ab and venus. Scale bars, 60 μm. (C) Percentages of MLN and LP DC subsets in Rag2+/− and Rag2−/− mice. Dead cells and doublets were eliminated by FSC/SSC gating and LIVE/DEAD staining, with live cells subjected to further gating as indicated. Results are representative of two independent experiments.
Figure 7
Figure 7. Hypothetical model of the possible crosstalk between XCR1+ DCs and intestinal T cells.
Activated intestinal T cells produce XCL1, which attracts nearby XCR1+ DC; the ensuing DC-T cell crosstalk supports T cell survival, promotes upregulation of CD103 expression with downregulation of CD62L expression, and leads to maintenance of intraepithelial and LP T cells; continuing XCL1 expression by the T cells in turn enables DC maturation, including upregulated CCR7 expression to enable migration from the LP to the MLN.
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