Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces


Epithelial tissues continually undergo apoptosis. Commensal organisms that inhabit the epithelium influence tissue homeostasis, in which regulatory T cells (Treg cells) have a central role. However, the physiological importance of epithelial cell apoptosis and how the number of Treg cells is regulated are both incompletely understood. Here we found that apoptotic epithelial cells negatively regulated the commensal-stimulated proliferation of Treg cells. Gut commensals stimulated CX3CR1+CD103CD11b+ dendritic cells (DCs) to produce interferon-β (IFN-β), which augmented the proliferation of Treg cells in the intestine. Conversely, phosphatidylserine exposed on apoptotic epithelial cells suppressed IFN-β production by the DCs via inhibitory signaling mediated by the cell-surface glycoprotein CD300a and thus suppressed Treg cell proliferation. Our findings reveal a regulatory role for apoptotic epithelial cells in maintaining the number of Treg cell and tissue homeostasis.

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Figure 1: The abundance of Foxp3+ Treg cells is greater in CD300a-deficient mice raised under SPF conditions but not in CD300a-deficient GF mice.
Figure 2: Blockade of the interaction between CD300a and PS increases the abundance of Treg cells in the colon, skin and lungs.
Figure 3: Immunohistochemical analysis of CD300a-expressing cells and apoptotic epithelial cells.
Figure 4: CD300a on CD11b+ DCs suppress the IFN-β-dependent proliferation of Treg cells.
Figure 5: Cd300a−/− mice show attenuated DSS-induced colitis.
Figure 6: The increase in the size of the Treg cell population in the lamina propria of Cd300a−/− mice is dependent on TRIF and IFN-β.
Figure 7: IFN-β is involved in the CD300a-mediated suppression of the Treg cell population.

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We thank B. Malissen (UM2 Aix-Marseille Université) for Foxp3eGFP mice; E. Nakayama (Okayama University) for anti-CD25; M. Tanaka (Research Center for Allergy and Immunology, Yokohama, Japan) for MFG-E8(D89E) and MFG-E8(EPT); K. Honda and G. Nunez for discussions; S. Tochihara and Y. Nomura for secretarial assistance; and F. Abe and R. Hirochika for technical assistance. Supported by Japan Society for the Promotion of Science (KAKENHI), Core Research for Evolutional Science and Technology, Japan Agency for Medical Research and Development–Core Research for Evolutional Science and Technology and Uehara Memorial Foundation (A.S.).

Author information

C.N.-O. conducted the experiments, analyzed the data, and wrote the paper; K.G.S.U. performed immunohistochemical studies and analyzed dermatitis; Yo.N. performed in vivo studies of DSS-induced colitis; Yu.N. analyzed signal transduction via TLR4 and CD300a; N.T., H.M. and S.I. performed the experiments of S. typhimurium infection, allergic airway inflammation and DSS-induced colitis, respectively; S.T.-H., S.H. and K.S. analyzed the data; and A.S. supervised the overall project and wrote the paper.

Correspondence to Akira Shibuya.

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Integrated supplementary information

Supplementary Figure 1 Treg cell populations are comparable in wild-type and Cd300a−/− mice in organs that lack a resident microbiota.

Flow cytometry of Foxp3 expression in CD4+ T cells of the small intestine (a) and spleen and mesenteric and axillar lymph node (b) from Cd300 a−/− or wild-type (WT) mice raised under specific-pathogen-free (SPF) conditions. Error bars indicate SEM. *, P < 0.05. NS, not significant.

Supplementary Figure 2 Immunohistochemical staining of intestine, skin and lungs.

Flag-tagged D89E MFG-E8 or EPT MFG-E8 was infused rectally (colon) into, applied topically (skin) on, and injected intranasally (lung) into mice; fixed sections were stained by fluorescein isothiocyanate (FITC)-conjugated anti-FLAG mAb and DAPI, and then analyzed by fluorescence microscopy. White dashed lines indicate the surface of epithelium. White bars indicate scale (20μm). Data are representative of more than three experiments.

Supplementary Figure 3 CD300a+ cells interact with epithelial apoptotic cells.

(a) RAW267.4 transfectants stably expressing CD300a or MAIR-II directly fused with DS-Red (red) at the C-terminus were cocultured with apoptotic thymocytes that had been stained with PSVue 480 (green), and analysed by immunofluorescence microscopy. (b-e) Mice were infused rectally or applied to the dorsum or administered intranasally with Flag-tagged D89E MFG-E8 (b, e) or PSVue 480 (d). Tissue sections from the colon, skin and lung of these mice (b-d) or CD11c-GFPmice (c) were stained with Alexa 647-conjugated anti-E-cadherin mAb (b), Alexa 546-conjugated anti-CD300a mAb (c-e), FITC-conjugated anti-CD207 mAb (c), Alexa 488-conjugated anti-GFP Ab (c), and/or FITC-conjugated anti-Flag mAb (b, e), followed by the staining with DAPI (b-e) and then analyzed by fluorescence microscopy. White arrows indicate CD300a-expressing DC or LC (c) and possible interactions between CD300a-expressing cells and epithelial apoptotic cells (d, e). White bars indicate scale (10 μm). Data are representative of three (a) and five (b-e) experiments. (f) Scheme of in vivo imaging analysis using probe-based confocal laser endomicroscopy of colon.

Supplementary Figure 4 Characterization of CD300a+ cells.

(a, b) Cells of the colonic lamina propria from WT (a and b) and CD300a−/− (a) mice were stained with Alexa 700-conjugated anti-CD45.2, propidium iodide (PI), Horizon V500-conjugated MHC class II, APC-Cy7-conjugated anti-CD11b, PE-Cy7-conjugated anti-CD11c, FITC-conjugated anti-CD103, PE-conjugated F4/80, and either APC-conjugated anti-CD300a (a), FITC and PE-conjugated mAbs indicated (b), or control Ab (a, b) and analyzed by means of flow cytometry. Data are representative of three mice. (c) Cells obtained from the colonic lamina propria, peritoneal cavity (PEC) or spleen of WT, CD300a−/−, Cd300afl/fl and Cd300afl/flItgax-Cre mice were stained as described above or with PE-conjugated anti-c-Kit, FITC-conjugated anti-FcεRI and PE-conjugated anti-F4/80. CD300a expressions were analyzed by means of flow cytometry. (d) Microarray analysis performed on the mRNA of CD11b+ DCs sorted from the colonic lamina propria of WT and Cd300a−/− of germ-free (GF) and SPF mice (pooled from 4 mice each). The heat map demonstrates changes in the expression levels of the indicated genes.

Supplementary Figure 5 Cd300a−/− mice show attenuated DSS-induced colitis and allergic inflammation in the skin and lungs.

Rag1−/− (n = 10) and Rag1−/−Cd300a−/−mice (n = 8) were treated with 2.5% DSS and monitored for loss of body weight. Error bars, SEM. Data are representative of two independent experiments. (b-e) WT and Cd300a−/− mice were treated with 2.5% DSS for 7 days. Cells were isolated from the lamina propria before and during DSS-treatment and stained with anti-CD45.2, anti-CD4, and either PE-conjugated anti-Foxp3 (b), FITC-conjugated anti-IFN-γ, PE-conjugated anti-IL-17 or APC-conjugated anti-IL-4 mAb (c). The proportions of Foxp3+ cells in CD4+ T cells (b) and CD4+ T cells producing IFN-γ, IL-17, IL-4 or IL-10 were calculated (c). (d) Cells were isolated from the lamina propria beforeand 7 days after the start of DSS treatment and cultured overnight. Cytokine levels were determined by ELISA in the culture supernatants. (e) CD11b+ DCs were isolated from the colonia lamina propria cells by flow cytometry 7 days after DSS treatment and examined for the expressions of cytokine mRNAs by quantitative RT-PCR. (f) Mice were injected intravenously with 500 μg of control or anti-CD25 mAb on day 0 and treated orally with 2.5% DSS for 7 days. On day 7, Foxp3 expression in the CD4+ T cells of the colonic lamina propria was analyzed by means of flow cytometry. (g-j) WT and Cd300a−/− mice were treated topically with LPS and ovalbumin (OVA) on the dorsal skin to induce allergic inflammation (g). On day 14, skin samples were dissected, analyzed histologically after hematoxylin and eosin staining (b and d), and evaluated for epidermal thickness (h). Serum IgE levels were measured with an ELISA (i). (j) WT and Cd300a−/− mice were injected intraperitoneally with anti-CD25 mAb or control mAb to deplete Treg cells on days -4, 3, and 10. (k-n) WT and Cd300a−/− mice were intranasally treated with LPS and OVA on the indicated days (k), and lungs were analyzed histologically after staining with hematoxylin and eosin or periodic acid–Schiff on day 18 (l). The number of Siglec F+ eosinophils in the lung tissues was determined by means of flow cytometry (m), and serum IgE levels were analyzed with an ELISA (n). Error bars, SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant.

Supplementary Figure 6 The increase in the size of the Treg cell population in the skin and lungs of Cd300a−/− mice is dependent on TRIF.

Cells isolated from the skin and lung of Cd300a−/−, Ticam1−/− and Ticam1−/−Cd300a−/− mice were analyzed for Foxp3+ expression in CD4+ T cells by means of flow cytometry, as described in the legend to Figure 1. Error bars, SEM. NS, not significant. Data are representative of two independent experiments.

Supplementary Figure 7 Involvement of CD300a in the suppression of IFN-b production by BMDCs.

(a) Bone marrow-derived cultured DCs (BMDCs) induced by GM-CSF and IL4 for 7 d were stained with PE-conjugated anti-CD11c, PECy7-conjugated anti-MHC classII, FITC-conjugated anti-CD11b, and Alexa647-conjugated anti-CD300a. CD11c+MHC class II+ cells were analyzed for expression of CD11b and CD300a by means of flow cytometry. (b) The percentage of apoptotic cells in cultures of BMDCs was analyzed by means of flow cytometry. Phosphatidylserine (PS) was stained by using a FITC-conjugated anti-PS antibody. Data are representative of 3 mice. (c) BMDCs induced from bone marrow cells of WT, Tlr3−/−, and Tlr4−/− mice were stimulated with fecal contents and treated with a neutralizing anti-CD300a mAb. Subsequent mRNA expression of IFN-b was measured by means of quantitative PCR analysis. Error bars, SEM. *, P < 0.05; **, P < 0.01. NS, not significant. ND, not detected. Data are representative of two independent experiments.

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Nakahashi-Oda, C., Udayanga, K., Nakamura, Y. et al. Apoptotic epithelial cells control the abundance of Treg cells at barrier surfaces. Nat Immunol 17, 441–450 (2016). https://doi.org/10.1038/ni.3345

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