Research ArticleMUCOSAL IMMUNOLOGY

GPR55 regulates intraepithelial lymphocyte migration dynamics and susceptibility to intestinal damage

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Science Immunology  08 Dec 2017:
Vol. 2, Issue 18, eaao1135
DOI: 10.1126/sciimmunol.aao1135
  • Fig. 1 GPR55 restrains IEL accumulation in the small intestine.

    (A) Gpr55 transcript abundance in the indicated cell subsets relative to Hprt. Each point indicates cells sorted from an individual mouse (except for intestinal DCs and macrophages that were sorted from pooled samples of 10 mice), and lines indicate means ± SEM. EC, epithelial cell. (B) Flow cytometric analysis of CD8α and TCRγδ expression by small intestinal IELs from the indicated mice. Number shows fraction of cells in the indicated gate. (C) Frequency of IELs and LPLs (left and center) and number of IELs (right) in the indicated mice. Each population was pregated on CD45+CD3ε+ cells. Each point represents data from an individual mouse, and lines represent means ± SEM. Left: GPR55 Het, n = 20; GPR55 KO, n = 15. Center: GPR55 Het, n = 13; GPR55 KO, n = 9. Right: GPR55 Het, n = 16; GPR55 KO, n = 12. (D) Expression of Vγ7, Thy1.2, β7 integrin, and CD103 (αE integrin) on CD8α+CD8β GL3+ IELs from GPR55 Het and KO mice. (E) Percentage of GL3+ cells among the CD4 and CD8 double-negative thymocyte population (left) and the fraction of these cells that express Vγ7 (right). GPR55 Het, n = 7; GPR55 KO, n = 4. (F) Frequency of IELs of the indicated types that had incorporated BrdU after a 1-week labeling period. GPR55 Het, n = 6; GPR55 KO, n = 5. (G) Number (left) and frequency (right) of IELs of the indicated types in irradiated WT mice reconstituted with GPR55 Het or KO BM. GPR55 Het, n = 4; GPR55 KO, n = 4. Data are representative of two independent experiments. (H) Ratio of GPR55 or empty vector (EV)–transduced cells compared with untransduced cells of the indicated types in BM chimeras reconstituted with GPR55 KO BM transduced with the indicated retroviral constructs. Thy1.1 marks transduced cells. **P < 0.01, *P < 0.05, n.s. (not significant). P > 0.05 by Student’s t test.

  • Fig. 2 GPR55 mediates lymphocyte migration inhibition via Gα13.

    (A and B) Migration of GPR55 and control transduced WEHI231 cells to CXCL12 and the indicated amounts of LPI (mixture of 16:0, 18:0, and 18:2) (A) or 1 μM of the indicated types of LPI (B) in Transwell assays. (C) Migration of empty vector– or GPR55-transduced B cells that either express (Mb1Cre Gna13f/f) or lack (Mb1Cre+ Gna13f/f) Gα13 to CXCL12 and the indicated amounts of 20:4 LPI. (D) Migration of γδT IELs to vehicle or CXCL10 and the indicated amounts of LPI (16:0, 18:0, and 18:2) or the GPR55 antagonist CID16020046. (E) Migration of control (Het) or GPR55 KO γδT IELs to vehicle or CCL25 in the absence or presence of 20:4 LPI. (F) Flow cytometric analysis of pERM levels in WEHI231 cells (top) or CD8αα γδT IELs from control (Het) or GPR55 KO mice (bottom) after treatment for 5 min with vehicle (DMSO) or 20:4 LPI (1 μM). Each experiment was performed in duplicate and repeated at least twice. Numbers inside plots indicate mean ± SEM. GPR55 Het, n = 3; GPR55 KO, n = 3.

  • Fig. 3 Detection of GPR55 ligand in the small intestine.

    (A) Abundance of the indicated forms of LPI in spleen (n = 5) and small intestine (n = 5) tissue determined by LC-MS/MS. (B and C) Bioassay measurement of relative GPR55 ligand activity in small intestine, colon, and spleen extract (B) and in small intestinal epithelial cell (SI-EC) culture supernatants (C). WEHI231 cells transduced with GPR55 were tested for their response to the tissue extracts in a Transwell migration assay. Extract at the indicated dilution was mixed with CXCL12 (100 ng/ml). Each experiment was performed in duplicate and repeated three times. (D) Histograms showing Thy1.1 reporter expression on intestinal and spleen DCs in mice that had been reconstituted with empty vector– or GPR55-transduced BM. (E to G) Summary data from mice of the type in (D), shown as the ratio of Thy1.1+/Thy1.1 cells of the indicated types within the intestine (E and F) and spleen (G). n = 4 in each group. **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test.

  • Fig. 4 Altered distribution of γδT IELs in GPR55-deficient mice.

    (A) Immunofluorescence detection of GL3 (γδTCR), CD8α, laminin, and DAPI with examples of CD8α+GL3+ cells in the PMS and in close association with the epithelium highlighted. Scale bar, 20 μm. (B) Distribution of CD8α+ γδT cells in the small intestine. In the left panels, yellow arrows are used to highlight cells in close association with the epithelium, and orange arrows cells associated with the PMS. Images are representative of five mice of each type. Scale bar, 100 μm. Enlarged images from rectangles in left panels are shown on the right. Scale bar, 25 μm. (C) Percentage of γδ IELs closely associated with epithelium in GPR55 Het and KO mice. (D) Diagram explaining the mixed BM chimera approach used to track control and KO γδT IELs in the same mice. 2P, two-photon; FCM, flow cytometry; IF, immunofluorescence. (E) Immunofluorescence analysis of control (Het, purple arrowheads) and GPR55 KO (red arrowheads) γδT IELs in the small intestine of a mixed BM chimeric mouse (n = 10). Circles show regions where IELs are localized in close association with epithelial cells. Scale bar, 50 μm. (F) Fraction of GPR55 Het and KO γδT IEL, identified as in (E), located in close association with the epithelium. (G) Ratio of γδT cells of KO versus Het origin in the small intestinal IELs and spleen compartments determined by flow cytometry (n = 5). (H) Ratio of γδT cells of KO versus Het origin in the small intestine determined by microscopy as in (E) (n = 10). ***P < 0.001, **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test (C and F) or one-way ANOVA (G).

  • Fig. 5 Increased movement of GPR55 KO cells in association with epithelial cells.

    (A) Representative z-projection view of small intestinal villi of GPR55 Het (top) and KO (bottom) mice, showing CD8α-PE–labeled cells (red) and cell nuclei (Hoechst, blue). Lines show tracks of labeled IELs. Images are representative from more than six mice in each genotype. Scale bars, 20 μm. (B) Representative z-projection view of small intestinal villi of TCRδ-eGFP+ GPR55 Het (top) and KO (bottom) mice, showing eGFP (green) and cell nuclei (Hoechst, blue). Lines show tracks of GFP+ cells. Circles and ovals in (A) and (B) show regions of tracks that are localized in close association with epithelial cells. Images are representative from more than seven mice in each genotype. Scale bars, 40 μm. (C) Representative snapshots from single z-plane movies. Left two panels from GPR55 Het and right two panels from GPR55 KO. Close association movement along epithelium observed as overlap of eGFP signal with the fluorescence signal of the epithelial cell nuclei labeled with Hoechst (circles in top right). Scale bars, 10 μm. (D and E) Representative multiple z-stack projection view (D) and single z-plane view (E) of small intestinal villi of mixed BM chimeras of the type described in Fig. 4D. Red cells are Het (red tracks), whereas red and green double-positive cells are KO (green tracks). Ovals in (D) (yellow in Het and orange in KO) show regions of tracks that are localized in close association with epithelial cells. Dashed white lines in (E) indicate the boundaries of the Hoechst-labeled epithelial cell nuclei (ECN) and the PMS used for the quantitation. Images are representative from six mice. Scale bars, 20 μm. (F) Speed of GPR55 Het and KO γδT IELs in mixed BM chimeras. (G and H) Percentage of time in close association with epithelial cells and time of each association (G), frequency of lateral intercellular space (LIS) probing, and time of each probing (H) of GPR55 Het and KO cells in mixed BM chimeras. Combined data from six movies from six different chimeric mice. (I) Induction of Pkr and Usp18 mRNA in the small intestine of GPR55 Het and KO mice treated with anti-CD3 antibody (Ab) for 3 hours, plotted relative to Hprt. HPRT, hypoxanthine-guanine phosphoribosyltransferase. Combined data from two independent experiments (n = 9 in each group). ***P < 0.001, **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test.

  • Fig. 6 Increased resistance of GPR55-deficient mice to indomethacin-induced intestinal permeability.

    (A to D) Serum FITC-dextran in GPR55 Het [DMSO, n = 4; indomethacin (Indo), n = 8] and KO (DMSO, n = 4; Indo, n = 8) mice (A), WT mice treated with vehicle (DMSO, n = 6; Indo, n = 13) or GPR55 antagonist CID16020046 (DMSO, n = 6; Indo, n = 13) (B), CCR9 WT (n = 6) and KO (n = 6) mice (C), or in WT (n = 12), GPR55 Het TCRδ KO (n = 13), and GPR55 KO TCRδ KO (n = 11) mice (D), all treated for 5 hours with vehicle (DMSO) or Indo and for 4 hours with FITC-dextran. (E) Immunofluorescence detection of TCRδ-eGFP and DAPI in GPR55 Het and KO mice treated with carrier (DMSO) or Indo for 4 hours. Scale bars, 50 μm. (F) Number of γδT IELs (number per 100-μm length) beneath (in PMS) or closely associated with the epithelium or in both compartments of GPR55 Het (DMSO, n = 6; Indo, n = 7) or KO (DMSO, n = 8; Indo, n = 9) mice treated with DMSO on Indo as in (E). (G) Percentage of γδT IEL in close association with the epithelium. (H) Number of γδT IEL in GPR55 Het (DMSO, n = 7; Indo, n = 11) and KO (DMSO, n = 9; Indo, n = 9) mice 4 hours after DMSO or Indo treatment as determined by flow cytometry. ***P < 0.001, **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test (A to C and F to H) or one-way ANOVA (D).

  • Fig. 7 GPR55 regulates γδT cell egress from PP and homing of gut-tropic CD8 T cells to the small intestine.

    (A) Frequency of CD8αα γδT cells in PPs, mLN, and peripheral blood lymphocytes (PBLs) of GPR55 Het (DMSO, n = 5; Indo, n = 8) and KO (DMSO, n = 4; Indo, n = 8) mice after treatment with carrier (DMSO) or Indo for 4 hours. (B) Ratio of CD45.2 GPR55 KO over CD45.1/2 GPR55 WT CD8αα γδT cells in the indicated tissues of mixed BM chimeras 4 hours after treatment with DMSO or Indo (n = 4 in each group). (C) Percentage (left) or numbers (right) of CD8αα γδT cells in the indicated GPR55 Het and KO mouse tissues after treatment for 4 hours with Indo and FTY720. Left: GPR55 Het, n = 9; GPR55 KO, n = 8. Right: GPR55 Het, n = 6; GPR55 KO, n = 9. (D) Migration of the indicated cell types from PPs of control and GPR55 KO mice to nil, CXCL12, or CXCL12 and 20:4 LPI in Transwell assays. Each experiment was performed in duplicate and repeated twice. (E) Expression of Ccr9 and Gpr55 mRNA on T cells cultured for the indicated days in the presence of anti-CD3 and anti-CD28 and with IL-2 (100 U/ml) from day 3 and in the absence or presence of retinoic acid (RA), plotted relative to Hprt (n = 4 in each group from two independent experiments). Bars indicate mean ± SEM. (F) Homing of CD8 T cell cultured with RA as in (E) to the spleen, small intestine, and blood, shown as the ratio of CD45.2 GPR55 KO cells to CD45.1 WT cells, 1 day after transfer. Data (n = 4 in each group) are representative of two independent experiments. (G) Homing of cells cultured as in (E) and transduced with empty or GPR55-containing vector, plotted as the ratio of transduced (Thy1.1 reporter–positive) to untransduced (Thy1.1 reporter–negative) cells of each type, 1 day after transfer. “Injected cells” in (F) and (G) indicates the ratio at the time of injection. Data (n = 3 in each group) are representative of two independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, n.s. P > 0.05 by Student’s t test (A to C, F, and G) or one-way ANOVA (E).

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/18/eaao1135/DC1

    Fig. S1. T cell populations and phenotype of GPR55-deficient and control gut or lymphoid tissues.

    Fig. S2. GPR55 mediates migration inhibition.

    Fig. S3. Detection of LPI species in the small intestine and spleen by LC-MS/MS.

    Fig. S4. Movement of GPR55 KO T cells in close association with epithelial cells.

    Fig. S5. Quantitation of PGE2 in the small intestine.

    Fig. S6. Distribution of γδT IELs in indomethacin-treated mouse small intestine.

    Fig. S7. Frequencies of conventional T cells in PPs, mLN, and blood after indomethacin treatment, and induction of gut homing receptors on cultured CD8 T cells.

    Movie S1. Real-time imaging of CD8α+ IEL migration in Gpr55+/− small intestine.

    Movie S2. Real-time imaging of CD8α+ IEL migration in Gpr55−/− small intestine.

    Movie S3. Real-time imaging of TCRδ-eGFP IEL migration in Gpr55+/− small intestine.

    Movie S4. Real-time imaging of TCRδ-eGFP IEL migration in Gpr55−/− small intestine.

    Movie S5. Real-time imaging of TCRδ-eGFP IEL migration in Gpr55+/− small intestine (single z-plane movie).

    Movie S6. Real-time imaging of TCRδ-eGFP IEL migration in Gpr55−/− small intestine (single z-plane movie).

    Movie S7. Real-time imaging of TCRδ-eGFP IEL migration in the small intestine of a Rag1−/− mouse reconstituted with a mixture of Gpr55+/− Tcrβ−/− and Gpr55−/− Tcrβ−/− TCRδ-eGFP BM.

    Movie S8. Real-time imaging of TCRδ-eGFP IEL migration in the small intestine of a Rag1−/− mouse reconstituted with a mixture of Gpr55+/− Tcrβ−/− and Gpr55−/− Tcrβ−/− TCRδ-eGFP BM (single z-plane movie).

  • Supplemental Materials

    Supplementary Material for:

    GPR55 regulates intraepithelial lymphocyte migration dynamics and susceptibility to intestinal damage

    Hayakazu Sumida, Erick Lu, Hsin Chen, Qiyun Yang, Ken Mackie, Jason G. Cyster*

    *Corresponding author. Email: jason.cyster{at}ucsf.edu

    Published 8 December 2017, Sci. Immunol. 2, eaao1135 (2017)
    DOI: 10.1126/sciimmunol.aao1135

    This PDF file includes:

    • Fig. S1. T cell populations and phenotype of GPR55-deficient and control gut or lymphoid tissues.
    • Fig. S2. GPR55 mediates migration inhibition.
    • Fig. S3. Detection of LPI species in the small intestine and spleen by LC-MS/MS.
    • Fig. S4. Movement of GPR55 KO T cells in close association with epithelial cells.
    • Fig. S5. Quantitation of PGE2 in the small intestine.
    • Fig. S6. Distribution of γδT IELs in indomethacin-treated mouse small intestine.
    • Fig. S7. Frequencies of conventional T cells in PPs, mLN, and blood after indomethacin treatment, and induction of gut homing receptors on cultured CD8 T cells.
    • Legends for movies S1 to S8

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Real-time imaging of CD8α+ IEL migration in Gpr55−/− small intestine.
    • Movie S2 (.mov format). Real-time imaging of CD8α+ IEL migration in Gpr55−/− small intestine.
    • Movie S3 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in Gpr55+/− small intestine.
    • Movie S4 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in Gpr55−/− small intestine.
    • Movie S5 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in Gpr55+/− small intestine (single z-plane movie).
    • Movie S6 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in Gpr55−/− small intestine (single z-plane movie).
    • Movie S7 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in the small intestine of a Rag1−/− mouse reconstituted with a mixture of Gpr55+/− Tcrβ−/− and Gpr55−/− Tcrβ−/− TCRδ-eGFP BM.
    • Movie S8 (.mov format). Real-time imaging of TCRδ-eGFP IEL migration in the small intestine of a Rag1−/− mouse reconstituted with a mixture of Gpr55+/− Tcrβ−/− and Gpr55−/− Tcrβ−/− TCRδ-eGFP BM (single z-plane movie).
    • Source data (Microsoft Excel format)

    Files in this Data Supplement:

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