LRCH1 interferes with DOCK8-Cdc42-induced T cell migration and ameliorates experimental autoimmune encephalomyelitis

Abstract

Directional autoreactive CD4⁺ T cell migration into the central nervous system plays a critical role in multiple sclerosis. Recently, DOCK8 was identified as a guanine-nucleotide exchange factor (GEF) for Cdc42 activation and has been associated with human mental retardation. Little is known about whether DOCK8 is related to multiple sclerosis (MS) and how to restrict its GEF activity. Using two screening systems, we found that LRCH1 competes with Cdc42 for interaction with DOCK8 and restrains T cell migration. In response to chemokine stimulation, PKCα phosphorylates DOCK8 at its three serine sites, promoting DOCK8 separation from LRCH1 and translocation to the leading edge to guide T cell migration. Point mutations at the DOCK8 serine sites block chemokine- and PKCα-induced T cell migration. Importantly, Dock8 mutant mice or Lrch1 transgenic mice were protected from MOG (35-55) peptide-induced experimental autoimmune encephalomyelitis (EAE), whereas Lrch1-deficient mice displayed a more severe phenotype. Notably, DOCK8 expression was markedly increased in PBMCs from the acute phase of MS patients. Together, our study demonstrates LRCH1 as a novel effector to restrain PKCα-DOCK8-Cdc42 module-induced T cell migration and ameliorate EAE.

Figure 1.

DOCK8 expression is positively associated with the peak phase of murine EAE. (A) The relative mRNA expression levels of the candidate genes in the PBMCs from MS patients and healthy volunteers (top left; n = 4). DOCK8 mRNA levels in the PBMCs (top right) from healthy volunteers (n = 42), NMO patients (n = 24), or MS patients (n = 38). DOCK8 expression in the PBMCs from healthy volunteers and MS patients by immunoblotting (bottom). (B) The total number of CD4⁺ T cells circulating in the blood (left) or infiltrating in the CNS (right) at different stages of murine EAE. n = 6. (C) Dock8 mRNA levels in CD4⁺ T cells from murine EAE at the presyndrome, peak, or remission stages. n = 3. (D) Clinical scores (top) and EAE incidence (bottom) of the Dock8^pri/+ and Dock8^pri/pri mice immunized with MOG (35–55). n = 10. (E and F) H&E and Luxol blue staining of the representative tissue sections of the spinal cords from the Dock8^pri/+ and Dock8^pri/pri mice on day 18 after EAE induction. Bars, 70 µm. (G and H) Frequency of CD4⁺ T, CD8⁺ T, and B220⁺ cells in the CNS by flow cytometry from the EAE-induced mice. n = 3. (I) The percentages of BrdU⁺CD4⁺ T cells and Annexin V⁺ CD4⁺ T cells in draining LNs from Dock8^pri/+or Dock8^pri/pri mice after EAE induction. n = 3. (J) The percentages of CD4⁺ T cells in the draining LNs and spleen. n = 3. (K) The percentages of IFN-γ⁺, IL-17A⁺, or Foxp3⁺ cells in CD4⁺ T cells from the draining LNs of Dock8^pri/+ or Dock8^pri/pri mice when EAE was induced at peak stage. n = 3. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.005. Data are representative of three independent experiments (D, mean ± SEM; B, C, and G, mean ± SD) or two independent experiments (H, I, J, and K, mean ± SD). Statistical significance was determined using unpaired Student’s t test.

Figure 2.

CD4⁺ T cells from Dock8^pri/pri mice ameliorate EAE with reduced CNS infiltration and migration. (A and B) The encephalitogenic CD4⁺ T cells were purified from Dock8^pri/+ or Dock8^pri/pri mice to check surface expression of CD44 and CD25 by FACS (A). IL-2 concentrations were checked by ELISA in the supernatants of Dock8^pri/pri or Dock8^pri/+ encephalitogenic CD4⁺ T cells stimulation with MOG for 3 d (B). n = 6. (C–E) Adoptive transfer of encephalitogenic Dock8^pri/+ and Dock8^pri/priCD4⁺ T cells into the sublethally irradiated WT mice (n = 6) to assess clinical scores & EAE incidence (C). (D) H&E staining and Luxol blue staining of the representative tissue sections of the spinal cords. Bars, 70 μm. (E) Total number of CD4⁺ T cells in the CNS and in the blood by flow cytometry. n = 6. (F) The encephalitogenic CD4⁺ T cells were isolated from the Dock8^pri/+ and Dock8^pri/pri mice at day18 after EAE induction for SDF-1α– or CCL5-induced migration (left), or for a FACS assay to check the surface expression of CXCR4 and CCR5 (right). n = 4–6. (G) The percentages of IFN-γ⁺ cells and IL-17A⁺ cells in CD4⁺ T cells in spleen from the recipient mice presented in C. n = 5. (H–I) T8.1 cells were transfected with FLAG-tagged DOCK8 or the pri mutant and treated with or without SDF-1α, followed by immunostaining with anti-FLAG and anti-CD44 (H; bar, 5 µm; n = 50), or for a transwell assay (I; left; n = 3 per group), or for a FACS assay to check the surface expression of CXCR4 (I, right). n = 4. (J) The migration of CD4⁺ T cells from naive Dock8^pri/+ or Dock8^pri/pri mice was examined in response to SDF-1α (left). Or WT and Dock8^pri/pri CD4⁺ T cells were reconstituted with the vector or DOCK8 to assess T cell migration in response to CCL5 (right). n = 3. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.005. Data are representative of three independent experiments (C, mean ± SEM; E–F and H–I, mean ± SD), or two experiments (A, B, G, and J, mean ± SD). Statistical significance was determined using unpaired Student’s t test.

Figure 3.

Identification of LRCH1 as a new binding partner of DOCK8. (A) An anti-FLAG IP was performed with FLAG-DOCK8–transfected T8.1 cells for the mass spectrometry assay, and the Coomassie blue staining is shown. (B) DOCK8 interaction with LRCH1 in the yeast two-hybrid system. DOCK8 and the pDEST22 vector, LRCH1 and the pDEST32 vector, and DOCK8 and LRCH1 were cotransfected into the yeast strain Mav 203-activated expression of β-glycosidase. Krev-1 and RalGDS-WT were cotransfected as a positive control. Krev-1 and RalGDS-m2 were cotransfected as a negative control. (C–F) 293T cells were cotransfected with FLAG-DOCK8 and HA-LRCH1 (C); FLAG-DOCK8 and HA-LRCH1 or its deletion mutants (HA-LRCH1 1–238 aa, 1–304 aa, or 305–763 aa; D); HA-LRCH1 and FLAG-DOCK8 or its mutants (DOCK8 861–2099 aa, 1151-2099 aa, 1451-2099 aa, and 1687–2099 aa, or 1–1634 aa, 1635–2099 aa; E and F) for immunoprecipitation followed by immunoblotting with the indicated antibodies. (G) 293T cells were cotransfected with HA-tagged LRCH1 and FLAG-tagged the DHR2 domain of DOCK6 or DOCK7. Cell lysates were immunoprecipitated with anti-FLAG antibody and followed by immunoblotting with the indicated antibodies. Data are representative of three experiments.

Figure 4.

Lrch1 transgenic mice are resistant to EAE with reduced T cell migration. (A) T8.1 cells were transfected with the vector control, LRCH1, or its fragments (LRR_1-9, L305-763) for a transwell assay in response to SDF-1α (n = 3). FACS assay was performed to check the cell surface expression of CXCR4. The transfected exogenous human LRCH1 was detected at 130 kD and the endogenous murine LRCH1 in T8.1 cells was detected at 95 kD by Western blot. (B) The expression of FLAG-Lrch1 in thymus from Lrch1 transgenic mice was assessed by immunoblotting. (C) The clinical scores (left) and EAE incidence (right) of WT and Lrch1 transgenic mice induced by the MOG (35–55) peptide. n = 5. (D and E) The total numbers of CD4⁺ T cells in the CNS, blood (D), spleen, and draining LNs (E; middle right and right) of the WT or Lrch1 transgenic mice; percentages of BrdU⁺ or Annexin V⁺ CD4⁺ T cells in spleen were checked at the peak stage of EAE (E; left and middle left). n = 4–5. (F) The number of lymphocytes in spleen and draining LNs were counted from WT and Lrch1 Tg mice after EAE induction. n = 5. (G) The percentage of Foxp3⁺ CD4⁺ T reg cells in draining LNs from the WT or Lrch1 transgenic mice at the peak stage of EAE. n = 4. (H and I) The encephalitogenic CD4⁺ T cells were purified from the WT or Lrch1 transgenic mice for a transwell assay in response to SDF-1α (H), or for a FACS assay to check the surface expression of CXCR4 (I). n = 4–5. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of four experiments (A, mean ± SD), three experiments (C, mean ± SEM), or two experiments (D–I, mean ± SD). Statistical significance was determined using unpaired Student’s t test.

Figure 5.

Adoptive transfer of Lrch1 KO CD4⁺T cells accelerates EAE with enhanced T cell migration. (A) Generation of Lrch1 KO mice. The exon 1 of the Lrch1 gene was specifically targeted by TALEN, and DNA sequencing confirmed the nucleotide mutation in the Lrch1 locus adjacent to the FOKI cleavage site (arrow). (B) Numbers of CD4⁺ T cells in spleen and LNs from unimmunized mice. n = 3. (C) EAE incidence of WT and Lrch1 KO mice in response to MOG (35–55) treatment. n = 5. (D–F) Numbers of CD4⁺ T cells in spleen and draining LNs (D); TCR usage analyzed by anti-TCR Vα and anti-TCR Vβ antibodies (E); and percentages of BrdU⁺ CD4⁺ T cells, IL-2 secretion, and the surface expression of CD25 and CD44 in CD4⁺ T cells from the draining LNs (F) of WT and Lrch1 KO mice after EAE induction. n = 5. (G) The sublethally irradiated WT recipient mice were reconstituted with WT or Lrch1^−/− encephalitogenic CD4⁺ T cells to assess their clinical scores (left) and EAE incidence (right). n = 5. (H) The total numbers of WT or Lrch1^−/− encephalitogenic CD4⁺ T cells in the CNS from the sublethally irradiated WT recipient mice presented in G. The encephalitogenic CD4⁺ T cells were purified from the WT or Lrch1^−/− mice for a transwell assay in response to CCL5 (right). n = 5. (I) CD4⁺ T cells were reconstituted with LRCH1 or the vector control, for a transwell assay in response to CCL5 (left). The surface expression of CCR5 in the spleen CD4⁺ T cells from WT and Lrch1 KO mice after EAE induction (right). n = 3–4. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of three experiments (G, mean ± SEM), or two experiments (B–F and H­–I, mean ± SD). Statistical significance was determined using unpaired Student’s t test.

Figure 6.

LRCH1 attenuates DOCK8-mediated Cdc42 activation for T cell migration. (A) T8.1 cells were transfected with FLAG-DOCK8, incubated with Ttox peptide-pulsed L625.7 cells to form cell conjugates, followed by immunostaining to visualize DOCK8 and Cdc42. Bar, 5 µm. (B) The FRET efficiency of biosensor Raichu-Cdc42 between WT and Lrch1^−/−CD4⁺ T cells in response to SDF-1αtreatment. FRET efficiency was measured with donor dequenching approach, and was calculated as E = (Post −[Pre/Post]) × 100%, where Post and Pre represents the donor fluorescence before and after photo bleaching (left). n = 20. NS, not significant (P > 0.05); **, P < 0.01. Activated Cdc42 was precipitated by a GST-CRIB-PAK1 pull-down assay in T8.1 cells that overexpressed LRCH1 or the vector control (right). (C) 293T cells were transfected with FLAG-DOCK8 with or without HA-LRCH1, and the amount of DOCK8 binding to the GST-Cdc42G15A–coated beads was used to evaluate the GEF activity of DOCK8. (D) 293T cells were cotransfected with FLAG-DHR-2, HA-Cdc42 with or without HA-LRCH1. The cell lysates were subjected to a GST-CRIB-PAK1 pull-down assay to precipitate the active Cdc42. (E) 293T cells were transfected with HA-Cdc42, Myc-LRCH1, and FLAG-DHR-2, followed by immunoprecipitation with anti-HA and immunoblotting with anti-HA or anti-FLAG. (F) The purity of the purified His-DHR-2 (1632–2068 aa) and GST-Cdc42 G15A protein from E. coli was determined by Coomassie blue staining. (G and H) Increasing amounts of FLAG-LRR_1-9 (G) or FLAG-L305-763 (H) were added into the solution containing the purified recombinant proteins His-DHR-2 and GST-Cdc42G15A, incubated and then subjected for precipitation using anti-His antibody. (I) T8.1 cells, which were transfected with FLAG-DOCK8 and HA-LRCH1, were treated with or without SDF-1α to assess localization of DOCK8 (red, top) and LRCH1 (green, middle). Bar, 5 µm. (J and K) T8.1 cells expressing FLAG-DOCK8 and HA-LRCH1 were treated or untreated with SDF-1α and PMA (J). 293T cells were transfected with HA-LRCH1, FLAG-DOCK8, or the membrane-localized CVIM-FLAG-DOCK8 (K), followed by immunoprecipitation with anti-FLAG to detect the phosphorylation levels of DOCK8 and the amount of LRCH1. Data presented are representative of two independent experiments (A–D, G, H, and J), or three independent experiments (E, I, and K). Intensity of the immunoblots was quantified and shown at the bottom (mean ± SD). Statistical significance was determined using unpaired Student’s t test.

Figure 7.

PKCα phosphorylates DOCK8 for separation from LRCH1. (A) The amino acid sequence (2075–2089) of DOCK8 and its mutants depict the three key serine residues. (B) 293T cells expressing CVIM-FLAG-DOCK8 were untreated or treated with the AKT and PKCα inhibitors, followed by immunoprecipitation with anti-FLAG to detect DOCK8 phosphorylation levels. (C and D) Migration of the T8.1 cells expressing the vector control, DOCK8, or the mutant 3S/E or 3S/A were examined by a transwell assay in response to SDF-1α in the presence or absence of the PKC inhibitor. n = 3. (E) 293T cells were transfected with FLAG-DOCK8 or the 3S/A mutant, followed by a GST-Cdc42G15A pull-down assay to measure their GEF activity. (F) The migration of T8.1 cells coexpressing PKCα with GFP, DOCK8, or the 3S/A mutant was assessed by a transwell assay. n = 3. (G) 293T cells were transfected with HA-LRCH1, FLAG-DOCK8, or 3S/E, followed by immunoprecipitation with anti-FLAG to analyze their binding to LRCH1. (H) The vector control, DOCK8, or the mutant 3S/E were coexpressed with or without LRCH1 into T8.1 cells and migration was examined by a transwell assay in response to SDF-1α. n = 3. (I) The localization of LRCH1 (green), CVIM-DOCK8, or CVIM-DOCK8 3S/A (red) was examined in 293T cells by immunostaining. Bar, 5 µm. (J) 293T cells were transfected with FLAG-DOCK8 or 3S/A together with HA-LRCH1 and Myc-PKCα, stimulated with or without PMA, followed by immunoprecipitation with anti-FLAG to analyze DOCK8 phosphorylation levels and binding to LRCH-1. NS, not significant (P > 0.05); *, P < 0.05; **, P < 0.01. Data are representative of three experiments (C, D, F, H, mean ± SD) or two experiments (B, E, G, and J). Intensity of the immunoblots was quantified and shown at the bottom (mean ± SD). Statistical significance was determined using unpaired Student’s t test.