Plasmacytoid dendritic cells (pDCs) play a key role in antiviral immunity, but also contribute to the pathogenesis of certain autoimmune diseases, by producing large amounts of type I IFNs. Although activation of pDCs is triggered by engagement of nucleotide-sensing toll-like receptors (TLR) 7 and 9, type I IFN induction additionally requires IκB kinase (IKK) α–dependent activation of IFN regulatory factor (IRF) 7. However, the signaling pathway mediating IKK-α activation is poorly defined. We show that DOCK2, an atypical Rac activator, is essential for TLR7- and TLR9-mediated IFN-α induction in pDCs. We found that the exposure of pDCs to nucleic acid ligands induces Rac activation through a TLR-independent and DOCK2-dependent mechanism. Although this Rac activation was dispensable for induction of inflammatory cytokines, phosphorylation of IKK-α and nuclear translocation of IRF-7 were impaired in Dock2-deficient pDCs, resulting in selective loss of IFN-α induction. Similar results were obtained when a dominant-negative Rac mutant was expressed in wild-type pDCs. Thus, the DOCK2–Rac signaling pathway acts in parallel with TLR engagement to control IKK-α activation for type I IFN induction. Owing to its hematopoietic cell-specific expression, DOCK2 may serve as a therapeutic target for type I IFN–related autoimmune diseases.

Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

IntroductionDendritic cells (DCs) are specialized antigen-presenting cells found as sentinels in peripheral tissues and lymphoid organs. DCs are classified into 2 populations, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) with distinct expression patterns of costimulatory molecules and Toll-like receptors. 1,2 Although both mDCs and pDCs are produced in the bone marrow (BM) and migrate into lymphoid tissues to control immune response, the migratory pathways of these DC subsets are different. 3 As the migratory properties of DCs are of fundamental importance for their function, chemokines and their receptors have been extensively analyzed. However, the downstream signaling molecules critical for DC migration are largely unknown.Chemokine receptors are coupled with heterodimeric G i proteins that activate a variety of signaling pathways including Rac. DOCK2 is a novel member of the CDM family proteins, Caenorhabditis elegans CED-5, mammalian DOCK180, and Drosophila melanogaster Myoblast City, that are known to regulate the actin cytoskeleton by functioning upstream of Rac. 4,5 Although DOCK2 plays an important role in migration of lymphocytes and neutrophils, 6-10 the role of DOCK2 in DC migration remains unknown. In this study, we examined whether and how DOCK2-deficiency affects migration of mDCs and pDCs. MethodsMice DOCK2-deficient (DOCK2 Ϫ/Ϫ ) mice were backcrossed with C57BL/6 (B6) mice for more than 8 generations before use. All experiments were done in accordance with the guidelines of the committee of Ethics of Animal Experiments, Kyushu University. Cell preparationTo generate BM-derived pDCs and mDCs, BM cells were cultured for 7 days with Flt3 ligand (50 ng/mL; R&D Systems, Minneapolis, MN) or granulocyte-macrophage colony-stimulating factor (20 ng/mL; PeproTech, Rocky Hill, NJ), respectively. Cells were then purified with either anti-B220 or anti-CD11c microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). In some experiments, pDCs and mDCs were purified with a FACS Aria (BD Biosciences, Mountain View, CA) after staining the cells with anti-B220 and anti-CD11c antibodies (BD Biosciences). Flow cytometry and tissue stainingCells were stained with anti-B220, anti-CD11c, anti-mPDCA1 (Miltenyi Biotec), anti-CXCR3 (R&D Systems), anti-CXCR4 11 and/or anti-CCR7 11 antibodies, and analyzed on a FACS Calibur (BD Biosciences). For tissue staining, frozen sections were fixed in acetone or 4% paraformaldehyde, and incubated with anti-mPDCA1, anti-B220 and/or anti-MOMA1 (BMA Biomedicals, Augst, Switzerland) antibodies. Chemotaxis assayTranswell chemotaxis assay was performed as described, 6,9 with primary BM cells. EZ-Taxiscan chemotaxis assay was performed according to the manufacturer's protocol (GE Healthcare, Chalfont St Giles, United Kingdom) using BM-derived pDCs and mDCs. In vivo homing assayBM-derived pDCs were labeled with PKH fluorescent kit (Sigma-Aldrich, St Louis, MO) and injected intravenously into mice. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From Reverse transcriptas...

The lineage commitment of CD4+ T cells is coordinately regulated by signals through the T cell receptor and cytokine receptors, yet how these signals are integrated remains elusive. Here we find that mice lacking Dock2, a Rac activator in lymphocytes, developed allergic disease through a mechanism dependent on CD4+ T cells and the interleukin 4 receptor (IL-4R). Dock2-deficient CD4+ T cells showed impaired antigen-driven downregulation of IL-4Ralpha surface expression, resulting in sustained IL-4R signaling and excessive T helper type 2 responses. Dock2 was required for T cell receptor-mediated phosphorylation of the microtubule-destabilizing protein stathmin and for lysosomal trafficking and the degradation of IL-4Ralpha. Thus, Dock2 links T cell receptor signals to downregulation of IL-4Ralpha to control the lineage commitment of CD4+ T cells.