Th17 cells differentiated with mycelial membranes of Candida albicans prevent oral candidiasis.

Candida albicans is a human commensal that causes opportunistic infections. Th17 cells provide resistance against mucosal infection with C. albicans; however, the T cell antigens remain little known. Our final goal is to find effective T cell antigens of C. albicans that are responsible for immunotherapy against candidiasis. Here, we prepared fractions including cytosol, membrane and cell wall from yeast and mycelial cells. Proteins derived from a membrane fraction of mycelial cells effectively induced differentiation of CD4+ T cells into IL-17A-producing Th17 cells. To confirm the immunological response in vivo of proteins from mycelial membrane, we performed adoptive transfer experiments using ex vivo stimulated CD4+ T cells from IL-17A-GFP reporter mice. Mycelial membrane-differentiated CD4+ Th17 cells adoptively transferred intravenously prevented oral candidiasis by oral infection of C. albicans, compared with control anti-CD3-stimulated CD4+ T cells. This was confirmed by the clinical score and the number of neutrophils on the infected tissues. These data suggest that effective T cell antigens against candidiasis could be present in the membrane protein fraction of mycelial cells. The design of novel vaccination strategies against candidiasis will be our next step.

The AP-1 transcription factor JunB is required for Th17 cell differentiation.

Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.

Antifungal activity in vitro and in vivo of a salmon protamine peptide and its derived cyclic peptide against Candida albicans.

Protamine peptide (PP) derived from salmon is a 14-mer with 10 arginine residues. We investigated the in vitro and in vivo antifungal activity of PP against Candida albicans PP showed a concentration-dependent dual mode of action, with fungicidal activity and inhibitory activity for hyphal development in vitro. At lethal concentrations of PP, intracellular accumulation of PP was energy-dependent but independent of endocytosis, and resulted in ATP efflux and the generation of reactive oxygen species in the cells. PP at sublethal concentrations inhibited hyphal development in C. albicans by binding to the cell surface. Though antifungal activity of PP was inactivated by high concentrations of NaCl, the antifungal activity of the synthetic cyclic (via a disulfide bond) form of PP (cyclic PP) was not. Cyclic PP also showed the concentration-dependent dual mode of action, and had five-fold greater antifungal activity than PP. The advantage of antifungal activity of cyclic PP compared with PP in vitro resulted in a high in vivo efficacy in a murine oral candidiasis model. Oral treatment with cyclic PP inhibited hyphal development of C. albicans on mouse tongues and protected against the development of severe candidiasis. This study shows the therapeutic potential of cyclic PP as an antifungal peptide against C. albicans.

Intronic regulation of Aire expression by Jmjd6 for self-tolerance induction in the thymus.

The thymus has spatially distinct microenvironments, the cortex and the medulla, where the developing T-cells are selected to mature or die through the interaction with thymic stromal cells. To establish the immunological self in the thymus, medullary thymic epithelial cells (mTECs) express diverse sets of tissue-specific self-antigens (TSAs). This ectopic expression of TSAs largely depends on the transcriptional regulator Aire, yet the mechanism controlling Aire expression itself remains unknown. Here, we show that Jmjd6, a dioxygenase that catalyses lysyl hydroxylation of splicing regulatory proteins, is critical for Aire expression. Although Jmjd6 deficiency does not affect abundance of Aire transcript, the intron 2 of Aire gene is not effectively spliced out in the absence of Jmjd6, resulting in marked reduction of mature Aire protein in mTECs and spontaneous development of multi-organ autoimmunity in mice. These results highlight the importance of intronic regulation in controlling Aire protein expression.

DOCK5 functions as a key signaling adaptor that links FcεRI signals to microtubule dynamics during mast cell degranulation.

Mast cells play a key role in the induction of anaphylaxis, a life-threatening IgE-dependent allergic reaction, by secreting chemical mediators that are stored in secretory granules. Degranulation of mast cells is triggered by aggregation of the high-affinity IgE receptor, FcεRI, and involves dynamic rearrangement of microtubules. Although much is known about proximal signals downstream of FcεRI, the distal signaling events controlling microtubule dynamics remain elusive. Here we report that DOCK5, an atypical guanine nucleotide exchange factor (GEF) for Rac, is essential for mast cell degranulation. As such, we found that DOCK5-deficient mice exhibit resistance to systemic and cutaneous anaphylaxis. The Rac GEF activity of DOCK5 is surprisingly not required for mast cell degranulation. Instead, DOCK5 associated with Nck2 and Akt to regulate microtubule dynamics through phosphorylation and inactivation of GSK3ß. When DOCK5-Nck2-Akt interactions were disrupted, microtubule formation and degranulation response were severely impaired. Our results thus identify DOCK5 as a key signaling adaptor that orchestrates remodeling of the microtubule network essential for mast cell degranulation.

Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse.

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP3) in the synaptic membrane. PIP3 functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP3 is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP3-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.

The Rac activator DOCK2 regulates natural killer cell-mediated cytotoxicity in mice through the lytic synapse formation.

Natural killer (NK) cells play an important role in protective immunity against viral infection and tumor progression, but they also contribute to rejection of bone marrow grafts via contact-dependent cytotoxicity. Ligation of activating NK receptors with their ligands expressed on target cells induces receptor clustering and actin reorganization at the interface and triggers polarized movement of lytic granules to the contact site. Although activation of the small GTPase Rac has been implicated in NK cell-mediated cytotoxicity, its precise role and the upstream regulator remain elusive. Here, we show that DOCK2, an atypical guanine nucleotide exchange factor for Rac, plays a key role in NK cell-mediated cytotoxicity. We found that although DOCK2 deficiency in NK cells did not affect conjugate formation with target cells, DOCK2-deficienct NK cells failed to effectively kill leukemia cells in vitro and major histocompatibility complex class I-deficient bone marrow cells in vivo, regardless of the sorts of activating receptors. In DOCK2-deficient NK cells, NKG2D-mediated Rac activation was almost completely lost, resulting in a severe defect in the lytic synapse formation. Similar results were obtained when the Rac guanine nucleotide exchange factor activity of DOCK2 was selectively abrogated. These results indicate that DOCK2-Rac axis controls NK cell-mediated cytotoxicity through the lytic synapse formation.

Dimerization of DOCK2 is essential for DOCK2-mediated Rac activation and lymphocyte migration.

The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane.

Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2.

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.

Selective control of type I IFN induction by the Rac activator DOCK2 during TLR-mediated plasmacytoid dendritic cell activation.

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 IkappaB kinase (IKK) alpha-dependent activation of IFN regulatory factor (IRF) 7. However, the signaling pathway mediating IKK-alpha activation is poorly defined. We show that DOCK2, an atypical Rac activator, is essential for TLR7- and TLR9-mediated IFN-alpha 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-alpha and nuclear translocation of IRF-7 were impaired in Dock2-deficient pDCs, resulting in selective loss of IFN-alpha 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-alpha 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.

Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D-mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

Differential requirement for DOCK2 in migration of plasmacytoid dendritic cells versus myeloid dendritic cells.

The migratory properties of dendritic cells (DCs) are important for their functions. Although several chemokines and their receptors have been implicated in DC migration, the downstream signaling molecules are largely unknown. Here we show that DOCK2, a hematopoietic cell-specific CDM family protein, is indispensable for migration of plasmacytoid DCs (pDCs), but not myeloid DCs (mDCs). Although DOCK2-deficiency did not affect development of pDCs, DOCK2-deficient (DOCK2(-/-)) mice exhibited a severe reduction of pDCs in the spleen and lymph nodes. Adoptive transfer experiments revealed that DOCK2(-/-) pDCs failed to migrate into the periarteriolar lymphoid sheaths of the spleen. In DOCK2(-/-) pDCs, chemokine-induced Rac activation was severely impaired, resulting in the reduction of motility and the loss of polarity during chemotaxis. In contrast, DOCK2(-/-) mDCs did not show any defects in Rac activation and migration. These results indicate that pDCs and mDCs use distinct molecules to activate Rac during chemotaxis.

DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.

Neutrophils are highly motile leukocytes, and they play important roles in the innate immune response to invading pathogens. Neutrophil chemotaxis requires Rac activation, yet the Rac activators functioning downstream of chemoattractant receptors remain to be determined. We show that DOCK2, which is a mammalian homologue of Caenorhabditis elegans CED-5 and Drosophila melanogaster Myoblast City, regulates motility and polarity during neutrophil chemotaxis. Although DOCK2-deficient neutrophils moved toward the chemoattractant source, they exhibited abnormal migratory behavior with a marked reduction in translocation speed. In DOCK2-deficient neutrophils, chemoattractant-induced activation of both Rac1 and Rac2 were severely impaired, resulting in the loss of polarized accumulation of F-actin and phosphatidylinositol 3,4,5-triphosphate (PIP3) at the leading edge. On the other hand, we found that DOCK2 associates with PIP3 and translocates to the leading edge of chemotaxing neutrophils in a phosphatidylinositol 3-kinase (PI3K)-dependent manner. These results indicate that during neutrophil chemotaxis DOCK2 regulates leading edge formation through PIP3-dependent membrane translocation and Rac activation.

DOCK2 is required in T cell precursors for development of Valpha14 NK T cells.

Mouse CD1d-restricted Valpha14 NKT cells are a unique subset of lymphocytes, which play important roles in immune regulation, tumor surveillance and host defense against pathogens. DOCK2, a mammalian homolog of Caenorhabditis elegans CED-5 and Drosophila melanogaster myoblast city, is critical for lymphocyte migration and regulates T cell responsiveness through immunological synapse formation, yet its role in Valpha14 NKT cells remains unknown. We found that DOCK2 deficiency causes marked reduction of Valpha14 NKT cells in the thymus, liver, and spleen. When alpha-galactosylceramide (alpha-GalCer), a ligand for Valpha14 NKT cells, was administrated, cytokine production was scarcely detected in DOCK2-deficient mice, suggesting that DOCK2 deficiency primarily affects generation of Valpha14 NKT cells. Supporting this idea, staining with CD1d/alpha-GalCer tetramers revealed that CD44- NK1.1- Valpha14 NKT cell precursors are severely reduced in the thymuses of DOCK2-deficient mice. In addition, studies using bone marrow chimeras indicated that development of Valpha14 NKT cells requires DOCK2 expression in T cell precursors, but not in APCs. These results indicate that DOCK2 is required for positive selection of Valpha14 NKT cells in a cell-autonomous manner, thereby suggesting that avidity-based selection also governs development of this unique subset of lymphocytes in the thymus.

Impaired IL-4 and c-Maf expression and enhanced Th1-cell development in Vav1-deficient mice.

Although c-Maf is crucial for Th2 differentiation and production of interleukin 4 (IL-4), its regulation is poorly understood. We report that Vav1-/- CD4+ T cells display deficient T-cell receptor (TCR)/CD28-induced IL-4 and c-Maf expression and, conversely, enhanced interferon gamma (IFN-gamma) production and T-bet expression (even when cultured under Th2-polarizing conditions), but intact expression of other Th2 cytokines and GATA-3. Up-regulation of c-Maf was dependent on Ca2+/nuclear factor of activated T cell (NFAT) and, together with IL-4 production, could be rescued in Vav1-/- T cells by Ca2+ ionophore. Deficient IL-4 production was restored by retrovirus-mediated Vav1 expression, but only partially by retroviral c-Maf expression. Similar IL-4 --> IFN-gamma skewing was observed in intact, antigen-primed Vav1-/- mice. Thus, Vav1 is selectively required for IL-4 and c-Maf expression, a requirement reflecting, at least in part, the dependence of c-Maf expression on Ca2+/NFAT signaling.

Degenerate recognition and response of human CD4+ Th cell clones: implications for basic and applied immunology.

It was once considered that the T cell response is an all or nothing type event, but recent studies have clearly indicated that T cells show many different types of activation in recognition of altered ligands for T cell receptors (TCR). In this review, we summarize our recent findings on the response of human CD4+ helper T (Th) cell clones to altered peptide ligands (APL); peptides carrying single or multiple residue substitutions in antigenic peptides. The extensive analyses revealed that TCR-antagonism and partial agonism are frequently observed by the stimulation with APLs substituted at particular amino acid residues of antigenic peptides. We observed unique partially agonistic APLs inducing prolongation of T cell survival without cell proliferation. Superagonistic APLs stimulated enhanced proliferation and production of cytokines in Th cell clones reactive to tumor-associated antigens. The other APL induced enhanced production of interleukin-12 by antigen presenting cells and subsequent enhancement of IFN-gamma production by T cells reactive to allergens. By utilizing an HLA-DR-restricted T cell epitope library generated by mutated invariant chain genes, it was revealed that human Th cell clones recognize a more diverse array of peptides with multiple and simultaneous amino acid substitutions in an antigenic peptide. APLs also induced altered intracellular signaling events including intracellular calcium increase and phosphorylation of signaling molecules. This information provides basic knowledge regarding the characteristics of antigen recognition by human Th cells and the subsequent activation, and a novel method for manipulation of human Th cell responses by APLs, as a possible candidate for antigen-specific immuno-potentiating or immunosuppressive therapy.

Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney.

BACKGROUND: The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD. In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method. METHODS: Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method. Differentially expressed bands were re-amplified and subcloned. Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis. Subsequently, mouse kidney cDNA library was screened. RESULTS: The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No. AY005469). Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues. The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach. Mouse SULT1C2 was expressed successfully using pET plasmid vector and E. coli. The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor. CONCLUSIONS: Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components.

Vav-induced activation of the human IFN-gamma gene promoter is mediated by upregulation of AP-1 activity.

The role of Vav in the transcriptional regulation of the human interferon-gamma (IFN-gamma) promoter was investigated. Overexpression of Vav in Jurkat-TAg cells enhanced T cell receptor (TCR)-induced activation of a luciferase (Luc) reporter gene construct driven by cis-regulatory element of the IFN-gamma gene (-346 to +7). Electrophoresis mobility shift and Luc reporter assays demonstrated that the DNA-binding and transcriptional activity of the proximal AP-1-dependent NFAT site (positions -172 to -138), the AP-1/Ying-Yang 1 (YY1)-binding site (-209 to -184), and a consensus AP-1-binding site were upregulated by Vav. Vav enhanced TCR-induced activation of c-Jun N-terminal kinase (JNK) and its upstream regulator, Rho family GTPases. Finally, coexpression of a dominant-negative Rac1 mutant suppressed Vav-mediated upregulation of the transcriptional and DNA-binding activity of the proximal NFAT/AP-1 site and the AP-1/YY1 site, as well as the complete IFN-gamma promoter activity. Vav activates the IFN-gamma promoter via upregulation of AP-1-binding through a Rac1/JNK pathway.