A soy protein enzymatic digest mitigates Nrf2-related oxidative stress and attenuates depression-like behavior in a mouse model of sub-chronic restraint stress.

Continuous oxidative stress conditions have been identified as a major cause of various neuropsychiatric disorders, including depression. The present study investigated the potential antidepressant-like effects of a soy protein enzymatic digest (SPD) containing soy-deprestatin, which is a soy-derived peptide with reported antidepressant-like effects, as well as its ability to mitigate oxidative stress in the brain caused by sub-chronic restraint stress. Mice were divided into two groups: a control group and restraint stress group. The restraint stress group was further divided into two groups administered water or SPD. After repeated short-time restraints over five days, we evaluated immobility times in the tail suspension test, and antioxidant enzyme activities, glutathione levels, oxidative stress maker levels, and the gene expression levels of Nrf2 and antioxidant enzymes in the brain. The results obtained showed that the oral administration of SPD reduced immobility times in mice exposed to restraint stress. In comparisons with the water-treated restraint group, the administration of SPD restored superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and glutathione levels and prevented restraint stress-induced increases in malondialdehyde, carbonyl protein, and 8-OHdG levels in the restraint stress group. In addition, high expression levels of Nrf2, HO-1, NQO-1 and GCLC were observed in the SPD-treated restraint group. These results suggest that SPD attenuated repeated restraint stress-induced depression-like behaviors by mitigating oxidative stress through the activation of the Nrf2 signaling pathway.

Analyses of a Mutant Foxp3 Allele Reveal BATF as a Critical Transcription Factor in the Differentiation and Accumulation of Tissue Regulatory T Cells.

Foxp3 controls the development and function of regulatory T (Treg) cells, but it remains elusive how Foxp3 functions in vivo. Here, we established mouse models harboring three unique missense Foxp3 mutations that were identified in patients with the autoimmune disease IPEX. The I363V and R397W mutations were loss-of-function mutations, causing multi-organ inflammation by globally compromising Treg cell physiology. By contrast, the A384T mutation induced a distinctive tissue-restricted inflammation by specifically impairing the ability of Treg cells to compete with pathogenic T cells in certain non-lymphoid tissues. Mechanistically, repressed BATF expression contributed to these A384T effects. At the molecular level, the A384T mutation altered Foxp3 interactions with its specific target genes including Batf by broadening its DNA-binding specificity. Our findings identify BATF as a critical regulator of tissue Treg cells and suggest that sequence-specific perturbations of Foxp3-DNA interactions can influence specific facets of Treg cell physiology and the immunopathologies they regulate.

Intradermal Delivery of Antigens Enhances Specific IgG and Diminishes IgE Production: Potential Use for Vaccination and Allergy Immunotherapy.

Skin is protected by a tough but flexible multilayered barrier and is a front line for immune responses against invading particles. For many years now, skin has been a tissue where certain vaccines are injected for the prevention of infectious disease, however, the detailed mechanisms of the skin immune response are not yet well understood. Using thin and small injection needles, we carefully injected OVA into a restricted region of mouse skin, i.e., intradermal (ID), and examined the antibody response in comparison with subcutaneous (SC) injection or epicutaneous patch administration of OVA. Epicutaneous patches induced a high IgE response against OVA, but IgG production was low. High IgG production was induced by both ID and SC injection, moreover, ID injection induced higher IgG production without any adjutants. Furthermore, OVA-specific IgE production was diminished by ID injection. We found that ID injection could efficiently stimulate skin resident DCs, drive Th1-biased conditions and diminish IgE production. The ID injection response was regulated by Langerin+ dermal DCs, because OVA was taken up mainly by these cells and, after transiently deleting them, the IgE response was no longer diminished and IgG1 production was enhanced. We also tested whether ID injection might be an effective allergy treatment by attempting to inhibit ongoing IgE production in mice with experimentally induced high serum IgE levels. Multiple ID injections of OVA were shown to prevent elevation of serum OVA-specific IgE after repeated allergen challenge. In contrast, SC OVA injection could only transiently inhibit the OVA-specific IgE production. These findings indicated that ID injection results in higher induction of antigen-specific IgG, and thus may be useful for vaccine delivery with little or no adjuvant components. Moreover, the observed diminishment of IgE and induction of Th1-biased immune responses suggest that ID may be a useful injection route for allergy immunotherapy.

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis.

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis.

Requirement of Stat3 Signaling in the Postnatal Development of Thymic Medullary Epithelial Cells.

Thymic medullary regions are formed in neonatal mice as islet-like structures, which increase in size over time and eventually fuse a few weeks after birth into a continuous structure. The development of medullary thymic epithelial cells (TEC) is dependent on NF-κB associated signaling though other signaling pathways may contribute. Here, we demonstrate that Stat3-mediated signals determine medullary TEC cellularity, architectural organization and hence the size of the medulla. Deleting Stat3 expression selectively in thymic epithelia precludes the postnatal enlargement of the medulla retaining a neonatal architecture of small separate medullary islets. In contrast, loss of Stat3 expression in cortical TEC neither affects the cellularity or organization of the epithelia. Activation of Stat3 is mainly positioned downstream of EGF-R as its ablation in TEC phenocopies the loss of Stat3 expression in these cells. These results indicate that Stat3 meditated signal via EGF-R is required for the postnatal development of thymic medullary regions.

Zinc transporter SLC39A10/ZIP10 facilitates antiapoptotic signaling during early B-cell development.

The immune system is influenced by the vital zinc (Zn) status, and Zn deficiency triggers lymphopenia; however, the mechanisms underlying Zn-mediated lymphocyte maintenance remain elusive. Here we investigated ZIP10, a Zn transporter expressed in the early B-cell developmental process. Genetic ablation of Zip10 in early B-cell stages resulted in significant reductions in B-cell populations, and the inducible deletion of Zip10 in pro-B cells increased the caspase activity in parallel with a decrease in intracellular Zn levels. Similarly, the depletion of intracellular Zn by a chemical chelator resulted in spontaneous caspase activation leading to cell death. Collectively, these findings indicated that ZIP10-mediated Zn homeostasis is essential for early B-cell survival. Moreover, we found that ZIP10 expression was regulated by JAK-STAT pathways, and its expression was correlated with STAT activation in human B-cell lymphoma, indicating that the JAK-STAT-ZIP10-Zn signaling axis influences the B-cell homeostasis. Our results establish a role of ZIP10 in cell survival during early B-cell development, and underscore the importance of Zn homeostasis in immune system maintenance.

Zinc transporter SLC39A10/ZIP10 controls humoral immunity by modulating B-cell receptor signal strength.

The humoral immune response, also called the antibody-mediated immune response, is one of the main adaptive immune systems. The essential micronutrient zinc (Zn) is known to modulate adaptive immune responses, and dysregulated Zn homeostasis leads to immunodeficiency. However, the molecular mechanisms underlying this Zn-mediated modulation are largely unknown. Here, we show that the Zn transporter SLC39A10/ZIP10 plays an important role in B-cell antigen receptor (BCR) signal transduction. Zip10-deficiency in mature B cells attenuated both T-cell-dependent and -independent immune responses in vivo. The Zip10-deficient mature B cells proliferated poorly in response to BCR cross-linking, as a result of dysregulated BCR signaling. The perturbed signaling was found to be triggered by a reduction in CD45R phosphatase activity and consequent hyperactivation of LYN, an essential protein kinase in BCR signaling. Our data suggest that ZIP10 functions as a positive regulator of CD45R to modulate the BCR signal strength, thereby setting a threshold for BCR signaling in humoral immune responses.

Development and function of invariant natural killer T cells producing T(h)2- and T(h)17-cytokines.

There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in T(H)2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB⁺iNKT cells are present in the thymic CD44⁺/⁻ NK1.1⁻ population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB⁻iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB⁺ and IL-17RB⁻ subsets. The IL-17RB⁺iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4⁺ IL-17RB⁺iNKT cells produce T(H)2 (IL-13), T(H)9 (IL-9 and IL-10), and T(H)17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4⁻ IL-17RB⁺iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt⁺ subset producing T(H)17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB⁺iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB⁺iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.

Altered antibody production and helper T cell function in mice lacking chemokines CCL19 and CCL21-Ser.

The roles of chemokines CCL19 and CCL21 in Ab production were investigated using plt mutant mice, which lack expression of CCL19 and CCL21-ser in their lymphoid organs. In these mice, the Th response has been shown to tend towards the Th1 type because of accumulation of inflammatory dendritic cells. When plt mice were immunized with 100 µg OVA in CFA, the number of Ab-forming cells in the draining LN, and serum concentrations of OVA-specific IgM and IgG Ab, were very close to those of the control, yet IgG2a Ab in plt mice was increased. In vitro IFN-γ production by the draining LN cells of plt mice was increased. In addition, the ability of helper T cells from plt mice to stimulate Ab production in vitro was prolonged. Also, in the plt mice, in vivo challenge with OVA in incomplete Freund's adjuvant elicited a stronger IgG2a response and a weaker IgG1 response, which is suggestive of a Th1-dominant response. Similar findings were obtained when mice were immunized with 100 µg OVA in alum, except that with alum the increases observed in plt mice were IgG1 produced in vivo and IL-4 produced in vitro by draining LN cells. Furthermore, immunization with alum adjuvant also induced a prolonged in vitro recall response of IFN-γ and IL-4. These findings indicate that plt mice mount an anti-OVA Ab response, and suggest that CCL19 and CCL21 induce prompt Ab responses to antigen, and negatively regulate helper T cell responses in vivo.

xCT deficiency accelerates chemically induced tumorigenesis.

During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic materials, including reactive oxygen species. Thus, macrophages require a protective machinery against oxidative stress to survive at the inflammatory site. Here, we showed that xCT, a component of transport system x(c)(-), was significantly up-regulated in activated infiltrating cells, including macrophages and neutrophils at the inflammatory site. System x(c)(-) mediates the uptake of extracellular L-cystine and is consequently responsible for maintenance of intracellular glutathione levels. We established a loss-of-function mouse mutant line of xCT by N-ethyl-N-nitrosourea mutagenesis. Macrophages from xCT(mu/mu) mice showed cell death in association with the excessive release of high mobility group box chromosomal protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes unremitting inflammation because of the impaired survival of activated macrophages at the inflammatory site. Subcutaneous injection of 3-methylcholanthrene (3-MCA) induced the generation of fibrosarcoma in association with inflammation. When 3-MCA was injected s.c. into mice, xCT mRNA was up-regulated in situ. In xCT(mu/mu) mice, inflammatory cytokines (such as IL-1beta and TNFalpha) were overexpressed, and the generation of 3-MCA-induced fibrosarcoma was accelerated. These results clearly indicate that the defect of the protective system against oxidative stress impaired survival of activated macrophages and subsequently enhanced tumorigenecity.

Establishment of an improved mouse model for infantile neuroaxonal dystrophy that shows early disease onset and bears a point mutation in Pla2g6.

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

CCR7 ligands are required for development of experimental autoimmune encephalomyelitis through generating IL-23-dependent Th17 cells.

CCL19 and CCL21 are thought to be critical for experimental autoimmune encephalomyelitis (EAE) induction, but their precise role is unknown. We examined the role of these chemokines in inducing EAE. C57BL/6 mice lacking expression of these chemokines (plt/plt mice) or their receptor CCR7 were resistant to EAE induced with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG(35-55)) and pertussis toxin. However, passive transfer of pathogenic T cells from wild-type mice induced EAE in plt/plt mice, suggesting a defect independent of the role of CCR7 ligands in the migration of immune cells. Examination of draining lymph node (DLN) cells from MOG(35-55)-immunized plt/plt mice found decreased IL-23 and IL-12 production by plt/plt dendritic cells (DCs) and a concomitant defect in Th17 cell and Th1 cell generation. In contrast, production of the Th17 lineage commitment factors IL-6 and TGF-beta were unaffected by loss of CCR7 ligands. The adoptive transfer of in vitro-generated Th17 cells from DLN cells of MOG(35-55)-immunized plt/plt mice developed EAE in wild-type recipient mice, whereas that of Th1 cells did not. Pathogenic Th17 cell generation was restored in plt/plt DLNs with the addition of exogenous IL-23 or CCL19/CCL21 and could be reversed by inclusion of anti-IL-23 mAb in cultures. Exogenous CCL19/CCL21 induced IL-23p19 expression and IL-23 production by plt/plt or wild-type DCs. Therefore, CCR7 ligands have a novel function in stimulating DCs to produce IL-23 and are important in the IL-23-dependent generation of pathogenic Th17 cells in EAE induction.

A novel gene essential for the development of single positive thymocytes.

A critical step during intrathymic T-cell development is the transition of CD4(+) CD8(+) double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4(-) CD8(+) and MHC-II-restricted CD4(+) CD8(-) single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes.

Chemokines CCL19 and CCL21 promote activation-induced cell death of antigen-responding T cells.

Secondary lymphoid organs (SLOs) provide a niche for the initiation and regulation of T-cell responses, but the mechanisms have been poorly understood. We investigated the influence of chemokines CCL19 and CCL21 constitutively expressed in SLOs on activation-induced cell death (AICD) of CD4+ T cells. When paucity of lymph node T cells (plt) mutant mice lacking expression of CCL19/CCL21 were primed with OVA/CFA, both expansion of OVA-responding CD4+ T cells in the draining lymph nodes and an in vitro recall response were prolonged as compared with responses in wild-type (WT) mice. The apoptotic cell frequency among OVA-responding CD4+ T cells was similarly low in plt/plt and WT mice during the clonal expansion phase. However, during the clonal contraction phase, the frequency never increased in plt/plt mice, whereas in WT mice it continuously increased to a peak 18 days after immunization. The presence of CCL19/CCL21 during the in vitro stimulation of CD4+ T cells with anti-CD3 plus anti-CD28 significantly enhanced in vitro AICD induction of the restimulated T cells, partially through enhancing expression of Fas ligand. Our results suggest that CCL19/CCL21 produced by stromal cells and antigen-presenting cells regulate CD4+ T-cell immune responses in SLOs by promoting AICD.