Iron Supplementation for Hypoferritinemia-Related Psychological Symptoms in Children and Adolescents.

BACKGROUND: Although some studies have described the association between serum ferritin levels and specific disorders in child and adolescent psychiatry, few have focused on mental status per se with low serum ferritin levels in children and adolescents. This study examined the effects of iron administration on psychological status of children and adolescents with reduced serum ferritin concentration. METHODS: This prospective study evaluated 19 participants aged 6-15 years with serum ferritin levels <30 ng/mL who visited a mental health clinic and received oral iron administration for 12 weeks. The participants were assessed using the Clinical Global Impression Severity (CGI-S), Profile of Mood States 2nd Edition Youth-Short (POMS), Center for Epidemiologic Studies Depression Scale (CES-D), and Pittsburgh Sleep Quality Index (PSQI). In addition to serum ferritin, blood biochemical values such as hemoglobin (Hb) and mean corpuscular volume (MCV) were examined. School attendance was recorded. RESULTS: The most prevalent physical symptoms were fatigability and insomnia. The CGI-S, PSQI, and CES-D scores decreased significantly following iron supplementation, whereas the scores of almost all POMS subscales improved significantly at week 12. No participant had hemoglobin levels <12 g/dL. Serum ferritin concentration increased significantly, whereas Hb and MCV remained unchanged. At baseline, 74% of the participants did not attend school regularly; this number improved to varying degrees by week 12. CONCLUSIONS: Serum ferritin levels would be preferable to be measured in children and adolescents with insomnia and/or fatigability regardless of psychiatric diagnoses or gender. Iron supplementation can improve the hypoferritinemia-related psychological symptoms of children and adolescents, such as poor concentration, anxiety, depression, low energy and/or irritability.

mTORC1 underlies age-related muscle fiber damage and loss by inducing oxidative stress and catabolism.

Aging leads to skeletal muscle atrophy (i.e., sarcopenia), and muscle fiber loss is a critical component of this process. The mechanisms underlying these age-related changes, however, remain unclear. We show here that mTORC1 signaling is activated in a subset of skeletal muscle fibers in aging mouse and human, colocalized with fiber damage. Activation of mTORC1 in TSC1 knockout mouse muscle fibers increases the content of morphologically abnormal mitochondria and causes progressive oxidative stress, fiber damage, and fiber loss over the lifespan. Transcriptomic profiling reveals that mTORC1's activation increases the expression of growth differentiation factors (GDF3, 5, and 15), and of genes involved in mitochondrial oxidative stress and catabolism. We show that increased GDF15 is sufficient to induce oxidative stress and catabolic changes, and that mTORC1 increases the expression of GDF15 via phosphorylation of STAT3. Inhibition of mTORC1 in aging mouse decreases the expression of GDFs and STAT3's phosphorylation in skeletal muscle, reducing oxidative stress and muscle fiber damage and loss. Thus, chronically increased mTORC1 activity contributes to age-related muscle atrophy, and GDF signaling is a proposed mechanism.

SIRPα+ dendritic cells regulate homeostasis of fibroblastic reticular cells via TNF receptor ligands in the adult spleen.

In secondary lymphoid organs, development and homeostasis of stromal cells such as podoplanin (Pdpn)-positive fibroblastic reticular cells (FRCs) are regulated by hematopoietic cells, but the cellular and molecular mechanisms of such regulation have remained unclear. Here we show that ablation of either signal regulatory protein α (SIRPα), an Ig superfamily protein, or its ligand CD47 in conventional dendritic cells (cDCs) markedly reduced the number of CD4+ cDCs as well as that of Pdpn+ FRCs and T cells in the adult mouse spleen. Such ablation also impaired the survival of FRCs as well as the production by CD4+ cDCs of tumor necrosis factor receptor (TNFR) ligands, including TNF-α, which was shown to promote the proliferation and survival of Pdpn+ FRCs. CD4+ cDCs thus regulate the steady-state homeostasis of FRCs in the adult spleen via the production of TNFR ligands, with the CD47-SIRPα interaction in cDCs likely being indispensable for such regulation.

Role of SIRPα in Homeostatic Regulation of T Cells and Fibroblastic Reticular Cells in the Spleen.

Signal regulatory protein α (SIRPα), is an immunoglobulin superfamily protein that is predominantly expressed in macrophages and dendritic cells (DCs), especially CD4+ conventional DCs (cDCs). In this study, we demonstrated that, in addition to the reduced number of CD4+ cDCs, the number of T cells was significantly decreased in the spleen of Sirpa-/- mice, in which full-length of SIRPα protein was systemically ablated. The size of the T cell zone was markedly reduced in the spleen of Sirpa-/- mice. In addition, Sirpa-/- mice revealed a marked reduction of CCL19, CCL21, and IL-7 expression, which are thought to be important for homeostasis of T cells in the spleen. Consistently, the abundance of fibroblastic reticular cells (FRCs), a subset of stromal cells in the T cell zone, was markedly reduced in the spleen of Sirpa-/- mice compared with Sirpaf/f mice. Moreover, we demonstrated that the mRNA expression of Lymphotoxin (LT) α, LTß, and LIGHT was significantly reduced in the spleen of Sirpa-/- mice. These data thus suggest that SIRPα is essential for steady-state homeostasis of T cells and FRCs in the spleen.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

Regulation by gut commensal bacteria of carcinoembryonic antigen-related cell adhesion molecule expression in the intestinal epithelium.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 1 and CEACAM20, immunoglobulin superfamily members, are predominantly expressed in intestinal epithelial cells (IECs) and co-localized at the apical surface of these cells. We here showed that the expression of mouse CEACAM1 and CEACAM20 at both mRNA and protein levels was markedly reduced in IECs of the small intestine by the treatment of mice with antibiotics against Gram-positive bacteria. The expression of both proteins was also decreased in IECs of the small intestine from germ-free mice, compared with that from control specific-pathogen-free mice. Exposure of intestinal organoids to IFN-γ markedly increased the expression of either CEACAM1 or CEACAM20, whereas the exposure to TNF-α increased the expression of the former protein, but not that of the latter. In contrast, the expression of CEACAM20, but not of CEACAM1, in intestinal organoids was markedly increased by exposure to butyrate, a short-chain fatty acid produced by bacterial fermentation in the intestine. Collectively, our results suggest that Gram-positive bacteria promote the mRNA expression of CEACAM1 or CEACAM20 in the small intestine. Inflammatory cytokines or butyrate likely participates in such effects of commensal bacteria.

Dendritic cell SIRPα regulates homeostasis of dendritic cells in lymphoid organs.

Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is expressed predominantly in myeloid lineage cells such as dendritic cells (DCs) or macrophages, mediates cell-cell signaling. In the immune system, SIRPα is thought to be important for homeostasis of DCs, but it remains unclear whether SIRPα intrinsic to DCs is indeed indispensable for such functional role. Thus, we here generated the mice, in which SIRPα was specifically ablated in CD11c(+) DCs (Sirpa(Δ) (DC) ). Sirpa(Δ) (DC) mice manifested a marked reduction of CD4(+) CD8α(-) conventional DCs (cDCs) in the secondary lymphoid organs, as well as of Langerhans cells in the epidermis. Such reduction of cDCs in Sirpa(Δ) (DC) mice was comparable to that apparent with the mice, in which SIRPα was systemically ablated. Expression of SIRPα in DCs was well correlated with that of either endothelial cell-selective adhesion molecule (ESAM) or Epstein-Barr virus-induced molecule 2 (EBI2), both of which were also implicated in the regulation of DC homeostasis. Indeed, ESAM(+) or EBI2(+) cDCs were markedly reduced in the spleen of Sirpa(Δ) (DC) mice. Thus, our results suggest that SIRPα intrinsic to CD11c(+) DCs is essential for homeostasis of cDCs in the secondary lymphoid organs and skin.

Role of the protein tyrosine phosphatase Shp2 in homeostasis of the intestinal epithelium.

Protein tyrosine phosphorylation is thought to be important for regulation of the proliferation, differentiation, and rapid turnover of intestinal epithelial cells (IECs). The role of protein tyrosine phosphatases in such homeostatic regulation of IECs has remained largely unknown, however. Src homology 2-containing protein tyrosine phosphatase (Shp2) is a ubiquitously expressed cytoplasmic protein tyrosine phosphatase that functions as a positive regulator of the Ras-mitogen-activated protein kinase (MAPK) signaling pathway operative downstream of the receptors for various growth factors and cytokines, and it is thereby thought to contribute to the regulation of cell proliferation and differentiation. We now show that mice lacking Shp2 specifically in IECs (Shp2 CKO mice) develop severe colitis and die as early as 3 to 4 weeks after birth. The number of goblet cells in both the small intestine and colon of Shp2 CKO mice was markedly reduced compared with that for control mice. Furthermore, Shp2 CKO mice showed marked impairment of both IEC migration along the crypt-villus axis in the small intestine and the development of intestinal organoids from isolated crypts. The colitis as well as the reduction in the number of goblet cells apparent in Shp2 CKO mice were normalized by expression of an activated form of K-Ras in IECs. Our results thus suggest that Shp2 regulates IEC homeostasis through activation of Ras and thereby protects against the development of colitis.

Tyrosine Phosphorylation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 20 and Its Functional Role.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20 is an immunoglobulin-superfamily transmembrane protein that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region. However, the mechanism for tyrosine phosphorylation of, or the physiological function of, this protein remains largely unknown. Here we have shown that CEACAM20 is indeed tyrosine-phosphorylated by either treatment with pervanadate or forced expression of c-Src. In addition, Tyr522, Tyr559 or Tyr570, the latter two of which are within the ITAM, is likely important for such tyrosine phosphorylation. Forced expression of Myc-tagged wild-type CEACAM20 promoted the phagocytic activity of cultured cells for microbeads coupled with anti-Myc antibodies. By contrast, such phagocytic activity was markedly reduced when a mutant form of CEACAM20, in which Tyr559 and Tyr570 were substituted with phenylalanine, was expressed. Furthermore, the CEACAM20-mediated phagocytic activity was markedly prevented by the treatment with an inhibitor for either Src family kinases (SFKs), Syk, phosphoinositide 3-kinase (PI3K) or phospholipase C-γ (PLCγ). Inhibition of actin polymerization by Cytochalasin D significantly inhibited the CEACAM20-mediated phagocytosis. These results thus suggest that tyrosine phosphorylation of CEACAM20 likely promotes phagocytic activity of the cells. The CEACAM20-mediated phagocytic activity requires the activation of SFKs, Syk, PI3K or PLCγ.

Dendritic cell-specific ablation of the protein tyrosine phosphatase Shp1 promotes Th1 cell differentiation and induces autoimmunity.

Dendritic cells (DCs) promote immune responses to foreign Ags and immune tolerance to self-Ags. Deregulation of DCs is implicated in autoimmunity, but the molecules that regulate DCs to protect against autoimmunity have remained unknown. In this study, we show that mice lacking the protein tyrosine phosphatase Shp1 specifically in DCs develop splenomegaly associated with more CD11c(+) DCs. Splenic DCs from the mutant mice showed upregulation of CD86 and CCR7 expression and of LPS-induced production of proinflammatory cytokines. The mice manifested more splenic Th1 cells, consistent with the increased ability of their DCs to induce production of IFN-γ by Ag-specific T cells in vitro. The number of splenic CD5(+)CD19(+) B-1a cells and the serum concentrations of Igs M and G2a were also increased in the mutant mice. Moreover, aged mutant mice developed glomerulonephritis and interstitial pneumonitis together with increased serum concentrations of autoantibodies. Shp1 is thus a key regulator of DC functions that protects against autoimmunity.

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain.

Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37 °C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30 °C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.

Src family kinases: modulators of neurotransmitter receptor function and behavior.

Src family kinases (SFKs) are non-receptor-type protein tyrosine kinases that were originally identified as the products of proto-oncogenes and were subsequently implicated in the regulation of cell proliferation and differentiation in the developing mammalian brain. Recent studies using transgenic mouse models have demonstrated that SFKs that are highly expressed in the adult brain regulate neuronal plasticity and behavior through tyrosine phosphorylation of key substrates such as neurotransmitter receptors. Here, we provide an overview of these recent studies, as well as discussing how modulation of the endocytosis of neurotransmitter receptors by SFKs contributes, in part, to this regulation. Deregulation of SFK-dependent tyrosine phosphorylation of such substrates might underlie certain brain disorders.

Signal regulatory protein α regulates the homeostasis of T lymphocytes in the spleen.

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell-cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4(+) T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin ß receptor, as well as the in vivo response to lymphotoxin ß receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.

Essential roles of SIRPα in homeostatic regulation of skin dendritic cells.

Signal regulatory protein α (SIRPα) is an immunoglobulin superfamily protein that is predominantly expressed in dendritic cells (DCs). Its cytoplasmic region binds SHP-1 or SHP-2 protein tyrosine phosphatases, while its extracellular region interacts with CD47, another immunoglobulin superfamily protein, constituting cell-cell signaling. SIRPα was previously shown to be important for development of contact hypersensitivity, likely as a result of its positive regulation of the priming by DCs of CD4(+) T cells. However, the mechanism by which SIRPα regulates DC functions remains unknown. Here we found that the number of I-A(+) cells, which represent migratory DCs such as Langerhans cells (LCs) or dermal DCs from the skin, in the peripheral lymph nodes (LNs) was markedly decreased in mice expressing a mutant form of SIRPα that lacks the cytoplasmic region compared with that of wild-type (WT) mice. In addition, an increase of fluorescein isothiocyanate (FITC)-bearing I-A(+) cells in the draining lymph nodes (LNs) after skin-painting with FITC was markedly blunted in SIRPα mutant mice. However, migratory ability, as well as expression of CCR7, of bone marrow-derived DCs prepared from SIRPα mutant mice were not impaired. By contrast, the number of I-A(+) LCs in the epidermis of SIRPα mutant mice was markedly decreased compared with that of WT mice. In addition, the mRNA expression of transforming growth factor-ß receptor II in LCs of SIRPα mutant mice was markedly decreased compared with that of WT mice. These results suggest that SIRPα is important for homeostasis of LCs in the skin, as well as of migratory DCs in the LNs, but unlikely for migration of these cells from the skin to draining LNs.

Role of SIRPα in regulation of mucosal immunity in the intestine.

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c(+) CD11b(+) LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady-state homeostasis of mucosal immunity in the intestine, the flagellin-stimulated production of IL-17 or interferon (IFN)-γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild-type cells. Moreover, the flagellin-induced production of IL-6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL-10 deficiency. Our data thus suggest that SIRPα expressed on CD11c(+) LP cells is important for the production of IL-17 or IFN-γ in the LP as well as for the development of colitis induced by IL-10 deficiency.

Requirement of SIRPα for protective immunity against Leishmania major.

Signal regulatory protein α (SIRPα) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Wild-type (WT) C57BL/6 mice are known to be resistant to Leishmania major infection. We here found that C57BL/6 mice that express a mutant version of SIRPα lacking most of the cytoplasmic region manifested increased susceptibility to L. major infection, characterized by the marked infiltration of inflammatory cells in the infected lesions. The numbers of the parasites in footpads, draining lymph nodes and spleens were also markedly increased in the infected SIRPα mutant mice, compared with those for the infected WT mice. In addition, soluble leishmanial antigen-induced production of IFN-γ by splenocytes of the infected SIRPα mutant mice was markedly reduced. By contrast, the ability of macrophages of SIRPα mutant mice to produce nitric oxide in response to IFN-γ was almost equivalent to that of macrophages from WT mice. These results suggest that SIRPα is indispensable for protective immunity against L. major by the induction of Th1 response.

Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein alpha (SIRPalpha) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPalpha that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

Regulation by SIRPα of dendritic cell homeostasis in lymphoid tissues.

The molecular basis for regulation of dendritic cell (DC) development and homeostasis remains unclear. Signal regulatory protein α (SIRPα), an immunoglobulin superfamily protein that is predominantly expressed in DCs, mediates cell-cell signaling by interacting with CD47, another immunoglobulin superfamily protein. We now show that the number of CD11c(high) DCs (conventional DCs, or cDCs), in particular, that of CD8-CD4+ (CD4+) cDCs, is selectively reduced in secondary lymphoid tissues of mice expressing a mutant form of SIRPα that lacks the cytoplasmic region. We also found that SIRPα is required intrinsically within cDCs or DC precursors for the homeostasis of splenic CD4+ cDCs. Differentiation of bone marrow cells from SIRPα mutant mice into DCs induced by either macrophage-granulocyte colony-stimulating factor or Flt3 ligand in vitro was not impaired. Although the accumulation of the immediate precursors of cDCs in the spleen was also not impaired, the half-life of newly generated splenic CD4+ cDCs was markedly reduced in SIRPα mutant mice. Both hematopoietic and nonhematopoietic CD47 was found to be required for the homeostasis of CD4+ cDCs and CD8-CD4- (double negative) cDCs in the spleen. SIRPα as well as its ligand, CD47, are thus important for the homeostasis of CD4+ cDCs or double negative cDCs in lymphoid tissues.