Papers - Moriwaki, Yasuhiro

IgG-Binding Peptidomimetic Mixed-Charge Polymer-Modified Resins for Chromatographic Purification of Antibodies.

Deura K, Sakama A, Moriwaki Y, Citterio D, Hiruta Y.

ACS Appl Mater Interfaces. (American Chemical Society (United States))   2024.11

Research paper (scientific journal), Joint Work, Accepted,  ISSN  19448244

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The process of antibody purification using Fc affinity ligands such as protein A, G, and L faces several challenges including high cost, low stability, and loss of antibody activity due to harsh elution conditions. Here, we describe a chromatographic purification of antibodies utilizing a pH-responsive mixed-charge polymer that mimics the IgG-binding peptide (Z34C) derived from the B domain of protein A. The protein A mimetic resins were prepared by modifying the surface of a TOYOPEARL, methacrylate resin with a polymer that mimics the amino acid sequence of Z34C and the functions of histidine and acidic and neutral amino acids using histamine methacrylamide (HisMA), methacrylic acid, and neutral monomers. The therapeutic monoclonal antibody (mAb), rituximab, was retained on the column at pH 7 and eluted under mildly acidic conditions at pH 5 using a protein A mimetic resin (HisMA20-EEMA) optimized for antibody interaction. The injected antibodies were selectively captured on the column by hydrophobic and electrostatic interactions with the protein A mimetic polymer under neutral conditions and eluted by electrostatic repulsion under acidic conditions. The HisMA20-EEMA column successfully purified mAbs from mixtures with BSA, mouse ascites fluid, and hybridoma cell culture supernatant. In addition, the HisMA20-EEMA column consistently achieved 90% antibody recovery in 100 consecutive purifications from cell culture supernatant. The antibody purification method presented in this study is low cost, highly durable, easy to synthesize, and allows for mild elution conditions. The results demonstrate that the approach of mimicking IgG-binding peptides with mixed-charge polymers is useful for the development of column packing materials for antibody purification.

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Suppression of Neuroinflammation by Coffee Component Pyrocatechol via Inhibition of NF-κB in Microglia

Murata T, Tago K, Miyata K, Moriwaki Y, Misawa H, Kobata K, Nakazawa Y, Tamura H, Funakoshi-Tago M.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  25 ( 1 )  2023.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  16616596

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According to numerous studies, it has been epidemiologically suggested that habitual coffee intake seems to prevent the onset of neurodegenerative diseases. In this study, we hypothesized that coffee consumption suppresses neuroinflammation, which is closely related to the development of neurodegenerative diseases. Using microglial BV-2 cells, we first found that the inflammatory responses induced by lipopolysaccharide (LPS) stimulation was diminished by both coffee and decaffeinated coffee through the inhibition of an inflammation-related transcription factor, nuclear factor-κB (NF-κB). Pyrocatechol, a component of roasted coffee produced by the thermal decomposition of chlorogenic acid, also exhibited anti-inflammatory activity by inhibiting the LPS-induced activation of NF-κB. Finally, in an inflammation model using mice injected with LPS into the cerebrum, we observed that intake of pyrocatechol as well as coffee decoctions drastically suppressed the accumulation of microglia and the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), CCL2, and CXCL1 in the inflammatory brain. These observations strongly encourage us to hypothesize that the anti-inflammatory activity of pyrocatechol as well as coffee decoction would be useful for the suppression of neurodegeneration and the prevention of the onsets of Alzheimer’s (AD) and Perkinson’s diseases (PD).

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GTS-21 Enhances Regulatory T Cell Development from T Cell Receptor-Activated Human CD4⁺ T Cells Exhibiting Varied Levels of CHRNA7 and CHRFAM7A Expression

Mashimo M., Fujii T., Ono S., Moriwaki Y., Misawa H., Azami T., Kasahara T., Kawashima K.

International Journal of Molecular Sciences (International Journal of Molecular Sciences)  24 ( 15 )  2023.07

Research paper (scientific journal), Accepted,  ISSN  16616596

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Immune cells such as T cells and macrophages express α7 nicotinic acetylcholine receptors (α7 nAChRs), which contribute to the regulation of immune and inflammatory responses. Earlier findings suggest α7 nAChR activation promotes the development of regulatory T cells (Tregs) in mice. Using human CD4+ T cells, we investigated the mRNA expression of the α7 subunit and the human-specific dupα7 nAChR subunit, which functions as a dominant-negative regulator of ion channel function, under resting conditions and T cell receptor (TCR)-activation. We then explored the effects of the selective α7 nAChR agonist GTS-21 on proliferation of TCR-activated T cells and Treg development. Varied levels of mRNA for both the α7 and dupα7 nAChR subunits were detected in resting human CD4+ T cells. mRNA expression of the α7 nAChR subunit was profoundly suppressed on days 4 and 7 of TCR-activation as compared to day 1, whereas mRNA expression of the dupα7 nAChR subunit remained nearly constant. GTS-21 did not alter CD4+ T cell proliferation but significantly promoted Treg development. These results suggest the potential ex vivo utility of GTS-21 for preparing Tregs for adoptive immunotherapy, even with high expression of the dupα7 subunit.

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Regulation of immune functions by non-neuronal acetylcholine (ACh) via muscarinic and nicotinic ACh receptors.

Mashimo M, Moriwaki Y, Misawa H, Kawashima K, Fujii T.

International Journal of Molecular Sciences (MDPI)  22 ( 13 ) 6818 2021.06

Research paper (scientific journal), Joint Work, Accepted,  ISSN  16616596

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Acetylcholine (ACh) is the classical neurotransmitter in the cholinergic nervous system. However, ACh is now known to regulate various immune cell functions. In fact, T cells, B cells, and macrophages all express components of the cholinergic system, including ACh, muscarinic, and nicotinic ACh receptors (mAChRs and nAChRs), choline acetyltransferase, acetylcholinesterase, and choline transporters. In this review, we will discuss the actions of ACh in the immune system. We will first briefly describe the mechanisms by which ACh is stored in and released from immune cells. We will then address Ca2+ signaling pathways activated via mAChRs and nAChRs on T cells and B cells, highlighting the importance of ACh for the function of T cells, B cells, and macrophages, as well as its impact on innate and acquired (cellular and humoral) immunity. Lastly, we will discuss the effects of two peptide ligands, secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1) and hippocampal cholinergic neurostimulating peptide (HCNP), on cholinergic activity in T cells. Overall, we stress the fact that ACh does not function only as a neurotransmitter; it impacts immunity by exerting diverse effects on immune cells via mAChRs and nAChRs.

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New pathways for the skin´s stress response: the cholinergic neuropeptide SLURP-1 can activate mast cells and alter cytokine production in mice.

Ertle CM, Rommel FR, Tumala S, Moriwaki Y, Klein J, Kruse J, Gieler U, Peters EMJ.

Frontiers in Immunology (Frontiers in Immunology)  12   631881 2021.03

Research paper (scientific journal), Joint Work, Accepted

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Background: The alpha7 nicotinic acetylcholine receptor (Chrna7) plays an essential anti-inflammatory role in immune homeostasis and was recently found on mast cells (MC). Psychosocial stress can trigger MC hyperactivation and increases pro-inflammatory cytokines in target tissues such as the skin. If the cholinergic system (CS) and Chrna7 ligands play a role in these cascades is largely unknown. Objective: To elucidate the role of the CS in the response to psychosocial stress using a mouse-model for stress-triggered cutaneous inflammatory circuits. Methods: Key CS markers (ACh, Ch, SLURP-1, SLURP-2, Lynx1, Chrm3, Chrna7, Chrna9, ChAT, VAChT, Oct3, AChE, and BChE) in skin and its MC (sMC), MC activation, immune parameters (TNFα, IL1β, IL10, TGFβ, HIF1α, and STAT3) and oxidative stress were analyzed in skin from 24 h noise-stressed mice and in cultured MC (cMC) from C57BL/6 or Chrna7-Knockout mice. Results: First, Chrna7 and SLURP-1 mRNA were exclusively upregulated in stressed skin. Second, histomorphometry located Chrna7 and SLURP-1 in nerves and sMC and demonstrated upregulated contacts and increased Chrna7+ sMC in stressed skin, while 5 ng/mL SLURP-1 degranulated cMC. Third, IL1β+ sMC were high in stressed skin, and while SLURP-1 alone had no significant effect on cMC cytokines, it upregulated IL1β in cMC from Chrna7-KO and in IL1β-treated wildtype cMC. In addition, HIF1α+ sMC were high in stressed skin and Chrna7-agonist AR-R 17779 induced ROS in cMC while SLURP-1 upregulated TNFα and IL1β in cMC when HIF1α was blocked. Conclusions: These data infer that the CS plays a role in the regulation of stress-sensitive inflammatory responses but may have a surprising pro-inflammatory effect in healthy skin, driving IL1β expression if SLURP-1 is involved.

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Endogenous neurotoxin-like protein Ly6H inhibits alpha7 nicotinic acetylcholine receptor currents at the plasma membrane.

Moriwaki Y*, Kubo N, Watanabe M, Asano S, Shinoda T, Sugino T, Ichikawa D, Tsuji S, Kato F, Misawa H.

Scientific Reports (Scientific Reports)  10 ( 1 ) 11996 2020.07

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted

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© 2020, The Author(s). α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the central nervous system and regarded as potential therapeutic targets for neurodegenerative conditions, such as Alzheimer’s disease and schizophrenia. Yet, despite the assumed pathophysiological importance of the α7 nAChR, molecular physiological characterization remains poorly advanced because α7 nAChR cannot be properly folded and sorted to the plasma membranes in most mammalian cell lines, thus preventing the analyses in heterologous expression system. Recently, ER-resident membrane protein NACHO was discovered as a strong chaperone for the functional expression of α7 nAChR in non-permissive cells. Ly6H, a brain-enriched GPI-anchored neurotoxin-like protein, was reported as a novel modulator regulating intracellular trafficking of α7 nAChR. In this study, we established cell lines that stably and robustly express surface α7 nAChR by introducing α7 nAChR, Ric-3, and NACHO cDNA into HEK293 cells (Triple α7 nAChR/RIC-3/NACHO cells; TARO cells), and re-evaluated the function of Ly6H. We report here that Ly6H binds with α7 nAChRs on the cell membrane and modulates the channel activity without affecting intracellular trafficking of α7 nAChR.

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Minireview: Divergent roles of α7 nicotinic acetylcholine receptors expressed on antigen-presenting cells and CD4⁺ T cells in the regulation of T cell differentiation

Mashimo M., Fujii T., Ono S., Moriwaki Y., Misawa H., Kawashima K.

International Immunopharmacology (International Immunopharmacology)  82   106306 2020.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  15675769

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© 2020 Elsevier B.V. α7 nAChRs expressed on immune cells regulate antigen-specific antibody and proinflammatory cytokine production. Using spleen cells from ovalbumin (OVA)-specific T cell receptor transgenic DO11.10 mice and the α7 nAChR agonist GTS-21, investigation of (1) antigen processing-dependent and (2) -independent, antigen presenting cell (APC)-dependent, naïve CD4+ T cell differentiation, as well as (3) non-specific APC-independent, anti-CD3/CD28 mAbs-induced CD4+ T cell differentiation, revealed the differential roles of α7 nAChRs expressed on T cells and APCs in the regulation of CD4+ T cell differentiation. GTS-21 suppressed OVA-induced antigen processing- and APC-dependent differentiation into regulatory T cells (Tregs) and effector T cells (Th1, Th2 and Th17) without affecting OVA uptake or cell viability. By contrast, GTS-21 upregulated OVA peptide-induced antigen processing-independent T cell differentiation into all lineages. During anti-CD3/CD28 mAbs-induced T cell differentiation in the presence of polarizing cytokines, GTS-21 promoted wild-type T cell differentiation into all lineages, but did not affect α7 nAChR-deficient T cell differentiation. These results demonstrate (1) that α7 nAChRs on APCs downregulate T cell differentiation by inhibiting antigen processing and thereby interfering with antigen presentation; and (2) that α7 nAChRs on T cells upregulate differentiation into Tregs and effector T cells. Thus, the divergent roles of α7 nAChRs on APCs and T cells likely regulate the intensity of immune responses. These findings suggest the possibility of using α7 nAChR agonists to harvest greater numbers of Tregs and Th1 and Th2 cells for adoptive immune therapies for treatment of autoimmune diseases and cancers.

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Distinct roles of α7 nAChRs in antigen-presenting cells and CD4⁺ T cells in the regulation of T cell differentiation

Mashimo M., Komori M., Matsui Y., Murase M., Fujii T., Takeshima S., Okuyama H., Ono S., Moriwaki Y., Misawa H., Kawashima K.

Frontiers in Immunology (Frontiers in Immunology)  10 ( MAY ) 1102 2019.05

Research paper (scientific journal), Joint Work, Accepted

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Copyright © 2019 Mashimo, Komori, Matsui, Murase, Fujii, Takeshima, Okuyama, Ono, Moriwaki, Misawa and Kawashima. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. It is now apparent that immune cells express a functional cholinergic system and that α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating T cell differentiation and the synthesis of antigen-specific antibodies and proinflammatory cytokines. Here, we investigated the specific function α7 nAChRs on T cells and antigen presenting cells (APCs) by testing the effect of GTS-21, a selective α7 nAChR agonist, on differentiation of CD4+ T cells from ovalbumin (OVA)-specific TCR transgenic DO11.10 mice activated with OVA or OVA peptide323−339 (OVAp). GTS-21 suppressed OVA-induced antigen processing-dependent development of CD4+ regulatory T cells (Tregs) and effector T cells (Th1, Th2, and Th17). By contrast, GTS-21 up-regulated OVAp-induced antigen processing-independent development of CD4+ Tregs and effector T cells. GTS-21 also suppressed production of IL-2, IFN-γ, IL-4, IL-17, and IL-6 during OVA-induced activation but, with the exception IL-2, enhanced their production during OVAp-induced activation. In addition, during antigen-nonspecific, APC-independent anti-CD3/CD28 antibody-induced CD4+ polyclonal T cell activation in the presence of respective polarizing cytokines, GTS-21 promoted development of all lineages, which indicates that GTS-21 also acts via α7 nAChRs on T cells. These results suggest 1) that α7 nAChRs on APCs suppress CD4+ T cell activation by interfering with antigen presentation through inhibition of antigen processing; 2) that α7 nAChRs on CD4+ T cells up-regulate development of Tregs and effector T cells; and that α7 nAChR agonists and antagonists could be potentially useful agents for immune response modulation and enhancement.

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Identification of mesothelioma-specific sialylated epitope recognized with monoclonal antibody SKM9-2 in a mucin-like membrane protein HEG1.

Matsuura R. Kaji H, Tomioka A, Sato T, Narimatsu H, Moriwaki Y, Misawa H, Imai K, Tsuji S.

Sci Rep. (Scientific Reports)  8 ( 1 ) 14251 2018.09

Research paper (scientific journal), Joint Work, Accepted

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© 2018, The Author(s). The anti-mesothelioma mAb SKM9-2 recognizes the sialylated protein HEG homolog 1 (HEG1). HEG1 is a 400 kDa mucin-like membrane protein found on mesothelioma. SKM9-2 can detect mesothelioma more specifically and sensitively than other antibodies against current mesothelioma markers; therefore, SKM9-2 would be likely useful for the precise detection and diagnosis of malignant mesothelioma. In the present study, we investigated the epitope of SKM9-2. We analyzed the binding of SKM9-2 to truncated HEG1 and candidate epitope-fused glycosylphosphatidylinositol-anchor proteins. The epitope of SKM9-2 was identified as an O-glycosylated region, 893-SKSPSLVSLPT-903, in HEG1. An alanine scanning assay of the epitope showed that SKM9-2 bound to a simple epitope in HEG1, and the SKxPSxVS sequence within the epitope was essential for SKM9-2 recognition. Mass spectrometry analysis and lectin binding analysis of soluble epitope peptides indicated that the SKM9-2 epitope, in which Ser897 was not glycosylated, contained two disialylated core 1 O-linked glycan-modified serine residues, Ser893 and Ser900. Neuraminidase treatment analysis also confirmed that the epitope in mesothelioma cells contained a similar glycan modification. The specific detection of mesothelioma with SKM9-2 can thus be performed by the recognition of sialylated glycan modification in the specific region of HEG1.

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Dissociation of blood-brain barrier disruption and disease manifestation in an aquaporin-4-deficient mouse model of amyotrophic lateral sclerosis.

Watanabe-Matsumoto S, Moriwaki Y, Okuda T, Ohara S, Yamanaka K, Abe Y, Yasui M, Misawa H.

Neurosci Res. (Neuroscience Research)  133   48 - 57 2018.08

Research paper (scientific journal), Joint Work, Accepted,  ISSN  01680102

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© 2017 Elsevier Ireland Ltd and Japan Neuroscience Society Aquaporin-4 (AQP4) is abundantly expressed in the central nervous system and is involved in the water balance in the cellular environment. Previous studies have reported that AQP4 expression is upregulated in rat models of amyotrophic lateral sclerosis (ALS), a fatal disease that affects motor neurons in the brain and spinal cord. In this study, we report that astrocytic AQP4 overexpression is evident during the course of disease in the spinal cord of an ALS mouse model, as well as in tissue from patients with ALS. AQP4 overexpression appears to be specifically associated with ALS because it was not induced by other experimental manipulations that produced acute or chronic gliosis. In order to examine the contribution of AQP4 to ALS disease development, we crossed AQP4 knockout mice with a mouse model of ALS. Significant improvement in blood-brain barrier (BBB) permeability was observed in the AQP4-deficient ALS mouse model. However, the time to disease onset and total lifespan were reduced in the AQP4-deficient ALS mouse model. The contradictory results suggest that changes in AQP4 may be context-dependent and further studies are required to understand the precise contribution of brain water balance in ALS.

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SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosome.

Moriwaki Y*, Ohno Y, Ishii T, Takamura Y, Kita Y, Watabe K, Sango K, Tsuji S, Misawa H.

PLoS One. 13 ( 6 ) e0199829 2018.06

Research paper (scientific journal), Joint Work, Lead author, Corresponding author, Accepted

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Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes.

Komine O, Yamashita H, Fujimori-Tonou N, Koike M, Jin S, Moriwaki Y, Endo F, Watanabe S, Uematsu S, Akira S, Uchiyama Y, Takahashi R, Misawa H, Yamanaka K.

Cell Death Differ. (Cell Death and Differentiation)  25 ( 12 ) 2130 - 2146 2018.03

Research paper (scientific journal), Joint Work, Accepted,  ISSN  13509047

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© 2018, ADMC Associazione Differenziamento e Morte Cellulare. There is compelling evidence that glial-immune interactions contribute to the progression of neurodegenerative diseases. The adaptive immune response has been implicated in disease processes of amyotrophic lateral sclerosis (ALS), but it remains unknown if innate immune signaling also contributes to ALS progression. Here we report that deficiency of the innate immune adaptor TIR domain-containing adaptor inducing interferon-β (TRIF), which is essential for certain Toll-like receptor (TLR) signaling cascades, significantly shortens survival time and accelerates disease progression of ALS mice. While myeloid differentiation factor 88 (MyD88) is also a crucial adaptor for most TLR signaling pathways, MyD88 deficiency had only a marginal impact on disease course. Moreover, TRIF deficiency reduced the number of natural killer (NK), NK-T-lymphocytes, and CD8-T cells infiltrating into the spinal cord of ALS mice, but experimental modulation of these populations did not substantially influence survival time. Instead, we found that aberrantly activated astrocytes expressing Mac2, p62, and apoptotic markers were accumulated in the lesions of TRIF-deficient ALS mice, and that the number of aberrantly activated astrocytes was negatively correlated with survival time. These findings suggest that TRIF pathway plays an important role in protecting a microenvironment surrounding motor neurons by eliminating aberrantly activated astrocytes.

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Expression and Function of the Cholinergic System in Immune Cells.

Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K.

Front Immunol. 8   1085 2017.09

Research paper (scientific journal), Joint Work, Accepted

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Physiological functions of the cholinergic system in immune cells.

Fujii T, Mashimo M, Moriwaki Y, Misawa H, Ono S, Horiguchi K, Kawashima K.

J Pharmacol Sci. 134 ( 1 ) 1 - 21 2017.05

Research paper (scientific journal), Accepted

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HEG1 is a novel mucin-like membrane protein that serves as a diagnostic and therapeutic target for malignant mesothelioma.

Tsuji S, Washimi K, Kageyama T, Yamashita M, Yoshihara M, Matsuura R, Yokose T, Kameda Y, Hayashi H, Morohoshi T, Tsuura Y, Yusa T, Sato T, Togayachi A, Narimatsu H, Nagasaki T, Nakamoto K, Moriwaki Y, Misawa H, Hiroshima K, Miyagi Y, Imai K.

Sci Rep. 7   45768 2017.03

Research paper (scientific journal), Joint Work, Accepted

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A bis-malonic acid fullerene derivative significantly suppressed IL-33-induced IL-6 expression by inhibiting NF-κB activation.

Funakoshi-Tago M, Miyagawa Y, Ueda F, Mashino T, Moriwaki Y, Tago K, Kasahara T, Tamura H.

Int Immunopharmacol. 40   254 - 264 2016.09

Research paper (scientific journal), Joint Work, Accepted

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Reappraisal of VAChT-Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers.

Misawa H, Inomata D, Kikuchi M, Maruyama S, Moriwaki Y, Okuda T, Nukina N, Yamanaka T.

Genesis 54 ( 11 ) 568 - 572 2016.09

Research paper (scientific journal), Joint Work, Accepted

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Selective Expression of Osteopontin in ALS-resistant Motor Neurons is a Critical Determinant of Late Phase Neurodegeneration Mediated by Matrix Metalloproteinase-9.

Morisaki Y, Niikura M, Watanabe M, Onishi K, Tanabe S, Moriwaki Y, Okuda T, Ohara S, Murayama S, Takao M. Uchida S, Yamanaka K, Misawa H.

Sci. Rep. (Nature Publishing Group.)  6   27354 2016.06

Research paper (scientific journal), Accepted

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IL-22/STAT3-induced increases in SLURP1 expression within psoriatic lesions exerts antimicrobial effects against Staphylococcus aureus.

Moriwaki Y*, Takada K, Nagasaki T, Kubo N, Ishii T, Kose K, Kageyama T, Tsuji S, Kawashima K, Misawa H.

PLoS One. 10 ( 10 ) e0140750 2015.10

Research paper (scientific journal), Joint Work, Accepted

Transcriptional regulation of SLURP2, a psoriasis-associated gene, is under control of IL-22 in the skin.

Moriwaki Yasuhiro*, Takada Kiyoko, Tsuji Shoutaro, Kawashima Koichiro, Misawa Hidemi.

International Immunopharmacology 29 ( 1 ) 71 - 75 2015.02

Research paper (scientific journal), Joint Work,  ISSN  1567-5769

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<p>A novel nicotinic acetylcholine (ACh) receptor (nAChR)-mediated transduction pathway, regulating keratinocyte function, has been elucidated in studies of secreted mammalian Ly6/urokinase plasminogen activator receptor-related protein (SLURP)-1 and -2. SLURPs are members of Ly6/neurotoxin superfamily (Ly6SF) of proteins containing the unique three-finger domain in their three-dimensional structure. Some endogenously expressed Ly6SF proteins (such as LYNX1, SLURP-1, and SLURP-2) modulate the function of nAChR, either as allosteric and/or orthosteric modulators, or as antagonists. Although the expression and functions of SLURP-1 and SLURP-2 in keratinocytes are well documented, the expression and the modes of action of LYNX1 in keratinocytes are unknown. Additionally, a particular hybrid transcript, LYNX1-SLURP2, which contains both LYNX1 and SLURP-2 sequences, with unknown function, has been reported. Furthermore, although SLURP2 is a gene strongly induced in psoriatic skin lesions, the mechanisms controlling SLURP2 expression are largely unknown. To better understand the function of nAChRs in keratinocytes, we investigated the expression profiles of LYNX1, LYNX1-SLURP-2, and SLURP-2 in keratinocytes under various inflammatory conditions. We found that keratinocytes express LYNX1 and SLURP2, but not LYNX1-SLURP2, at mRNA and protein levels. IL-22 treatment increased SLURP2 expression in keratinocytes, but this effect was completely abolished by IFN-γ. Furthermore, the IL-22-induced up-regulation of SLURP2 was completely suppressed by the inhibitor or siRNA for STAT3, a major transcriptional factor downstream of IL-22. These findings provide new insights into the nAChR-mediated regulatory mechanism of SLURP-2 expression in keratinocytes.</p>

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Non-neuronal cholinergic system in regulation of immune function with a focus on α7 nAChRs.

Kawashima Koichiro, Fujii Takeshi, Moriwaki Yasuhiro, Misawa Hidemi, Horiguchi Kazuhide.

International Immunopharmacology 29 ( 1 )  2015.02

Research paper (scientific journal), Joint Work,  ISSN  1567-5769

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<p>In 1929, Dale and Dudley described the first reported natural occurrence of acetylcholine (ACh) in an animal's body. They identified this ACh in the spleens of horses and oxen, which we now know suggests possible involvement of ACh in the regulation of lymphocyte activity and immune function. However, the source and function of splenic ACh were left unexplored for several decades. Recent studies on the source of ACh in the blood revealed ACh synthesis catalyzed by choline acetyltransferase (ChAT) in CD4&lt;sup&gt;+&lt;/sup&gt; T cells. T and B cells, macrophages and dendritic cells (DCs) all express all five muscarinic ACh receptor subtypes (mAChRs) and several subtypes of nicotinic AChRs (nAChRs), including α7 nAChRs. Stimulation of these mAChRs and nAChRs by their respective agonists causes functional and biochemical changes in the cells. Using AChR knockout mice, we found that M&lt;inf&gt;1&lt;/inf&gt;/M&lt;inf&gt;5&lt;/inf&gt; mAChR signaling up-regulates IgG&lt;inf&gt;1&lt;/inf&gt; and pro-inflammatory cytokine production, while α7 nAChR signaling has the opposite effect. These findings suggest that ACh synthesized by T cells acts in an autocrine/paracrine fashion at AChRs on various immune cells to modulate immune function. In addition, an endogenous allosteric and/or orthosteric α7 nAChR ligand, SLURP-1, facilitates functional development of T cells and increases ACh synthesis via up-regulation of ChAT mRNA expression. SLURP-1 is expressed in CD205&lt;sup&gt;+&lt;/sup&gt; DCs residing in the tonsil in close proximity to T cells, macrophages and B cells. Collectively, these findings suggest that ACh released from T cells along with SLURP-1 regulates cytokine production by activating α7 nAChRs on various immune cells, thereby facilitating T cell development and/or differentiation, leading to immune modulation.</p>

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T cells downregulate macrophage TNF production by IRAK1-mediated IL-10 expression and control innate hyperinflammation.

Inoue M, Arikawa T, Chen YH, Moriwaki Y, Price MS, Brown M, Perfect JR, Shinohara ML.

Proc Natl Acad Sci U S A 111 ( 14 ) 5295 - 5300 2014.04

Research paper (scientific journal), Joint Work, Accepted

SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, is expressed in CD205+ dendritic cells in human tonsils and potentiates lymphocytic cholinergic activity.

Fujii T, Horiguchi K, Sunaga H, Moriwaki Y, Misawa H, Kasahara T, Tsuji S, Kawashima K.

J Neuroimmunol 267 ( 1-2 ) 43 - 49 2014.02

Research paper (scientific journal), Joint Work, Accepted

Effect of secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor- related peptide-1 (SLURP-1) on airway epithelial cells.

Narumoto O, Nikura Y, Ishii S, Morihara H, Okashiro S, Nakahari T, Nakano T, Matsumura H, Shimamoto C, Moriwaki Y, Misawa H, Yamashita N, Nagase T, Kawashima K, Yamashita N.

Biochem Biophys Res Commun. 438 ( 1 ) 175-9 2013.08

Research paper (scientific journal), Joint Work, Accepted

Localization of acetylcholine-related molecules in the retina: implication of the communication from photoreceptor to retinal pigment epithelium.

Matsumoto H, Shibasaki K, Uchigashima M, Koizumi A, Kurachi M, Moriwaki Y, Misawa H, Kawashima K, Watanabe M, Kishi S, Ishizaki Y.

PLoS One. 7 ( 8 ) e48241 2012.08

Research paper (scientific journal), Joint Work, Accepted

α-Synuclein BAC transgenic mice as a model for Parkinson's disease manifested decreased anxiety-like behavior and hyperlocomotion.

Yamakado H, Moriwaki Y, Yamasaki N, Miyakawa T, Kurisu J, Uemura K, Inoue H, Takahashi M, Takahashi R.

Neurosci Res 73 ( 2 ) 173-177 2012.06

Research paper (scientific journal), Joint Work, Accepted

Osteopontin is an alpha motor neuron marker in the mouse spinal cord.

Misawa H, Hara M, Tanabe S, Niikura M, Moriwaki Y, Okuda T.

J Neurosci Res 90 ( 4 ) 732-742 2012.04

Research paper (scientific journal), Joint Work, Accepted

Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in regulation of immune function.

Kawashima K, Fujii T, Moriwaki Y, Misawa H.

Life Sci. 91 ( 21-22 ) 1027-32 2012

Research paper (scientific journal), Joint Work, Accepted

Reconciling neuronally and non-neuronally derived acetylcholine in the regulation of immune function

Kawashima K, Fujii T, Moriwaki Y, Misawa H, Horiguchi K.

Ann N Y Acad Sci. 1261 ( 1 ) 7-17 2012

Research paper (scientific journal), Joint Work, Accepted

Critical role of intracellular osteopontin in anti-fungal innate immune responses.

Inoue M, Moriwaki Y, Arikawa T, Chen YH, Oh YJ, Oliver T, Shinohara ML.

J Immunol 186 ( 1 ) 19-23 2011.01

Research paper (scientific journal), Joint Work

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila.

Imai Y, Kanao T, Sawada T, Kobayashi Y, Moriwaki Y, Ishida Y, Takeda K, Ichijo H,　Lu B, Takahashi R.

PLoS Gent 6 ( 12 ) e1001229 2010.12

Research paper (scientific journal), Joint Work, Accepted

Down-regulation of secreted lymphocyte antige-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), an endogenous allosteric alpha7 nicotinic acetylcholine receptor modulator, in murine and human asthmatic conditions.

Narumoto O, Horiguchi K, Horiguchi S, Moriwaki Y, Takano-Ohmuro H, Soji S, Misawa H, Yamashita N, Nagase T, Kawashima K, Yamashita N.

Biochem Biophys Res Commun 398 ( 4 ) 713-8 2010.08

Research paper (scientific journal), Joint Work, Accepted

Acetylcholine synthesis and release in NIH3T3 cells co-expressing the high-affinity choline transporter and choline acetyltransferase.

Fujii T, Masai M, Misawa H, Okuda T, Takada-Takatori Y, Moriwaki Y, Haga T, Kawashima K.

J Neurosci Res 87 ( 13 ) 3024-32 2009.10

Research paper (scientific journal), Joint Work, Accepted

Expression of SLURP-1, an endogenous α7 nicotinic acetylcholine receptor allosteric ligand, in murine bronchial epithelial cells.

Horiguchi K, Yamashita N, Horiguchi S, Irie K, Masuda J, Takano-Ohmuro H, Himi T, Miyazawa M, Moriwaki Y, Okuda T, Misawa H, Ozaki H, Kawashima K.

J Neurosci Res 87 ( 12 ) 2740-7 2009.09

Research paper (scientific journal), Joint Work, Accepted

Primary sensory neuronal expression of SLURP-1, an endogenous nicotinic acetylcholine receptor ligand.

Moriwaki Y, Watanabe Y, Shinagawa T, Kai M, Miyazawa M, Okuda T, Kawashima K, Yabashi A, Waguri S, Misawa H.

Neurosci Res 64 ( 4 ) 403-12 2009.08

Research paper (scientific journal), Joint Work, Accepted

Aberrant trafficking of the high-affinity choline transporter in AP-3-deficient mice.

Misawa H*, Fijigaya H, Nishimura T, Moriwaki Y, Okuda T, Kawashima K, Nakata K, Ruggiero AM, Blakely RD, Nakatsu F, Ohno H.

Eur J Neurosci 27   3109-3117 2008.06

Research paper (scientific journal), Joint Work, Accepted

L347P PINK1 mutant that fails to bind to Hsp90/cdc37 chaperones is rapidly degraded in a proteasome-dependent manner.

Moriwaki Y, Kim YJ, Ido Y, Misawa H, Kawashima K, Endo S, Takahashi R.

Neurosci Res 61 ( 1 ) 43-48 2008.05

Research paper (scientific journal), Joint Work, Accepted

Production and regulation of eotaxin-2/CCL24 in a differentiated human leukemic cell line, HT93.

Yoshida N, Aizu-Yokota E, Sonoda Y, Moriwaki Y, Kishi K, Kasahara T*.

Biol Pharm Bull 30(10)   1826-1832 2007.10

Research paper (scientific journal), Joint Work, Accepted

Enhanced serum antigen-specific IgG(1) and proinflammatory cytokine production in nicotinic acetylcholine receptor a7 subunit gene knockout mice.

Fujii YX, Fujigaya H, Moriwaki Y, Misawa H, Kasahara T, Grando SA, Kawashima K*.

J Neuroimmunol 189(1-2)   69-74 2007.09

Research paper (scientific journal), Joint Work, Accepted

Diminished antigen-specific IgG(1) and interleukin-6 production and acetylcholinesterase expression in combined M(1) and M(5) muscarinic acetylcholine receptor knockout mice.

Fujii YX, Tashiro A, Arimoto K, Fujigaya H, Moriwaki Y, Misawa H, Fujii T, Matsui M, Kasahara T, Kawashima K.

J Neuroimmunol 188   80-85 2007.08

Research paper (scientific journal), Joint Work, Accepted

PINK1, a gene product of PARK6, accumulates in alpha-synucleinopathy brains.

Murakami T, Moriwaki Y, Kawarabayashi T, Nagai M, Ohta Y, Deguchi K, Kurata T, Morimoto N, Takehisa Y, Matsubara E, Ikeda M, Harigaya Y, Shoji M, Takahashi R, Abe K.

J Neurol Neurosurg Psychiatry 78   653-654 2007.06

Research paper (scientific journal), Joint Work, Accepted

Expression and function of genes encoding cholinergic components in murine immune cells.

Kawashima K, Yoshikawa K, Fujii YX, Moriwaki Y, Misawa H.

Life Sciences 80   2314-2319 2007.05

Research paper (scientific journal), Joint Work, Accepted

Immune system expression of SLURP-1 and SLURP-2, two endogenous nicotinic acetylcholine receptor ligands.

Moriwaki Y, Yoshikawa K, Fukuda H, Fujii YX, Misawa H, Kawashima K.

Life Sciences 80   2365-2368 2007.05

Research paper (scientific journal), Joint Work, Accepted

Ubiquitous expression of acetylcholine and its biological functions in life forms without nervous systems.

Kawashima K, Misawa H, Moriwaki Y, Fujii YX, Fujii T, Horiuchi Y, Yamada T, Imanaka T, Kamekura M.

Life Sciences 80   2206-2209 2007.05

Research paper (scientific journal), Joint Work, Accepted

Conditional knockout of Mn superoxide dismutase in postnatal motor neurons reveals resistance to mitochondrial generated superoxide radicals.

Misawa H*, Nakata K, Matsuura J, Moriwaki Y, Kawashima K, Shimizu T, Shirasawa T, Takahashi R.

Neurobiol Dis 23   169-177 2006.07

Research paper (scientific journal), Joint Work, Accepted

Mycobacterium bovis BCG Cell Wall-Specific Differentially Expressed Genes Identified by Differential Display and cDNA Subtraction in Human Macrophages.

Begum NA, Ishii K, Kurita-Taniguchi M, Tanabe M, Kobayashi M, Moriwaki Y, Matsumoto M, Fukumori Y, Azuma I, Toyoshima K, Seya T.

Infect Immun 72 ( 2 ) 937 - 48 2004.02

Research paper (scientific journal), Joint Work, Accepted

Mycobacterium bovis BCG cell wall and lipopolysaccharide induce a novel gene, BIGM103, encoding a 7-TM protein: identification of a new protein family Zn-transporter and Zn-metalloprotease signatures.

Begum NA, Kobayashi M, Moriwaki Y, Matsumoto M, Toyoshima K, Seya T.

Genomics 80 ( 6 ) 630 - 45 2002.12

Research paper (scientific journal), Joint Work, Accepted

Mycobacterium bovis Bacillus Calmette-Guerin and its cell wall complex induce a novel lysosomal membrane protein, SIMPLE, that bridges the missing link between lipopolysaccharide and p53-inducible gene, LITAF(PIG7), and estrogen-inducible gene, EET-1.

Moriwaki Y, Begum NA, Kobayashi M, Matsumoto M, Toyoshima K, Seya T.

J Biol Chem 276 ( 25 ) 23065 - 76 2001.06

Research paper (scientific journal), Joint Work, Accepted