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Lack of Galanin Receptor 3 Alleviates Psoriasis by Altering Vascularization, Immune Cell Infiltration, and Cytokine Expression

  • Felix Locker
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Silvia Vidali
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Barbara S. Holub
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria

    Department of Dermatology, Paracelsus Medical University, Salzburg, Austria
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  • Julia Stockinger
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Susanne M. Brunner
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Sabine Ebner
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Andreas Koller
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Andrea Trost
    Affiliations
    Department of Ophthalmology/Optometry, Research Program Experimental Ophthalmology, Paracelsus Medical University Salzburg, Austria
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  • Herbert A. Reitsamer
    Affiliations
    Department of Ophthalmology/Optometry, Research Program Experimental Ophthalmology, Paracelsus Medical University Salzburg, Austria
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  • David Schwarzenbacher
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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  • Roland Lang
    Affiliations
    Department of Dermatology, Paracelsus Medical University, Salzburg, Austria
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  • Barbara Kofler
    Correspondence
    Correspondence: Barbara Kofler, Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Müllner Hauptstraße 48, 5020 Salzburg, Austria.
    Affiliations
    Laura Bassi Centre of Expertise–THERAPEP, Research Program for Receptor Biochemistry and Tumor Metabolism, Department of Pediatrics, Paracelsus Medical University, Salzburg, Austria
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Open ArchivePublished:August 24, 2017DOI:https://doi.org/10.1016/j.jid.2017.08.015
      The neuropeptide galanin is distributed in the central and peripheral nervous systems and in non-neuronal peripheral organs, including the skin. Galanin acts via three G protein-coupled receptors which, except galanin receptor 1, are expressed in various skin structures. The galanin system has been associated with inflammatory processes of the skin and of several other organs. Psoriasis is an inflammatory skin disease with increased neovascularization, keratinocyte hyperproliferation, a proinflammatory cytokine milieu, and immune cell infiltration. In this study, we showed that galanin receptor 3 is present in endothelial cells in human and murine dermal vessels and is co-expressed with nestin in neo-vessels of psoriatic patients. Moreover, in a murine psoriasis model, we showed that C57/BL6 mice lacking galanin receptor 3 display a milder course of psoriasis upon imiquimod treatment, leading to decreased disease severity, delayed neo-vascularization, reduced infiltration of neutrophils, and significantly lower levels of proinflammatory cytokines compared with wild-type mice. In contrast, galanin receptor 2-knockout animals did not differ significantly from wild type mice at both the macroscopic and molecular levels in their inflammatory response to imiquimod treatment. Our data indicate that galanin receptor 3, but not galanin receptor 2, plays an important role in psoriasis-like skin inflammation.

      Abbreviations:

      GAL (galanin), GAL2-KO (GAL2-R knockout mouse), GAL3-KO (GAL3-R knockout mouse), GAL2-R (galanin receptor 2), GAL3-R (galanin receptor 3), IMQ (imiquimod), KO (knockout), MPO (myeloperoxidase), TNF-α (tumor necrosis factor-α), WT (wild-type)

      Introduction

      The mammalian skin forms the largest organ of the body and includes a network of cutaneous nerves, cells of the immune system, and mediators of the neuro-endocrine axis (
      • Theoharides T.C.
      • Stewart J.M.
      • Taracanova A.
      • Conti P.
      • Zouboulis C.C.
      Neuroendocrinology of the skin.
      ). Analogous to the hypothalamic-pituitary-adrenal axis, neuropeptides and their receptors are present in the skin, including galanin (GAL) (
      • Lang R.
      • Gundlach A.L.
      • Holmes F.E.
      • Hobson S.A.
      • Wynick D.
      • Hokfelt T.
      • et al.
      Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.
      ,
      • Theoharides T.C.
      • Stewart J.M.
      • Taracanova A.
      • Conti P.
      • Zouboulis C.C.
      Neuroendocrinology of the skin.
      ).
      GAL is a neuropeptide with a length of 30 amino acids (29 in rodents) that is distributed in the central and peripheral nervous systems and in non-neuronal organs such as the skin (
      • Lang R.
      • Gundlach A.L.
      • Holmes F.E.
      • Hobson S.A.
      • Wynick D.
      • Hokfelt T.
      • et al.
      Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.
      ). GAL is localized in afferent sensory neurons and specialized cutaneous sensory structures such as Merkel cells and Meissner corpuscles and in non-neuronal structures like eccrine sweat glands, in follicular and epidermal keratinocytes, and in smooth muscle cells of dermal blood vessels (
      • Fantini F.
      • Johansson O.
      Neurochemical markers in human cutaneous Merkel cells. An immunohistochemical investigation.
      ,
      • Johansson O.
      • Fantini F.
      • Hu H.
      Neuronal structural proteins, transmitters, transmitter enzymes and neuropeptides in human Meissner's corpuscles: a reappraisal using immunohistochemistry.
      ,
      • Johansson O.
      • Vaalasti A.
      • Tainio H.
      • Ljungberg A.
      Immunohistochemical evidence of galanin in sensory nerves of human digital skin.
      ,
      • Kofler B.
      • Berger A.
      • Santic R.
      • Moritz K.
      • Almer D.
      • Tuechler C.
      • et al.
      Expression of neuropeptide galanin and galanin receptors in human skin.
      ,
      • Pincelli C.
      • Fantini F.
      • Massimi P.
      • Girolomoni G.
      • Seidenari S.
      • Giannetti A.
      Neuropeptides in skin from patients with atopic dermatitis: an immunohistochemical study.
      ,
      • Xu X.J.
      • Hao J.X.
      • Wiesenfeld-Hallin Z.
      • Hakanson R.
      • Folkers K.
      • Hokfelt T.
      Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hindpaw.
      ). In addition, in a murine model of allergic contact dermatitis, a significant elevation of GAL-positive nerve fibers was observed compared with healthy skin. However, the concentration of the peptide, as measured by radioimmune assay of skin extracts, was markedly reduced (
      • El-Nour H.
      • Lundeberg L.
      • Boman A.
      • Theodorsson E.
      • Hokfelt T.
      • Nordlind K.
      Galanin expression in a murine model of allergic contact dermatitis.
      ). Carrageenan-induced inflammation in the rat hind paw led to elevated GAL-like immunoreactivity and GAL mRNA in the epidermis, as well as GAL-like immunoreactivity in ED-1–positive immunocytes in the dermis (
      • Ji R.R.
      • Zhang X.
      • Zhang Q.
      • Dagerlind A.
      • Nilsson S.
      • Wiesenfeld-Hallin Z.
      • et al.
      Central and peripheral expression of galanin in response to inflammation.
      ).
      GAL exerts its functions via three G protein-coupled receptors (GAL1-R, GAL2-R, and GAL3-R) (
      • Fathi Z.
      • Cunningham A.M.
      • Iben L.G.
      • Battaglino P.B.
      • Ward S.A.
      • Nichol K.A.
      • et al.
      Cloning, pharmacological characterization and distribution of a novel galanin receptor.
      ,
      • Habert-Ortoli E.
      • Amiranoff B.
      • Loquet I.
      • Laburthe M.
      • Mayaux J.F.
      Molecular cloning of a functional human galanin receptor.
      ,
      • Howard A.D.
      • Tan C.
      • Shiao L.L.
      • Palyha O.C.
      • McKee K.K.
      • Weinberg D.H.
      • et al.
      Molecular cloning and characterization of a new receptor for galanin.
      ). The first evidence for GAL receptor expression in human skin was provided by receptor autoradiography, which detected GAL binding around dermal blood vessels and sweat gland cells (
      • Kofler B.
      • Berger A.
      • Santic R.
      • Moritz K.
      • Almer D.
      • Tuechler C.
      • et al.
      Expression of neuropeptide galanin and galanin receptors in human skin.
      ). Analysis of human skin showed GAL2-R–like immunoreactivity in the basal layer of the epidermis, around dermal blood vessels, and in sweat gland cells (
      • Bovell D.L.
      • Holub B.S.
      • Odusanwo O.
      • Brodowicz B.
      • Rauch I.
      • Kofler B.
      • et al.
      Galanin is a modulator of eccrine sweat gland secretion.
      ,
      • Dallos A.
      • Kiss M.
      • Polyanka H.
      • Dobozy A.
      • Kemeny L.
      • Husz S.
      Galanin receptor expression in cultured human keratinocytes and in normal human skin.
      ). GAL3-R–like immunoreactivity was detected in sweat gland cells, and GAL3-R mRNA was shown to be expressed by outer root sheath keratinocytes and hair follicles (
      • Bovell D.L.
      • Holub B.S.
      • Odusanwo O.
      • Brodowicz B.
      • Rauch I.
      • Kofler B.
      • et al.
      Galanin is a modulator of eccrine sweat gland secretion.
      ,
      • Holub B.S.
      • Kloepper J.E.
      • Toth B.I.
      • Biro T.
      • Kofler B.
      • Paus R.
      The neuropeptide galanin is a novel inhibitor of human hair growth.
      ). In addition, GAL2-R and GAL3-R are expressed on immune cells such as neutrophils, macrophages, and monocytes, which are, inter alia, important in maintaining skin immunity (
      • Chiu I.M.
      • Heesters B.A.
      • Ghasemlou N.
      • Von Hehn C.A.
      • Zhao F.
      • Tran J.
      • et al.
      Bacteria activate sensory neurons that modulate pain and inflammation.
      ,
      • Locker F.
      • Lang A.A.
      • Koller A.
      • Lang R.
      • Bianchini R.
      • Kofler B.
      Galanin modulates human and murine neutrophil activation in vitro.
      ). In contrast, there is no evidence so far for GAL1-R expression in human skin (
      • Bovell D.L.
      • Holub B.S.
      • Odusanwo O.
      • Brodowicz B.
      • Rauch I.
      • Kofler B.
      • et al.
      Galanin is a modulator of eccrine sweat gland secretion.
      ,
      • Dallos A.
      • Kiss M.
      • Polyanka H.
      • Dobozy A.
      • Kemeny L.
      • Husz S.
      Galanin receptor expression in cultured human keratinocytes and in normal human skin.
      ). It is well known that GAL receptors are expressed in rodent skin as well; however, the lack of specific GAL receptor antibodies for this species explains the deficit of data regarding the cellular distribution of GAL receptors on mouse and rat skin (
      • Lu X.Y.
      • Barr A.M.
      • Bartfai T.
      Galanin receptors as novel drug targets for the treatment of depression and anxiety.
      ). As a vasoactive peptide, GAL is able to block plasma extravasation induced by different stimuli such as histamine, substance P, or antidromic C fiber stimulation in rodent and pigeon skin (
      • Jancso G.
      • Santha P.
      • Horvath V.
      • Pierau F.
      Inhibitory neurogenic modulation of histamine-induced cutaneous plasma extravasation in the pigeon.
      ,
      • Xu X.J.
      • Hao J.X.
      • Wiesenfeld-Hallin Z.
      • Hakanson R.
      • Folkers K.
      • Hokfelt T.
      Spantide II, a novel tachykinin antagonist, and galanin inhibit plasma extravasation induced by antidromic C-fiber stimulation in rat hindpaw.
      ). Furthermore, after cutaneous treatment with tumor necrosis factor-α (TNF-α), heat, or a combination of substance P and calcitonin gene-related peptide, GAL-knockout (KO) mice lack neutrophil accumulation in the skin (
      • Schmidhuber S.M.
      • Starr A.
      • Wynick D.
      • Kofler B.
      • Brain S.D.
      Targeted disruption of the galanin gene attenuates inflammatory responses in murine skin.
      ). In the K/B×N serum transfer model of autoimmune arthritis, an increase in clinical severity and vascular hyperpermeability in GAL3-R–knockout (GAL3-KO) animals compared with wild-type (WT) littermates was observed (
      • Botz B.
      • Kemeny A.
      • Brunner S.M.
      • Locker F.
      • Csepregi J.
      • Mocsai A.
      • et al.
      Lack of galanin 3 receptor aggravates murine autoimmune arthritis.
      ), suggesting an anti-inflammatory role of GAL3-R. However, GAL3-KO mice showed no change in the progression of oxazolone-induced contact dermatitis compared with WT mice (
      • Botz B.
      • Brunner S.M.
      • Kemeny A.
      • Pinter E.
      • McDougall J.J.
      • Kofler B.
      • et al.
      Galanin 3 receptor-deficient mice show no alteration in the oxazolone-induced contact dermatitis phenotype.
      ). The absence of an obvious phenotype of GAL3-KO mice in the dermatitis model and the discrepancy between the arthritis and oxazolone models highlight the complexity of the role of GAL3-R in inflammation, which seems to depend on the type of inflammatory disease and the tissue affected.
      Psoriasis is an inflammatory skin disease characterized by epidermal hyperplasia; micro-abscesses; neo-angiogenesis; a proinflammatory cytokine environment involving alterations of IL-17A, IL-23, and TNF-α; and activation of and infiltration by macrophages, mast cells, neutrophils, CD11c+ dendritic cells, and T cells (
      • Elder J.T.
      • Bruce A.T.
      • Gudjonsson J.E.
      • Johnston A.
      • Stuart P.E.
      • Tejasvi T.
      • et al.
      Molecular dissection of psoriasis: integrating genetics and biology.
      ,
      • Ha H.L.
      • Wang H.
      • Pisitkun P.
      • Kim J.C.
      • Tassi I.
      • Tang W.
      • et al.
      IL-17 drives psoriatic inflammation via distinct, target cell-specific mechanisms.
      ). Topical application of imiquimod (IMQ), an innate toll like receptor 7/8 ligand, induces a dermatitis closely resembling human psoriasis. Similar to psoriasis observed in the clinic, inflamed, scaly, and plaque skin lesions; epidermal hyperproliferation; abnormal differentiation; and neo-vascularization occur upon IMQ treatment. This is caused by the infiltration of neutrophils and macrophages and the activation of mast cells, followed by recruitment of specific dendritic cells and T cells (
      • Brembilla N.C.
      • Stalder R.
      • Senra L.
      • Bohencke W.H.
      IL-17A localizes in the exocytic compartment of mast cells in psoriatic skin [e-pub ahead of print].
      ,
      • Cai Y.
      • Shen X.
      • Ding C.
      • Qi C.
      • Li K.
      • Li X.
      • et al.
      Pivotal role of dermal IL-17-producing gammadelta T cells in skin inflammation.
      ,
      • Pantelyushin S.
      • Haak S.
      • Ingold B.
      • Kulig P.
      • Heppner F.L.
      • Navarini A.A.
      • et al.
      Rorgammat+ innate lymphocytes and gammadelta T cells initiate psoriasiform plaque formation in mice.
      ,
      • Schon M.P.
      • Schon M.
      Imiquimod: mode of action.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). There is clinical evidence that absence of neural input resulting from denervation injury leads to improvement of psoriasis, which suggests a role of the nervous system in the pathophysiology of this skin disease (
      • Zhu T.H.
      • Nakamura M.
      • Farahnik B.
      • Abrouk M.
      • Lee K.
      • Singh R.
      • et al.
      The role of the nervous system in the pathophysiology of psoriasis: a review of cases of psoriasis remission or improvement following denervation injury.
      ). For example, α-melanocyte–stimulating hormone ameliorates inflammation in an IMQ-induced psoriasiform-inflammation model (
      • Auriemma M.
      • Brzoska T.
      • Klenner L.
      • Kupas V.
      • Goerge T.
      • Voskort M.
      • et al.
      alpha-MSH-stimulated tolerogenic dendritic cells induce functional regulatory T cells and ameliorate ongoing skin inflammation.
      ). Recently, a topical formulation of α-melanocyte–stimulating hormone was developed that reduced IMQ-induced psoriasiform inflammation in mice (
      • Shah P.P.
      • Desai P.R.
      • Boakye C.H.
      • Patlolla R.
      • Kikwai L.C.
      • Babu R.J.
      • et al.
      Percutaneous delivery of alpha-melanocyte-stimulating hormone for the treatment of imiquimod-induced psoriasis.
      ).
      The aim of this study was to determine GAL receptor expression in human psoriatic skin and to elucidate progression of inflammation in murine models of psoriasis in GAL2-KO and GAL3-KO animals.

      Results

      Co-expression of GAL3-R and CD31 in human vessels

      Although GAL binding sites have been detected around dermal blood vessels (
      • Kofler B.
      • Berger A.
      • Santic R.
      • Moritz K.
      • Almer D.
      • Tuechler C.
      • et al.
      Expression of neuropeptide galanin and galanin receptors in human skin.
      ), evidence concerning which GAL receptor is expressed in the dermal vasculature is missing. Laser microdissection of human dermal blood vessels showed expression of GAL3-R but not GAL1-R and GAL2-R mRNA (see Supplementary Figure S1 online). To verify GAL3-R expression in the vasculature of human skin at the protein level and to determine whether GAL3-R expression differs in healthy versus psoriatic skin samples, we performed immunofluorescence staining of normal and psoriatic human skin samples. We used antibodies against GAL3-R, CD31, and α-smooth muscle actin to discriminate between CD31-positive endothelial cells (Figure 1a ) and α-smooth muscle actin–positive pericytes (Figure 1b). GAL3-R staining (Figure 1c) was observed around large, mature vessels of male and female donors in CD31-positive endothelial cells, whereas the α-smooth muscle actin–positive pericytes remained GAL3-R–negative (Figure 1d).
      Figure 1
      Figure 1GAL3-R, α-SMA, and CD31 staining of human skin. Representative images of immunofluorescent staining of (a) CD31 (white), (b) α-SMA (red), (c) GAL3-R (green), and (d) merge of all channels, in normal male skin. Scale bar = 50 μm. GAL3-R, galanin receptor 3; SMA, smooth muscle actin.
      In the skin we also detected GAL2-R– and GAL3-R–positive structures such as sweat gland cells and structures exclusively positive for GAL2-R such as epidermal keratinocytes, which is in line with the literature (
      • Bovell D.L.
      • Holub B.S.
      • Odusanwo O.
      • Brodowicz B.
      • Rauch I.
      • Kofler B.
      • et al.
      Galanin is a modulator of eccrine sweat gland secretion.
      ,
      • Dallos A.
      • Kiss M.
      • Polyanka H.
      • Dobozy A.
      • Kemeny L.
      • Husz S.
      Galanin receptor expression in cultured human keratinocytes and in normal human skin.
      ). A few GAL2-R– and GAL3-R–positive neutrophils and macrophages were observed in healthy skin. In addition, no GAL2-R expression was found in dermal blood vessels. An overview of GAL2-R and GAL3-R staining in different skin structures is depicted in Supplementary Figure S2 online.

      GAL3-R overlaps with nestin staining in psoriatic microvessels

      To analyze whether GAL3-R might also be present in proliferating, newly formed vessels, we stained skin biopsy samples of healthy and psoriatic individuals with anti-GAL3-R and nestin antibodies. Nestin serves as a marker for neural stem cells and angiogenesis, the latter a key pathogenic factor in psoriasis (
      • Heidenreich R.
      • Rocken M.
      • Ghoreschi K.
      Angiogenesis drives psoriasis pathogenesis.
      ,
      • Schonthaler H.B.
      • Huggenberger R.
      • Wculek S.K.
      • Detmar M.
      • Wagner E.F.
      Systemic anti-VEGF treatment strongly reduces skin inflammation in a mouse model of psoriasis.
      ,
      • Teranishi N.
      • Naito Z.
      • Ishiwata T.
      • Tanaka N.
      • Furukawa K.
      • Seya T.
      • et al.
      Identification of neovasculature using nestin in colorectal cancer.
      ). In normal skin, GAL3-R (Figure 2a, 2c ) but no nestin staining (Figure 2b, 2c) was observed in large, mature vessels. However, in newly formed microvessels, GAL3-R (Figure 2d) and nestin staining (Figure 2e) co-localized (Figure 2f), suggesting GAL3-R involvement in neo-angiogenesis.
      Figure 2
      Figure 2Immunohistochemical staining for GAL3-R and nestin in human skin. (a, d) Representative images of immunofluorescent staining of GAL3-R (red) and (b, e) nestin (green). (c, f) Merge of channels. (ac) Normal male skin, (df) psoriatic male skin. Arrows in a and c indicate GAL3-R–positive blood vessels in normal skin negative for nestin. Arrows in d indicate GAL3-R–positive blood vessels in psoriatic skin, arrows in e indicate nestin-positive blood vessels, and arrows in f indicate overlapping staining of GAL3-R and nestin in capillaries of the papillary dermis of psoriatic skin (psoriasis vulgaris). Scale bar = 50 μm. GAL3-R, galanin receptor 3.
      The immunohistochemical staining of GAL2-R and GAL3-R of psoriatic skin shows similar expression patterns as nonpsoriatic skin. Immune cells such as neutrophils and macrophages were stained positive for both GAL2-R and GAL3-R (see Supplementary Figure S3 online), indicating that GAL receptor signaling on immune cells could contribute to psoriasis progression.

      Altered response toward IMQ in GAL3-KO but not GAL2-KO mice

      Because neo-angiogenesis plays an important role in psoriasis, and because GAL2-R and GAL3-R are expressed in skin (
      • Schmidhuber S.M.
      • Rauch I.
      • Kofler B.
      • Brain S.D.
      Evidence that the modulatory effect of galanin on inflammatory edema formation is mediated by the galanin receptor 3 in the murine microvasculature.
      ) and GAL3-R was shown to influence inflammation in different inflammatory diseases, we compared the progression of psoriasis in GAL2-KO and GAL3-KO versus WT control mice using the IMQ model (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ).
      Psoriasis clinical severity was evaluated by semiquantitative scoring of erythema, scaling, and skin thickening. Erythema, which is an indicator of increased vascular permeability and inflammation, was significantly reduced in GAL3-KO compared with WT mice on day 5 (P = 0.0011) (Figure 2a), whereas GAL2-KO mice showed no differences in erythema development compared with WT mice (see Supplementary Figure S4a online). Psoriasis-typical scaling, which occurs due to hyperproliferation of keratinocytes, was also reduced in the GAL3-KO group on days 5 (P = 0.0015), 6 (P = 0.0001), and 7 (P = 0.0118) (Figure 2b) but not in GAL2-KO mice (see Supplementary Figure S4b). Thickening of the skin, which results from combined edema formation and immune cell infiltration as well as from acanthosis, was significantly lower on day 4 (P = 0.0020) in GAL3-KO mice (Figure 3c ). This was the only parameter decreased in GAL2-KO mice (day 5, P = 0.0001) (see Supplementary Figure S4c). The cumulative score showed that GAL3-KO animals exhibited a reduced psoriatic response toward IMQ treatment compared with WT animals on days 4 (P = 0.0144), 5 (P = 0.0003), and 6 (P = 0.0341) (Figure 3d), as displayed in Figure 3g (representative images). GAL2-KO showed no differences in the overall cumulative disease severity score compared with WT mice at any time point (see Supplementary Figure S4d).
      Figure 3
      Figure 3Severity of IMQ induced inflammation in GAL3-KO mice. Semiquantitative scoring of (a) erythema, (b) scaling, (c) thickening of the skin, and (d) cumulative scoring over time after IMQ treatment of WT and GAL3-KO mice. (e) The thickness of epidermis was measured in hematoxylin and eosin-stained skin samples of vehicle and IMQ-treated mice on the indicated time points. (f) Splenomegaly, indicated as total spleen weight, was analyzed in vehicle (ctrl) and IMQ-treated mice on days 4 and 7. (g) Representative images from days 4, 5, and 6. Values represent mean ± standard error of the mean. (a–d) n = 18–23, (e–f) n = 13–22 per group. P < 0.05, ∗∗P < 0.001, ∗∗∗P < 0.001 versus WT mice, °°°P < 0.001 vs. vehicle-treated (ctrl) mice. d, days; GAL3-KO, galanin receptor 3 knockout mice; IMQ, imiquimod; WT, wild type.
      Epidermal hyperplasia produces scaling; therefore, we analyzed epidermal thickening in skin sections upon IMQ treatment. GAL3-KO animals showed a reduced thickness of the epidermis after IMQ treatment compared with WT mice on day 4 (66.3 ± 8.8 μm vs. 53.5 ± 12.3 μm, P < 0.01) (Figure 3e, Supplementary Figure S5 online). In contrast, GAL2-KO animals displayed no differences in epidermal thickening compared with their corresponding WT mice on days 4 and 7 (see Supplementary Figure S4e). Upon psoriasiform inflammation, it has been reported that splenic mass increases upon IMQ treatment because of a systemic effect of the compound (
      • Flutter B.
      • Nestle F.O.
      TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
      ). GAL3-KO and WT mice displayed marked splenomegaly upon IMQ treatment, resulting in equal enlargement in WT mice (0.084 ± 0.020 g to 0.154 ± 0.018 g) and GAL3-KO mice (0.102 ± 0.036 g to 0.156 ± 0.022 g) on day 4. The splenic swelling progressed in both genotypes; however, we observed a smaller increase in spleen weight in GAL3-KO mice (0.217 ± 0.042 g) compared with WT mice (0.244 ± 0.045 g) on day 7 (P < 0.05) (Figure 3f). In GAL2-KO and their corresponding WT mice, IMQ-induced spleen enlargement was increased similarly (see Supplementary Figure S4f), indicating that although GAL2-R is expressed in the mouse skin, only GAL3-R seems to influence the progression of psoriasis.

      GAL3-KO mice show delayed neovascularization

      Angiogenesis is a key pathogenic factor in psoriatic disease progression (
      • Heidenreich R.
      • Rocken M.
      • Ghoreschi K.
      Angiogenesis drives psoriasis pathogenesis.
      ). Thus, we quantified CD31-positive vessels in tissue sections of control and IMQ-treated dorsal mouse skin. We observed an increase in vessel density in WT animals 4 and 7 days after IMQ treatment compared with vehicle-treated control skin (P < 0.001). GAL3-KO animals showed no IMQ-induced up-regulation of vessel number on day 4. However, on day 7, the vessel density was increased in IMQ-treated GAL3-KO mice compared with untreated GAL3-KO mice (P < 0.001). The difference in number of vessels between IMQ-treated GAL3-KO mice and WT mice on day 4 was significant (P < 0.01); however, the difference was diminished on day 7 (Figure 4a ). No differences in vessel densities between GAL2-KO and WT mice upon vehicle or IMQ treatment were observed on days 4 and 7 (see Supplementary Figure S6 online). This finding is in line with the absence of a macroscopic difference between GAL2-KO and WT animals upon IMQ treatment.
      Figure 4
      Figure 4Determination of vascular density and vascularization-related transcripts. (a) CD31-positive vessels in skin sections of vehicle-treated (ctrl) and IMQ-treated WT and GAL3-KO mice on days 4 and 7. GAL3-KO and WT mice show no differences in mRNA levels of (b) VEGF, (c) ANGPT I, and (d) ANGPT II in skin sections of IMQ-treated WT and GAL3-KO mice. GAL3-KO mRNA was calculated relative to the levels in vehicle-treated WT animals. Values represent mean ± standard error of the mean. (a) n = 11–15, (b) n = 12–15 per group. ∗∗P < 0.01 versus WT mice, °°°P < 0.001 versus vehicle-treated (ctrl) mice. ANGPT I, angiopoietin I; ANGPT II, angiopoietin II; ctrl, control; d, day; GAL3-KO, galanin receptor 3-knockout mice; IMQ, imiquimod; VEGF, vascular endothelial growth factor; WT, wild type.
      To investigate whether GAL3-R deficiency influences angiogenesis directly or via regulation of the expression of vascularization factors, we performed an expression analysis of vascularization-related genes. The mRNA levels of vascular endothelial growth factor, angiopoietin I, and angiopoietin II were not different between genotypes in IMQ-treated skin on days 4 or 7 (Figure 4b–d). Because the amount of gene products related to vascularization is not different, this could be an indication of a direct involvement of GAL3-R signaling in neovascularization. This is strengthened by the finding that GAL3-R mRNA was present in vessels of healthy and inflamed skin (day 4 after IMQ treatment) (see Supplementary Figure S7 online).

      GAL3-KO mice show reduced myeloperoxidase activity and reduced neutrophil infiltration

      Because we observed differences in vessel densities and because angiogenesis is necessary for the sustained survival and accumulation of inflammatory cells under chronic inflammatory conditions (
      • Prescott S.M.
      • Zimmerman G.A.
      • McIntyre T.M.
      Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin.
      ), we hypothesized that neutrophil influx might be affected in psoriatic GAL3-KO animals. Therefore, we quantified the amount of neutrophil-related myeloperoxidase (MPO) in control and IMQ-treated skin biopsy samples. We detected an increase on day 4 followed by a return to control levels on day 7 in both WT and GAL3-KO animals. On day 4 the amount of MPO was lower in the GAL3-KO compared with the WT group (0.098 ± 0.047 U/ml vs. 0.143 ± 0.055 U/ml, respectively; P < 0.01) (Figure 5a ).
      Figure 5
      Figure 5MPO activity and the amount of macrophages, neutrophils, and mast cells in murine psoriasiform skin. (a) MPO concentration (U/ml) in 8-mm skin punch biopsy samples from vehicle-treated (ctrl) and IMQ-treated mice on days 4 and 7. Number of (b) F4/80+, (c) NIMP-R14+, and (d) toluidine blue-stained cells in paraffin-embedded skin sections of vehicle-treated and IMQ-treated WT and GAL3-KO mice on day 4 and day 7, respectively. Values represent mean ± standard error of the mean. (a) n = 13–18, (b–d) n = 11–14 per group. ∗∗P < 0.01 versus WT mice, °°°P < 0.001 versus vehicle-treated (ctrl) mice. ctrl, control; d, day; GAL3-KO, galanin receptor 3 knockout mice; IMQ, imiquimod; MPO, myeloperoxidase; n.q., no quantification possible due to sparse presence of neutrophils in healthy skin; WT, wild type mice.
      To determine if the lower amount of MPO was a consequence of different amounts or subtypes of neutrophils or neutrophil-attracting macrophages, we quantified NIMP-R14+ neutrophils and F4/80-positive macrophages in the skin sections. No differences in macrophage abundance (Figure 5b) or expression of macrophage M1 and M2 subtype-related genes (
      • Jablonski K.A.
      • Amici S.A.
      • Webb L.M.
      • Ruiz-Rosado Jde D.
      • Popovich P.G.
      • Partida-Sanchez S.
      • et al.
      Novel markers to delineate murine M1 and M2 macrophages.
      ) (i.e., Arg1, Il12b, Fpr2, and Egr2 [see Supplementary Figure S8 online]) were observed in the skin sections of the two different genotypes of untreated and IMQ-treated mice on day 4 and day 7. However, a trend toward reduced NIMP-R14+ neutrophils was observed in the skin sections of GAL3-KO compared with WT mice upon IMQ treatment on day 4 (P = 0.08). In vehicle-treated skin no quantifiable amount of neutrophils was detected (Figure 5c). In line with the lack of a macroscopic phenotype, GAL2-KO and corresponding WT animals showed no difference in the number of NIMP-R14+ neutrophils on day 4 of IMQ treatment (see Supplementary Figure S9a online). Because neutrophils are key effector cells in IMQ-induced psoriasis, the reduced MPO amount, together with the trend toward reduced numbers of neutrophils in the GAL3-KO mice, supports the observed reduced psoriasis severity score in this transgenic mouse strain.

      Genotype-specific modulation of cytokine expression

      We next examined if the reduced severity of the IMQ-induced psoriasis in GAL3-KO animals could be due to alterations in the expression of psoriasis-related cytokines. We detected lower expression of IL-17A (65% reduction, P < 0.001), IL-22 (80% reduction, P < 0.001), IL-23 (64% reduction, P < 0.01), and TNF-α (49% reduction, P < 0.05) mRNA in GAL3-KO compared with WT animals on day 4 (Figure 6a ). On day 7, after the climax of inflammation, a decline in cytokine mRNA levels was noticed, and the significant differences between the genotypes disappeared (Figure 6b). In GAL2-KO animals, no differences in the mRNA expressions of IL-17A, IL-22, IL-23, and TNF-α on day 4 (see Supplementary Figure S10a online) or day 7 were detected compared with the WT (see Supplementary Figure S10b). The PCR primers and reaction conditions are provided in Supplementary Tables S1 and S2 online. To confirm that the reduced amount of IL-17 and IL-22 observed in the GAL3-KO animals is originating from a reduced number of IL-17– and IL-22–producing neutrophils and not mast cells (
      • Mashiko S.
      • Bouguermouh S.
      • Rubio M.
      • Baba N.
      • Bissonnette R.
      • Sarfati M.
      Human mast cells are major IL-22 producers in patients with psoriasis and atopic dermatitis.
      ), we determined the amount of toluidine blue-stained mast cells in the skin sections. No differences in mast cell numbers in control and IMQ-treated skin sections of GAL3-KO and WT animals on day 4 were detected (Figure 5d). As expected between GAL2-KO and their corresponding WT animals, the amount of mast cells did not differ (see Supplementary Figure S9b).
      Figure 6
      Figure 6mRNA expression analysis. Relative mRNA levels of IL-17A, IL-22, IL-23, and TNF-α in skin sections of IMQ-treated GAL3-KO mice on days (a) 4 and (b) 7; mRNA levels of GAL3-KO mouse skin are delineated relative to the expression levels in IMQ-treated WT animals. Values represent mean ± standard error of the mean, n = 12–15 per group. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus WT mice. GAL3-KO, galanin receptor 3 knockout mice; TNF, tumor necrosis factor; WT, wild type mice.
      Consequently, the amount of mast cells does not account for the altered expression of IL-17A and IL-22. Possibly, neutrophils that are also capable of producing these interleukins (
      • Taylor P.R.
      • Roy S.
      • Leal Jr., S.M.
      • Sun Y.
      • Howell S.J.
      • Cobb B.A.
      • et al.
      Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORgammat and dectin-2.
      ) are responsible for the alterations in cytokine levels.

      Discussion

      In this study, we showed that endothelial cells of the dermal vasculature of healthy individuals express GAL3-R and that nestin-positive neovessels in psoriatic skin co-express GAL3-R. Nestin, a type VI intermediate filament protein, is a selective marker for bone marrow-derived mesenchymal stem cells and is expressed on perivascular and endothelial cells during angiogenesis in different tissues (
      • Suzuki S.
      • Namiki J.
      • Shibata S.
      • Mastuzaki Y.
      • Okano H.
      The neural stem/progenitor cell marker nestin is expressed in proliferative endothelial cells, but not in mature vasculature.
      ,
      • Xie L.
      • Zeng X.
      • Hu J.
      • Chen Q.
      Characterization of Nestin, a selective marker for bone marrow derived mesenchymal stem cells.
      ). GAL2-R and GAL3-R mRNA is expressed in mouse embryonic cells, and GAL expression was observed during embryonic development (
      • Jones M.
      • Perumal P.
      • Vrontakis M.
      Presence of galanin-like immunoreactivity in mesenchymal and neural crest origin tissues during embryonic development in the mouse.
      ,
      • Tarasov K.V.
      • Tarasova Y.S.
      • Crider D.G.
      • Anisimov S.V.
      • Wobus A.M.
      • Boheler K.R.
      Galanin and galanin receptors in embryonic stem cells: accidental or essential?.
      ). These data indicate a potential role of GAL3-R in stem cell function and suggest that it may have a neo-angiogenic effect.
      A recent study reported the distribution of GAL receptors in the human eye, which is a model organ for studying vascularization: all three GAL receptor types were found to be expressed on choroidal blood vessels, GAL1-R and GAL3-R on iris vessels, and GAL3-R alone on ciliary vessels, indicating localization and tissue-specific expression patterns of GAL receptors (
      • Schrodl F.
      • Kaser-Eichberger A.
      • Trost A.
      • Strohmaier C.
      • Bogner B.
      • Runge C.
      • et al.
      Distribution of galanin receptors in the human eye.
      ). The abundance of GAL receptors in the eye could indicate a role in vascularization-related events.
      In psoriasis development, a small population of mast cells and macrophages residing in the skin can act as sentinel cells that initiate neutrophil recruitment by increasing the permeability of local blood vessels and the release of chemokines (
      • Ajuebor M.N.
      • Das A.M.
      • Virag L.
      • Flower R.J.
      • Szabo C.
      • Perretti M.
      Role of resident peritoneal macrophages and mast cells in chemokine production and neutrophil migration in acute inflammation: evidence for an inhibitory loop involving endogenous IL-10.
      ,
      • Vieira S.M.
      • Lemos H.P.
      • Grespan R.
      • Napimoga M.H.
      • Dal-Secco D.
      • Freitas A.
      • et al.
      A crucial role for TNF-alpha in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5.
      ). The first wave of infiltrating immune cells after IMQ treatment consists of neutrophils. On days 2 to 4 a second wave of neutrophils is accompanied by macrophages (
      • Flutter B.
      • Nestle F.O.
      TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
      ). We observed an increase in macrophages and neutrophils after 4 days of IMQ treatment and a reduction of both cells types by day 7. Molecular analysis of the skin showed no abnormalities in M1- and M2-related genes between WT and GAL3-KO animals. In skin sections of GAL3-KO animals, a trend toward a lower amount of Fpr2 expression was observed on day 4. Because Fpr2 is also present on neutrophils (
      • Hartt J.K.
      • Barish G.
      • Murphy P.M.
      • Gao J.L.
      N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR.
      ) and plays a role in homing of endothelial progenitor cells (
      • Heo S.C.
      • Kwon Y.W.
      • Jang I.H.
      • Jeong G.O.
      • Yoon J.W.
      • Kim C.D.
      • et al.
      WKYMVm-induced activation of formyl peptide receptor 2 stimulates ischemic neovasculogenesis by promoting homing of endothelial colony-forming cells.
      ), which show stemness characteristics and are crucially involved in the formation of vascular networks, we hypothesize that the lower Fpr2 levels in GAL3-KO animals may contribute to the vascular phenotype in vivo.
      Neovascularization upon IMQ treatment was observed to different degrees in WT and GAL3-KO mice, whereas the increase in vascular endothelial growth factor mRNA levels was genotype independent. Because we observed lower levels of the cytokines IL-17A, IL-22, IL-23, and TNF-α in IMQ-treated skin of GAL3-KO mice, we addressed the question of whether the lower cytokine levels could be linked to IL-17– and IL-22–expressing mast cells or neutrophils (
      • Mashiko S.
      • Bouguermouh S.
      • Rubio M.
      • Baba N.
      • Bissonnette R.
      • Sarfati M.
      Human mast cells are major IL-22 producers in patients with psoriasis and atopic dermatitis.
      ,
      • Taylor P.R.
      • Roy S.
      • Leal Jr., S.M.
      • Sun Y.
      • Howell S.J.
      • Cobb B.A.
      • et al.
      Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORgammat and dectin-2.
      ). Because mast cells in the skin originate from progenitors but neutrophils are recruited from blood into the skin, altered neovascularization occurring during the course of disease could contribute to variations in neutrophil number between WT and GAL3-KO mice. Indeed, we observed lower numbers of neutrophils but not mast cells in GAL3-KO compared with WT mice, which is in line with the reduced MPO activity. Furthermore, this supports our hypothesis that the expression of GAL3-R on blood vessels is important for disease progression. Accordingly, we detected GAL3-R mRNA in human and murine dermal vessels.
      Recent advances in the GAL field led to the development of specific peptidergic agonists and antagonists with higher specificity and increased stability (
      • Lang R.
      • Gundlach A.L.
      • Holmes F.E.
      • Hobson S.A.
      • Wynick D.
      • Hokfelt T.
      • et al.
      Physiology, signaling, and pharmacology of galanin peptides and receptors: three decades of emerging diversity.
      ). Despite the major effort of several research groups to generate non-peptidergic ligands (
      • Bulaj G.
      • Green B.R.
      • Lee H.K.
      • Robertson C.R.
      • White K.
      • Zhang L.
      • et al.
      Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities.
      ,
      • Reyes-Alcaraz A.
      • Lee Y.N.
      • Son G.H.
      • Kim N.H.
      • Kim D.K.
      • Yun S.
      • et al.
      Development of spexin-based human galanin receptor type II-specific agonists with increased stability in serum and anxiolytic effect in mice.
      ), the only available non-peptidergic GAL3-R ligands are SNAP 37889 and SNAP 398299, a less soluble derivative of SNAP 37889 (
      • Swanson C.J.
      • Blackburn T.P.
      • Zhang X.
      • Zheng K.
      • Xu Z.Q.
      • Hokfelt T.
      • et al.
      Anxiolytic- and antidepressant-like profiles of the galanin-3 receptor (Gal3) antagonists SNAP 37889 and SNAP 398299.
      ). In the skin it was shown that SNAP 37889 blocks plasma extravasation induced by substance P and calcitonin gene-related peptide injection (
      • Schmidhuber S.M.
      • Rauch I.
      • Kofler B.
      • Brain S.D.
      Evidence that the modulatory effect of galanin on inflammatory edema formation is mediated by the galanin receptor 3 in the murine microvasculature.
      ). Further research with this antagonist showed unspecific toxic effects on immune cells when used in high concentrations (
      • Koller A.
      • Rid R.
      • Beyreis M.
      • Bianchini R.
      • Holub B.S.
      • Lang A.
      • et al.
      In vitro toxicity of the galanin receptor 3 antagonist SNAP 37889.
      ). Therefore, local administration, especially on the skin, could potentially be toxic to immune cells, and thereby mimic an anti-inflammatory effect. However, intraperitoneal administration of SNAP 37889 has been reported to have anti-inflammatory effects in a model of murine pancreatitis and to suppress alcohol drinking and morphine self-administration in mice (
      • Barreto S.G.
      • Bazargan M.
      • Zotti M.
      • Hussey D.J.
      • Sukocheva O.A.
      • Peiris H.
      • et al.
      Galanin receptor 3, a potential target for acute pancreatitis therapy.
      ,
      • Scheller K.J.
      • Williams S.J.
      • Lawrence A.J.
      • Djouma E.
      The galanin-3 receptor antagonist, SNAP 37889, suppresses alcohol drinking and morphine self-administration in mice.
      ). In these studies no adverse effects were reported, possibly because of the intraperitoneal route of administration. Thus, further studies should investigate if a dermal application of SNAP 37889 is feasible and could be used to treat psoriasis.
      Future studies should investigate the direct effects of the GAL system on endothelial cells because they represent a link between nerve-derived neuropeptides and immune cells. Furthermore, studies should aim at developing stable, nontoxic GAL analogs for the treatment of psoriasis and other inflammatory diseases.

      Materials and Methods

      Human tissue samples

      Experiments were performed in accordance with the Helsinki Declaration of 1975 (revised in 1983) and the guidelines of the Salzburg State Ethics Research Committee, because this was neither a clinical drug trial nor an epidemiological investigation. Upon hospital admission, all patients (white, male and female, age range = 25–62 years) signed informed consent forms concerning the surgical removal of the skin biopsy samples. The study did not extend to examination of individual case records. The anonymity of the patients has been ensured.

      Psoriasis in vivo model

      Male and female mice 8–10 weeks old received a daily topical dose of 62.5 mg of the commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals, Neuss, Germany) applied to a shaved and depilated back for 3 or 6 consecutive days, translating to a daily dose of 3.125 mg of the active compound, as published previously (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Control mice were treated similarly with a control vehicle cream (Vaseline Lanette cream, Fagron, Barsbüttel, Germany).
      Additional information on experimental animals, the psoriasis in vivo model, evaluation of skin inflammation (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ), MPO assay (
      • Schierwagen C.
      • Bylund-Fellenius A.C.
      • Lundberg C.
      Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity.
      ), RNA isolation, expression analysis, immunohistochemistry, data analysis, and statistics are provided in the Supplementary Materials online.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The authors greatly acknowledge support by the EC FP7 Capacities Specific Program-funded EMMA service project, which provided B6;129S5-Galr3tm1Lex/Orl mice. We thank Marina Picciotto for providing GAL2-KO mice. The study was supported by the Austrian Research Promotion Agency (FFG, 822782/THERAPEP) and the Paracelsus Medical University Salzburg (PMU-FFF E-13/17/087-LAN).

      Supplementary Material

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