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Human endogenous retrovirus W in brain lesions: Rationale for targeted therapy in multiple sclerosis

Open AccessPublished:April 15, 2016DOI:https://doi.org/10.1016/j.msard.2016.04.006

      Highlights

      • HERV-W Endogenous Retroviral protein MSRV-Env is detected in all MS Brains.
      • Intense staining of MSRV-Env protein coincides with areas of active demyelination.
      • It has lifelong expression from earliest demyelination to late progressive lesions.
      • It is pathogenic, inducing inflammation, autoimmunity and remyelination blockade.
      • MSRV-Env is a pathogenic target in MS for new and specific therapeutic strategies.

      Abstract

      Background objective

      Attempts to identify a causative agent of Multiple Sclerosis (MS) among environmental viruses have consistently failed suggesting that development of MS is a result from gene-environment interactions. A new pathogenic player within human genes, a human endogenous retrovirus (HERV) was identified from MS cells, named MS-associated retrovirus element (MSRV) and unveiled homologous multicopy HERVs (HERV-W). As independent studies revealed biological features of HERV-W on immune-mediated inflammation and on remyelinating cells, the present study characterized the presence of HERV-W envelope protein (MSRV-Env) at the cellular level, in different MS lesion stages to extend and validate previous studies.

      Methods

      Immunohistological analysis of HERV-W envelope cellular expression in different lesion stages from a cohort of MS brains versus controls, using well-characterized and highly specific monoclonal antibodies.

      Results

      HERV-W envelope protein was detected in all MS brains and quite essentially in lesions. Immunohistochemistry showed dominant expression in macrophages and microglia, coinciding with areas of active demyelination, spread over the active lesions, or limited to the rim of active microglia in chronic active lesions or in few surviving astrocytes of inactive plaques. Weak expression was seen in MS normal appearing white matter. In active plaques, few lymphoid cells and astrocytes were also stained. This HERV-W expression was not observed in control brains.

      Interpretation

      HERV-W was expressed in demyelinated lesions from MS brains, which were all positive for this endogenous pathogenic protein. Pronounced HERV-W immunoreactivity in active MS lesions was intimately associated with areas of active demyelination throughout the successive stages of lesion evolution in MS brains. Based on its pathogenic potential, this HERV-W (MSRV) endogenous toxin thus appears to be a novel therapeutic target in MS. It also has a unique positioning as an early and lifelong expressed pathogenic agonist, acting upstream the pathways in which dysregulated physiological effectors are usually targeted by present therapeutic strategies for MS.

      Keywords

      1. Introduction

      Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Histopathological evaluation of MS brain tissue has provided insights into key pathological features of MS, including inflammation, demyelinated lesions in the white and the grey matter, oligodendrocyte loss, defects in remyelination by oligodendrocyte precursor cells (OPC), and axonal and neuronal degeneration (
      • Bien C.G.
      • Vincent A.
      • Barnett M.H.
      • Becker A.J.
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      • et al.
      Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis.
      ,
      • Chang A.
      • Tourtellotte W.W.
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      • Trapp B.D.
      Premyelinating oligodendrocytes in chronic lesions of multiple sclerosis.
      ). To date, a global understanding of etiological factors that could directly and/or indirectly be involved in MS, in its onset, in the lifelong mechanisms of inflammation-driven CNS damage and in the impairment of lesion remyelination, is likely to rely upon a gene-environment interplay (
      • van der Mei I.
      • Lucas R.M.
      • Taylor B.V.
      • Valery P.C.
      • Dwyer T.
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      Population attributable fractions and joint effects of key risk factors for multiple sclerosis.
      ,
      • Hedstrom A.K.
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      ,
      • Koch M.W.
      • Metz L.M.
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      Epigenetic changes in patients with multiple sclerosis.
      ).
      It therefore appears relevant that the search for pathogenic players in MS led to peculiar elements with both genomic and viral characteristics: Human Endogenous RetroViruses (HERVs), which have repeatedly entered the genome of species through germ-line infections during evolution of species and are now known to represent about 8% of the human genome (
      • Feschotte C.
      • Gilbert C.
      Endogenous viruses: insights into viral evolution and impact on host biology.
      ).
      The idea that a retrovirus could play a role in MS pathogenesis was evoked, but only a classically exogenous and infectious retrovirus such as a Human T-Lymphotropic virus (HTLV) was envisaged (
      • Koprowski H.
      • DeFreitas E.C.
      • Harper M.E.
      • Sandberg-Wollheim M.
      • Sheremata W.A.
      • et al.
      Multiple sclerosis and human T-cell lymphotropic retroviruses.
      ). So, when a new human retrovirus was identified in cultures from MS cells (MSRV, for Multiple sclerosis associated RetroVirus) but also unveiled a previously unknown family of homologous and endogenous retroviral sequences (HERV-W) (
      • Blond J.L.
      • Beseme F.
      • Duret L.
      • Bouton O.
      • Bedin F.
      • et al.
      Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family.
      ,
      • Perron H.
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      • Bedin F.
      • Beseme F.
      • Paranhos-Baccala G.
      • et al.
      Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis.
      ,
      • Perron H.
      • Lalande B.
      • Gratacap B.
      • Laurent A.
      • Genoulaz O.
      • et al.
      Isolation of retrovirus from patients with multiple sclerosis.
      ), these findings were initially regarded as anecdotal. Nonetheless, as studies further explored and progressively unveiled unexpected genetic and biological features of these HERVs, it became apparent that HERVs were not merely fossils of past infection and inert relics of ancient genomic invasions, but could display activities with a substantial impact on cellular function, in both health and disease (
      • Feschotte C.
      • Gilbert C.
      Endogenous viruses: insights into viral evolution and impact on host biology.
      ,
      • Perron H.
      • Lang A.
      The human endogenous retrovirus link between genes and environment in multiple sclerosis and in multifactorial diseases associating neuroinflammation.
      ,
      • Kassiotis G.
      Endogenous retroviruses and the development of cancer.
      ,
      • Sokol M.
      • Jessen K.M.
      • Pedersen F.S.
      Utility of next-generation RNA-sequencing in identifying chimeric transcription involving human endogenous retroviruses.
      ,
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,
      • Varela M.
      • Spencer T.E.
      • Palmarini M.
      • Arnaud F.
      Friendly viruses: the special relationship between endogenous retroviruses and their host.
      ). This also highlighted the existence of a new category of pathogens within our genomes behaving as the “enemy within” (
      • Engel M.E.
      • Hiebert S.W.
      The enemy within: dormant retroviruses awaken.
      ,
      • Volkman H.E.
      • Stetson D.B.
      The enemy within: endogenous retroelements and autoimmune disease.
      ). Thus, cumulated results from numerous studies during past decades progressively led to understand how their unique genetic positioning may confer upon them the potential to drive pathogenic cascades leading to MS in response to environmental triggers. HERV-W sequences isolated from MS were shown to produce a bioactive envelope protein (MSRV-Env) engaging pathways leading to final pathognomonic features of Multiple Sclerosis (MS).
      In particular, immunological effects of MSRV particles revealed to induce superantigen-like activation of T-lymphocytes (
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ), itself conditioned to an upstream activation of innate immunity causing pro-inflammatory effects through the Toll-like receptor 4 (TLR4) pathway (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ). These effects were shown to be specifically due to the HERV-W encoded envelope protein (MSRV-Env) (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ), which displays original and different biological effects compared to another known HERV-W full-length envelope protein, now named Syncytin (
      • Duperray A.
      • Barbe D.
      • Raguenez G.
      • Weksler B.B.
      • Romero I.A.
      • et al.
      Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.
      ). MSRV-Env was also shown to promote macrophage and dendritic cells differentiation, anti-myelin oligodendrocyte glycoprotein autoimmunity and experimental autoimmune encephalomyelitis (EAE) in C57/BL/6 mice, an animal model of MS, (
      • Perron H.
      • Dougier-Reynaud H.L.
      • Lomparski C.
      • Popa I.
      • Firouzi R.
      • et al.
      Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice.
      ). Parallel studies on glial cells had showed gliotoxicity induced by MSRV particles with reverse-transcriptase activity (
      • Menard A.
      • Amouri R.
      • Michel M.
      • Marcel F.
      • Brouillet A.
      • et al.
      Gliotoxicity, reverse transcriptase activity and retroviral RNA in monocyte/macrophage culture supernatants from patients with multiple sclerosis.
      ), but its molecular origin and mechanisms of action long remained unknown. However, it was recently discovered that transiently expressed TLR4 in early differentiating oligodendrocyte precursor cells (OPC) exposed them to an interaction with MSRV-Env causing a defect of myelin production through a differentiation blockade of OPC (
      • Kremer D.
      • Schichel T.
      • Forster M.
      • Tzekova N.
      • Bernard C.
      • et al.
      Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation.
      ). These observations indicated potential therapeutic application when a specific monoclonal antibody was shown to neutralize the immune pathogenic activity of MSRV-Env in vitro and in vivo (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,

      Curtin, F., Perron, H., Kromminga, A., Porchet, H., Lang, A.B., 2015. Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein.MAbs. 7 (1), 265–275, 10.4161/19420862.2014.985021.

      ) and, also, to neutralize its neuroglial pathogenic effects by reversing the inhibition of myelin protein production by OPC (

      D Kremer, M Förster, T Schichel, P Göttle, HP Hartung, et al. (2014) The neutralizing Antibody GNbAC1 Abrogates HERV-W Envelope protein-mediated oligodendroglial Maturation Blockade Mul Scler.

      ).
      This appeared relevant when considering that other studies confirmed the enhanced d expression of HERV-W/MSRV elements in patients with MS and the ex-vivo and post-mortem detection of its envelope protein (
      • Dolei A.
      • Serra C.
      • Mameli G.
      • Pugliatti M.
      • Sechi G.
      • et al.
      Multiple sclerosis-associated retrovirus (MSRV) in Sardinian MS patients.
      ,
      • Garson J.A.
      • Tuke P.W.
      • Giraud P.
      • Paranhos-Baccala G.
      • Perron H.
      Detection of virion-associated MSRV-RNA in serum of patients with multiple sclerosis.
      ,
      • Mameli G.
      • Astone V.
      • Arru G.
      • Marconi S.
      • Lovato L.
      • et al.
      Brains and peripheral blood mononuclear cells of multiple sclerosis (MS) patients hyperexpress MS-associated retrovirus/HERV-W endogenous retrovirus, but not Human herpesvirus 6.
      ,
      • Sotgiu S.
      • Mameli G.
      • Serra C.
      • Zarbo I.R.
      • Arru G.
      • et al.
      Multiple sclerosis-associated retrovirus and progressive disability of multiple sclerosis.
      ).
      Importantly, the HERV-W envelope was detected in MS pathognomonic lesions, i.e. within brain demyelinated lesions, using monoclonal antibodies (mAbs) raised against the MSRV-Env protein encoded by virion RNA isolated from MS cell cultures (
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ,
      • Komurian-Pradel F.
      • Paranhos-Baccala G.
      • Bedin F.
      • Ounanian-Paraz A.
      • Sodoyer M.
      • et al.
      Molecular cloning and characterization of MSRV-related sequences associated with retrovirus-like particles.
      ). Successive studies have repeatedly and independently confirmed the presence of HERV-W envelope (here named MSRV-Env) within MS lesions with various specific mAbs, as detailed in Table 1. Such results obtained after examination of brain regions and lesions from a total of 55 MS cases from 6 different centers and countries have consolidated the evidence for the presence of HERV-W Env protein expression in MS.
      Table 1Previous studies on HERV-W envelope Immunohistological detection in MS and control brains.
      YearNumber of MS casesNeurological controls and othersHERV-W Env antibodiesHERV/Virus antibodiesMulticenterRef.
      20041618: HIV encephalitis (8); Alzheimer's Disease (6)6A2B2NoNo
      • Antony J.M.
      • van Marle G.
      • Opii W.
      • Butterfield D.A.
      • Mallet F.
      • et al.
      Human endogenous retrovirus glycoprotein-mediated induction of redox reactants causes oligodendrocyte death and demyelination.
      )
      2005Cohort 1: 8 Cohort 2: 16 (with7 Marburg’s acute MS) Cohort 3: 2 Total: 26Cohort 1: progressive multifocal leukoencephalopathy (2), amyotrophic lateral sclerosis (2), Alzheimer's Disease (1), primary brain tumors (3), and non-neurological cases (3). Cohort 2: progressive multifocal leukoencephalopathy (4). Cohort 3: non-neurological cases (2)13H5A5, 3C1D5, 6A2B2, 5E9H9, 2A12A5HERV-K: (Negative in MS)Yes(
      • Perron H.
      • Lazarini F.
      • Ruprecht K.
      • Pechoux-Longin C.
      • Seilhean D.
      • et al.
      Human endogenous retrovirus (HERV)-W ENV and GAG proteins: physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions.
      )
      2007510: Alzheimer's Disease (2), astrocytoma (4), non-neurological cases (4)6A2B2HHV-6: (Negative in MS)No(
      • Mameli G.
      • Astone V.
      • Arru G.
      • Marconi S.
      • Lovato L.
      • et al.
      Brains and peripheral blood mononuclear cells of multiple sclerosis (MS) patients hyperexpress MS-associated retrovirus/HERV-W endogenous retrovirus, but not Human herpesvirus 6.
      )
      20128non-neurological cases (3)GN-mAb03,−04 and −05 (3 distant epitopes detected in same lesions)NoNo(
      • Perron H.
      • Germi R.
      • Bernard C.
      • Garcia-Montojo M.
      • Deluen C.
      • et al.
      Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease.
      )
      Total55 MS cases
      These observations reported its detection in astroglial or macrophage-like cells within MS lesions and in endothelial cells within Marburg's type hyperacute lesions. However, which immune or glial cells expressed this protein in the CNS and where, during the development of the lesions and according to their different stages of activity until the end of MS patient's life (when autopsy material is collected), still remained open questions. Further determining when such an expression arose and localized according to MS lesion development required a systematic analysis of MS brains, from first perivascular inflammatory infiltrates to late burnt out plaques.
      To address these questions, the present study performed a systematic analysis of HERV-W envelope protein (MSRV-Env) distribution and cellular localization in different stages of MS lesion development, using a large cohort of well-characterized MS samples with one of the 3 highly specific monoclonal antibodies that were previously used to examine its expression in sections of MS lesions (
      • Perron H.
      • Germi R.
      • Bernard C.
      • Garcia-Montojo M.
      • Deluen C.
      • et al.
      Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease.
      ).
      Here we show that MSRV-Env protein is abundantly present in MS brain lesions and is intimately associated with active areas of demyelination where macrophages and microglia represent the predominant cell type expressing MSRV Env. Only moderate expression was observed in reactive astrocytes throughout active and chronic active lesion areas, while rarely in inactive lesion areas. Expression in perivascular macrophages and in neighboring endothelial cells appeared quite essentially in early demyelinated areas.

      2. Materials and methods

      2.1 Autopsy material

      Brain samples from 20 patients with clinically diagnosed and neuropathologically confirmed MS were obtained at rapid autopsy and immediately fixed in buffered formalin (in collaboration with The Netherlands Brain Bank, Amsterdam; Dr. I. Huitinga, coordinator). The Netherlands Brain Bank received permission to perform autopsies for the use of tissue and for access to medical records for research purposes from the ethics committee of the VU Medical Center (Amsterdam, The Netherlands). Six cases without neurological disease were selected as controls. Tissue samples from control cases were taken from the subcortical white matter or corpus callosum, regions where most MS lesions were encountered and therefore analyzed. MS tissue samples were selected on the basis of postmortem MRI and lesions were classified according to validated histopathological criteria as previously published (
      • De Groot C.J.
      • Bergers E.
      • Kamphorst W.
      • Ravid R.
      • Polman C.H.
      • et al.
      Post-mortem MRI-guided sampling of multiple sclerosis brain lesions: increased yield of active demyelinating and (p)reactive lesions.
      ). MRI-guided white matter lesions were thus obtained from different locations in every case. Hence, cases with different type of lesion stages was included in the present MS cohort. However, different types of lesions were also obtained from individual brains as lesional activity can still be detected at autopsy in most cases (
      • van Horssen J.
      • Singh S.
      • van der Pol S.
      • Kipp M.
      • Lim J.L.
      • et al.
      Clusters of activated microglia in normal-appearing white matter show signs of innate immune activation.
      ). Relevant clinical information was retrieved from the medical records and is summarized in Table 2. All patients and controls, or their next of kin, had given informed consent for autopsy and the use of their brain tissue for research purposes.
      Table 2Detailed information on MS and control cases included in the immunohistochemical study.
      casegenderage (years)Post-mortem delay (h:m)disease duration (years)Type of MSType of lesion
      MS 1m441216NDA
      MS 2m511110SPA
      MS 3m548:509NDA
      MS 4f66622SPA
      MS 5m767:3055NDA
      MS 6f887:5525PPA
      MS 7m4410:1021NDCA
      MS 8f474:25NDNDCA
      MS 9m417:2314PPCA
      MS 10m49825SPCA
      MS 11m667:3026PPCA
      MS 12m56827SPCA
      MS 13f817:1758SPCA
      MS 14m577:5028SPCIA
      MS 15f769:4519SPCIA
      MS 16m509:3024NDCIA
      MS 17f706:5540NDCIA
      MS 18f697:3053SPCIA
      MS 19m556:2031SPCIA
      MS 20f4099PPCIA
      (Controls)Cause of death
      Control 1m569:15MI
      Control 2f648:25Urosepsis and pneumonia
      Control 3m718:55unknown
      Control 4f734Respiratory failure with heart failure
      Control 5f667cachexia
      Control 6m629:35unknown
      m: male, f: female, ND: not determined, SP: secondary progressive, PP: primary progressive, A: active, CA: chronic active, CIA: chronic inactive, MI: myocardial infarction.

      2.2 Antibodies

      All antibodies were mAb mouse IgGs obtained after immunization with either recombinant protein (Env) or immunization plasmid. Selected hybridomas were cultured in vitro for antibody production. mAb purification was made with protein-A sepharose columns and purity of the final product controlled by HPLC analysis. Their specificity was controlled by using MSRV-Env proteins produced in E. coli (non-glycosylated) or in transfected human cell cultures (glycosylated) and further purified as endotoxin-free recombinant full-length proteins for ELISA and WB analyses. Transfected cells were used for Immunofluorescence and immunocytochemistry analyses of mAb specificity (Cf. Supplementary material).

      2.3 Immunohistochemistry

      Formalin-fixed paraffin-embedded tissue from each selected region was serially sectioned at 5 µm within large and successive areas covering the entire lesion and stained for proteolipid protein (PLP; clone plpc1; Serotec) and major histocompatibility complex (MHC) class II (DAKO) to characterize lesion stage. HERV-W envelope protein (MSRV-Env) was detected using GN-mAb_03 at a concentration of 10 µg/ml. Sections were deparaffinized in xylene and rehydrated through graded alcohol into distilled water and endogenous peroxidase activity was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol. Serial sections of each tissue sample were incubated overnight with appropriate antibodies and subsequently incubated with horseradish peroxidase-labeled anti-mouse/rabbit from the EnVision kit (Dako) for 30 min at room temperature and finally diaminobenzidine tetrachloride. Between incubation steps, the sections were thoroughly washed with PBS. After a short rinse in tap water the sections were incubated with hematoxylin for 1 min and extensively washed with tap water for 10 min. Finally, the sections were dehydrated with ethanol followed by xylol and mounted with Entellan (Merck, Darmstadt, Germany) for systematic examination of all lesion areas. All antibodies were diluted in PBS containing 0.1% bovine serum albumin (Boehringer Mannheim). For cellular localization sections were incubated overnight with antibodies directed against HERV-W Env protein followed by incubation with Alexa-594-labeled goat anti-mouse (1:400; Molecular Probes). Astrocytes were visualized using rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Dako) and microglia/macrophages using either rabbit anti-Iba1 (1:100; Wako, ref. 019-19741) or rabbit anti-p22phox (Santa Cruz Biotechnology, ref. sc-20781). Both antibodies were applied for 1 h and followed by incubation with Alexa-488-labeled goat anti-rabbit (1:400; Molecular Probes). After being washed, the slides were covered with Vectashield (Vector Laboratories) supplemented with 0.4% DAPI to stain nuclei. Microscopical analysis was performed with a Leica TCS SP2 AOBS confocal laser-scanning microscope (Leica Microsystems, Heidelberg, Germany).

      3. Results

      Sections were prepared from tissue blocks of 20 MS patients and classification of lesion staging was based on immunohistochemical detection of inflammatory cells expressing MHC class II/HLA-DR antigens and on the detection pattern of PLP, which reveal areas of myelin loss or the presence of myelin in phagocytic cells as previously described (
      • Laman J.D.
      • Visser L.
      • Maassen C.B.
      • de Groot C.J.
      • de Jong L.A.
      • et al.
      Novel monoclonal antibodies against proteolipid protein peptide 139–151 demonstrate demyelination and myelin uptake by macrophages in MS and marmoset EAE lesions.
      ). Based on these stainings we selected 6 active, 7 chronic active and 7 chronic inactive lesions in studied sections. “Active” lesion areas were classified as lesions with myelin loss and abundant parenchymal macrophages. “Chronic active” lesion areas were characterized by a hypocellular demyelinated gliotic center with astrogliosis and a hypercellular rim containing activated microglia and macrophages. “Chronic inactive” lesion areas, in contrast to “active” and “chronic active” lesion areas, were devoid of macrophages and only sporadically contained some activated microglia. White matter samples from all control samples did not show any sign of changes in myelin distribution and lacked activated microglia and infiltrated macrophages.
      In control white matter samples, anti-Env antibody only weakly decorated isolated glial cells (Fig. 1). Glial cells with weak Env-positivity were seen in NAWM surrounding MS lesions (data not shown).
      Fig. 1.
      Fig. 1HERV-W Env protein was rarely detected in control white matter samples (A; proteolipid protein-PLP). Example of cellular expression restricted to rare isolated glial cells (B, inset). The magnification is indicated by the bars representing 50 µm.
      With marked contrast, when compared to control white matter samples and NAWM of MS brains, MSRV-Env was abundantly present in demyelinated areas with ongoing inflammation (Fig. 2 (A)–(C)). Particularly, infiltrating macrophages and activated microglia were markedly labeled with antibodies directed against MSRV-Env (Fig. 2(E) and (F)). Dense lymphoid infiltrates in the vicinity of this active plaque were observed and MSRV-Env was detected in a proportion of these lymphoid cells. Interestingly, it not only involved monocyte cells but also T-cells, as shown by co-staining of few CD3 positive cells (Fig. 2(G)). MSRV-Env-positive reactive astrocytes were also detected throughout active lesions (Fig. 2(C) and (F) with GFAP double staining).
      Fig. 2.
      Fig. 2Active MS lesions (A; PLP staining) with leukocyte influx (B; HLA-DR staining with anti-LN3 antibody). HERV-W Env protein was expressed by macrophages (C-magnified from boxed area in A; dotted arrow) and reactive astrocytes (C; plain arrow). HERV-W Env protein is weakly expressed in perivascular cuffs (d-the magnified region in D was distant from what had to be presented and correctly seen in A and is therefore not shown in A). With immunofluorescent labelling, HERV-W Env protein was observed in Iba-1-positive microglia (E) and GFAP-positive astrocytes (F). It was detected in few CD3-positive lymphocytes within perivascular cuff shown in D (G-magnification from D). In Figures A-D, the specific staining appears in brown with counter staining of cells in purple/blue. In Figures E-G, the specific staining appears in red for HERV-W Env, and in green for Iba-1 (E), for GFAP (F) and for CD3 (G). Co-stained areas or cells, for each combination of two markers, appear in yellow. The cell nuclei are counter stained in dark blue. The magnification is indicated by the bars representing 25 µm in A-D, 20 µm in C&G and 10 µm in E&F.
      In a very early perivascular demyelination process (Fig. 3(A)-(B)), perivascular macrophages infiltrating from the contiguous blood vessel were quite exclusively stained with anti-Env antibody as well as neighboring endothelial cells (Fig. 3(C)-(D)). Moreover, this MSRV-Env expression was restricted to the area of demyelination, as superposed with the small area with decreased or absence of PLP staining in the upper left part next to the blood vessel wall (Fig. 3(A)).Conversely, activated microglia (MHC-II positive in Fig. 3(B)) extended far beyond this initial demyelinated area, probably reflecting activation by secreted pro-inflammatory cytokines with large intraparenchymal diffusion. Thus, immunoinflammatory activation of microglia did not directly cause MSRV-Env expression, which was localized within the actively demyelinating area.
      Fig. 3.
      Fig. 3Newly forming lesion. Early perivascular demyelination is evidenced by the white area, without PLP staining, next to the top left part of the central vascular element (A). It is associated with Env-positive infiltrating perivascular macrophages only (C & d-magnification of C, centered on the blood vessel with demyelinated area on its left), whereas microglia is already activated to a longer distance from the blood vessel and from the PLP-depleted area, as shown with LN3 labelling of HLA-II expression (B). The magnification is indicated by the bars representing 25 µm in A-C and 5 µm in D.
      The cellular expression pattern of this HERV-W protein in chronic active lesion areas, while showing similarities with the distribution observed in active areas as MSRV-Env protein was markedly upregulated in activated microglia, was strikingly coinciding with the rim of a large demyelinating lesion as shown in Fig. 4. Here, the edge of an expanding lesion is decorated by an Env-positive line (4 C) coinciding with HLA DR-positive detection (4B). The fact that both activated microglia (actively demyelinating cells) and bipolar fusiform microglia (migrating cells) showed strong expression of MSRV-Env protein (4D), suggests that this “line of microglia” represents the “line of progression” of the demyelinating cells that expand the lesions in such chronic active plaques.
      Fig. 4.
      Fig. 4Chronic active demyelinated MS lesion showing a large area of completely demyelinated tissue without PLP staining (pale/white in bottom-right part in A, versus brown PLP-positive myelinated upper part), with a rim of HLA-DR-positive activated microglia (LN3-staining in B). HERV-W Env protein expression was markedly increased along the same line at the rim of the lesion (C) and co-localized with activated and migrating microglia (d-magnification of Box in C). The magnification is indicated by the bars representing 1 mm in A-C and 20 µm in D.
      In chronic inactive lesion areas, the intensity of the staining was strikingly reduced and limited to some sporadic HERV-W Env-immunoreactive astrocytes and to faint dust-like staining of fibrotic extracellular structures where secreted MSRV-Env protein could remain present (Fig. 5).
      Fig. 5.
      Fig. 5Chronic inactive demyelinated MS lesion are characterized by dominantly unstained sections with anti-PLP antibody (A, only showing a small PLP positive area in the bottom-right part) and by very few and scattered HLA-DR-positive microglia (B). In chronic inactive lesions HERV-W Env protein expression was predominantly detected in hypertrophic astrocytes and their processes (C). The magnification is indicated by the bars representing 1 mm in A-B and 20 µm in C.
      Taking together all data from this extensive immunohistochemical survey of these different types of lesions from 20 different MS brains, it became obvious that HERV-W envelope protein expression is markedly upregulated within all inflammatory lesions or within actively demyelinating microglia at the rim of chronic lesions. It predominantly localizes to macrophages, activated microglia and reactive astrocytes, intimately coinciding with actively demyelinating localization as detected in all examined MS brains (n=20).

      4. Discussion

      The HERV-W envelope protein (Env) is known to provoke immunoinflammatory and glial cythopathic effects on human cells in culture and in animal models. This was shown for MSRV-Env, as encoded by clones isolated from MS virus-like particles (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ,
      • Perron H.
      • Dougier-Reynaud H.L.
      • Lomparski C.
      • Popa I.
      • Firouzi R.
      • et al.
      Human endogenous retrovirus protein activates innate immunity and promotes experimental allergic encephalomyelitis in mice.
      ,
      • Kremer D.
      • Schichel T.
      • Forster M.
      • Tzekova N.
      • Bernard C.
      • et al.
      Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation.
      ), and even for the physiologically domesticated HERV-W Env “Syncytin” copy involved in placental syncytiotrophoblast fusion (
      • Bonnaud B.
      • Bouton O.
      • Oriol G.
      • Cheynet V.
      • Duret L.
      • et al.
      Evidence of selection on the domesticated ERVWE1 env retroviral element involved in placentation.
      ), when abnormally expressed in transgenic mouse astrocytes (
      • Antony J.M.
      • van Marle G.
      • Opii W.
      • Butterfield D.A.
      • Mallet F.
      • et al.
      Human endogenous retrovirus glycoprotein-mediated induction of redox reactants causes oligodendrocyte death and demyelination.
      ). The pathogenic effects of HERV-W envelope therefore coincide with the major pathways underlying hallmarks of MS pathology.
      Detailed immunohistochemical analysis showed that the corresponding envelope protein was detected in the successive typical stages of lesion from MS in strict association with cells and histopathological characteristics constituting hallmarks of active demyelination.
      HERV-W (MSRV-Env) expression appeared to be associated with active demyelination from the early stage, involving endothelial cells, perivascular macrophages and microglia neighboring macrophage infiltration within brain parenchyma, to the late lesion progression at the rim of reactive microglia figuring the limit between a large demyelinated plaque and the NAWM. Thus, a persisting weak detection in few surviving astrocytes within late inactive lesion also appeared consistent. At the cellular level, results of systematic observation from early to burnt-out demyelinated areas in different patients showed that (i) HERV-W Env protein production in perivascular macrophage and in microglia appear as a pivotal continuum of active demyelination, that (ii) reactive astrocytes were significantly stained in all lesion stages but to a lesser extent, and only in rare surviving astroglial cells of chronic inactive lesions, which may also correspond to Env protein capture at their surface, that (iii) occasional lymphoid infiltrates comprised positive T-cells, which may rather represent membrane bound protein than active production, and that (iv) along with perivascular macrophages, endothelial cells were stained in early demyelinated lesions. In the latter case, previous observations of similar endothelial staining in hyperacute lesions from Marburg's MS types (
      • Perron H.
      • Lazarini F.
      • Ruprecht K.
      • Pechoux-Longin C.
      • Seilhean D.
      • et al.
      Human endogenous retrovirus (HERV)-W ENV and GAG proteins: physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions.
      ) altogether with recent results showing TLR-4 mediated effects of MSRV-Env in endothelial cells (
      • Duperray A.
      • Barbe D.
      • Raguenez G.
      • Weksler B.B.
      • Romero I.A.
      • et al.
      Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.
      ) suggest an interplay between these cell types and MSRV-Env expression during the early and/or active phase of lesion formation at the blood-brain-barrier level. This is supported by negative detection in vascular elements in areas of older lesion as in NAWM. Concerning lymphocytes, as peripheral blood T-lymphocytes were reported not to express HERV-W RNA in MS, when natural killer (NK) cells did (
      • Mameli G.
      • Poddighe L.
      • Mei A.
      • Uleri E.
      • Sotgiu S.
      • et al.
      Expression and activation by Epstein Barr virus of human endogenous retroviruses-W in blood cells and astrocytes: inference for multiple sclerosis.
      ), further studies are needed to elucidate the phenotype of this T-cell subtype expressing HERV-W MSRV-Env protein in MS-brain. Nonetheless, we may simply observe what would be expected from the binding and/or the presentation by neighboring antigen-presenting cells of this endogenous retroviral protein to certain V-beta chains of the T-cell receptor, which is known to induce potent superantigen-like T-lymphocyte activation as previously shown (
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ). Since this revealed dependent upon TLR4-driven upstream activation of innate immune pathways (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ), the present observations of macrophage and/or microglial activation with MSRV-Env expression in earlier stages of demyelinated areas are consistent with their role in a subsequent recruitment of T-cells, along with induced overexpression of integrins at the surface of neighboring endothelial cells (
      • Duperray A.
      • Barbe D.
      • Raguenez G.
      • Weksler B.B.
      • Romero I.A.
      • et al.
      Inflammatory response of endothelial cells to a human endogenous retrovirus associated with multiple sclerosis is mediated by TLR4.
      ). Interestingly, this could illustrate how the activation of a disproportionate number of T-cells may occur in MS CNS, thus releasing unexpectedly huge quantities of Interferon gamma and of other pro-inflammatory molecules.
      When considering all sections studied in brains from the present cohort, 100% of MS cases were found positive for MSRV-Env protein. Since all MS cases tested in the previous studies also had shown positivity for this HERV-W envelope protein, the total number of MS cases with MSRV-Env positive brain lesions in dedicated studies is 75.
      Taken altogether, our data indicate that HERV-W envelope may be a key factor in lesion progression but this endogenous toxin is still present in the end-stage of lesions with dust-like staining of extracellular fibrotic structures or of rare surviving astrocytes. A key point is that HERV-W, at least its MSRV-Env protein, is expressed in post-mortem lesion tissue implying that it is implicated in active demyelination from the early development to the chronic expansion of lesions, until the end of MS patients’ life. It is thus likely to be produced from the onset of the disease throughout its whole course. This does not correspond to “hit-and run” pathogenic triggers nor to transiently disease-activated factors. It neither correspond to a non-specific activation of mostly defective HERV copies that can be transcriptionally upregulated, impacting various RNA detection (
      • Johnston J.B.
      Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases.
      ), but are not translated into protein production (
      • Perron H.
      • Lazarini F.
      • Ruprecht K.
      • Pechoux-Longin C.
      • Seilhean D.
      • et al.
      Human endogenous retrovirus (HERV)-W ENV and GAG proteins: physiological expression in human brain and pathophysiological modulation in multiple sclerosis lesions.
      ).
      Since HERV-W envelope has been shown to have potent immunopathogenic and gliotoxic properties (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,
      • Perron H.
      • Jouvin-Marche E.
      • Michel M.
      • Ounanian-Paraz A.
      • Camelo S.
      • et al.
      Multiple sclerosis retrovirus particles and recombinant envelope trigger an abnormal immune response in vitro, by inducing polyclonal Vbeta16 T-lymphocyte activation.
      ,
      • Kremer D.
      • Schichel T.
      • Forster M.
      • Tzekova N.
      • Bernard C.
      • et al.
      Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation.
      ), the lifelong expression of HERV-W Env protein appears to be a sufficient condition for fueling corresponding pathogenic cascades both initiated by TLR4 (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,
      • Kremer D.
      • Schichel T.
      • Forster M.
      • Tzekova N.
      • Bernard C.
      • et al.
      Human endogenous retrovirus type W envelope protein inhibits oligodendroglial precursor cell differentiation.
      ). As the resulting effects were demonstrated to be efficiently neutralized or reversed by a specific anti MSRV-Env antibody (
      • Rolland A.
      • Jouvin-Marche E.
      • Viret C.
      • Faure M.
      • Perron H.
      • et al.
      The envelope protein of a human endogenous retrovirus-W family activates innate immunity through CD14/TLR4 and promotes Th1-like responses.
      ,

      Curtin, F., Perron, H., Kromminga, A., Porchet, H., Lang, A.B., 2015. Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein.MAbs. 7 (1), 265–275, 10.4161/19420862.2014.985021.

      ,

      D Kremer, M Förster, T Schichel, P Göttle, HP Hartung, et al. (2014) The neutralizing Antibody GNbAC1 Abrogates HERV-W Envelope protein-mediated oligodendroglial Maturation Blockade Mul Scler.

      ), this confirms the specificity of MSRV-Env in the observed pathogenicity. HERV-W envelope in MS (MSRV-Env) is thus more likely to correspond to a central element of the pathogenesis and to a potential therapeutic target in this disease. Its association with demyelinating cells in the lesions support the rationale of a therapeutic strategy using a neutralizing antibody targeting this endogenous protein. A humanized neutralizing antibody is now available as a recombinant human IgG4, GNbAC1 (

      Curtin, F., Perron, H., Kromminga, A., Porchet, H., Lang, A.B., 2015. Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein.MAbs. 7 (1), 265–275, 10.4161/19420862.2014.985021.

      ). Phase I clinical trials in healthy volunteers and phase IIa in MS patients, a one-year study with monthly GNbAC1 infusions, have provided good results (

      Curtin, F., Perron, H., Kromminga, A., Porchet, H., Lang, A.B., 2015. Preclinical and early clinical development of GNbAC1, a humanized IgG4 monoclonal antibody targeting endogenous retroviral MSRV-Env protein.MAbs. 7 (1), 265–275, 10.4161/19420862.2014.985021.

      ,
      • Curtin F.
      • Lang A.B.
      • Perron H.
      • Laumonier M.
      • Vidal V.
      • et al.
      GNbAC1, a humanized monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus: a first-in-humans randomized clinical study.
      ,
      • Derfuss T.
      • Curtin F.
      • Guebelin C.
      • Bridel C.
      • Rasenack M.
      • et al.
      A phase IIa randomised clinical study of GNbAC1, a humanised monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus in multiple sclerosis patients.
      ) and confirmed that GNbAC1 did not affect patients‘ immune physiology (
      • Zimmermann M.
      • Sanderson N.S.
      • Rasenack M.
      • Lalive P.H.
      • Lang A.B.
      • et al.
      Immunologic monitoring during a phase 2a trial of the GNbAC1 antibody in patients with MS.
      ). They now await results from a large multi-center Phase IIb study, which should provide results allowing to conclude on what remains to be observed now, at the therapeutic level.

      Conflict of interest

      HP is employed by Geneuro and is co-author on patents.

      Acknowledgments

      The Immunohistological analysis was supported by Geneuro.

      Appendix A. Supplementary material

      .

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