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Thymosins in multiple sclerosis and its experimental models: moving from basic to clinical application

Published:October 03, 2018DOI:https://doi.org/10.1016/j.msard.2018.09.035

      Highlights

      • Thymosins are thymic hormone-like peptides that can mediate immune and non-immune physiological processes.
      • Thymosin-α1 (Tα1) displays powerful pleiotropic activities by promoting anti-inflammatory and regulatory milieu in different settings.
      • Thymosin-β4 (Tβ4) provides neuroprotection, immunosuppression, and neurorestoration in the central nervous system.
      • This review describes what is known on the effects and the potential mechanisms of action of treatment with Tβ4 or Tα1 in Multiple Sclerosis and its experimental models.

      Abstract

      Background: Multiple sclerosis (MS) afflicts more than 2.5 million individuals worldwide and this number is increasing over time. Within the past years, a great number of disease-modifying treatments have emerged; however, efficacious treatments and a cure for MS await discovery.
      Thymosins, soluble hormone-like peptides produced by the thymus gland, can mediate immune and non-immune physiological processes and have gained interest in recent years as therapeutics in inflammatory and autoimmune diseases.
      Methods: Pubmed was searched with no time constraints for articles using a combination of the keywords “thymosin/s” or “thymus factor/s” AND “multiple sclerosis”, mesh terms with no language restriction.
      Results: Here, we review the state-of-the-art on the effects of thymosins on MS and its experimental models. In particular, we describe what is known in this field on the roles of thymosin-α1 (Tα1) and -β4 (Tβ4) as potential anti-inflammatory as well as neuroprotective and remyelinating molecules and their mechanisms of action.
      Conclusion: Based on the data that Tα1 and Tβ4 act as anti-inflammatory molecules and as inducers of myelin repair and neuronal protection, respectively, a possible therapeutic application in MS for Tα1 and Tβ4 alone or combined with other approved drugs may be envisaged. This approach is reasonable in light of the current clinical usage of Tα1 and data demonstrating the safety, tolerability and efficacy of Tβ4 in clinical practice.

      Keywords

      Abbreviations

      1. Introduction

      1.1 Multiple sclerosis and treatment strategies

      A complex genetic component with multiple susceptibility genes as well as several environmental factors, including UV light exposure or viral infections, have been implicated in Multiple Sclerosis (MS) risk and development (
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part II: noninfectious factors..
      ,
      • Ascherio A.
      • Munger K.L.
      Environmental risk factors for multiple sclerosis. Part I: the role of infection..
      ).
      This enigmatic immune-mediated disease of the central nervous system (CNS) displays a progressive neurodegeneration due to immune attacks directly against myelin constituents that cause myelin destruction (
      • Lassmann H.
      • Bruck W.
      • Lucchinetti C.F.
      The immunopathology of multiple sclerosis: an overview.
      ) in genetically susceptible individuals.
      Treatment development in the past years has been extremely active and a great number of potential new therapies have emerged for patients with MS.
      MS therapeutic approaches focused for the past three decades on strategies to reduce inflammation and immune activation, acting on immunomodulation, on migration of specific inflammatory cell subsets or on agents directly mediating neuroprotection and neurorestoration.
      Disease-modifying therapies have shown beneficial effects in patients with relapsing-remitting MS (RRMS). The majority of these drugs are, however, of little benefit during progressive MS where axonal degeneration following demyelination outweighs inflammation. For obvious reasons, this discrepancy in therapeutic efficacy of approved drugs has sparked great interest in the development of new remyelination therapies aimed at providing neuroprotection and functional recovery.
      The monotherapeutic strategy to suppress immune cells from attacking the CNS is insufficient in preventing or ameliorating permanent and accumulating MS deficits (
      • Kremer D.
      • Kury P.
      • Dutta R.
      Promoting remyelination in multiple sclerosis: current drugs and future prospects.
      ). Additional therapies for MS are urgently required to enhance remyelination and to reduce axonal damage to improve functional recovery.
      Demyelination is a loss of myelin sheaths from axons, which results from damage and death of myelin producing oligodendrocytes (OLs). Remyelination requires oligodendrocyte progenitor cells (OPCs) to differentiate into mature myelinating OLs, because mature OLs in the adult mammalian CNS are post mitotic and, thus, unable to proliferate in response to injury and to form new myelin sheaths (
      • McTigue D.M.
      • Tripathi R.B.
      The life, death, and replacement of oligodendrocytes in the adult CNS.
      ,
      • Keirstead H.S.
      • Blakemore W.F.
      Identification of post-mitotic oligodendrocytes incapable of remyelination within the demyelinated adult spinal cord.
      ). The failure of newly generated OLs to remyelinate axons and to preserve axonal integrity impedes functional recovery after MS (
      • Kremer D.
      • Kury P.
      • Dutta R.
      Promoting remyelination in multiple sclerosis: current drugs and future prospects.
      ). Cell therapies, including stem cell transplantation, have a potential for CNS repair and may be able to provide protection from inflammatory damage caused after injury (
      • Rice C.M.
      • et al.
      Cell therapy for multiple sclerosis: an evolving concept with implications for other neurodegenerative diseases.
      ).
      The glial cell communication network, including astrocytes and microglia, also plays important roles in de/remyelination (
      • Domingues H.S.
      • et al.
      Oligodendrocyte, astrocyte, and microglia crosstalk in myelin development, damage, and repair.
      ). Thus, it is important to investigate a treatment strategy for MS that would include immunomodulation paired to neuroprotection and neurorestoration. Many new molecules or previously approved drugs developed by high-throughput screens appear to have a positive impact on remyelination and neuroprotection and are in the therapeutic pipeline envisaging their use alone or as part of a combination therapy, including immunomodulatory drugs for the treatment of MS (
      • Harlow D.E.
      • Honce J.M.
      • Miravalle A.A.
      Remyelination therapy in multiple sclerosis.
      ). Although the beneficial effects of these drugs on CNS cells are encouraging, careful study of off-target effects will need to be undertaken, given that many of these drugs were originally utilized for non-CNS targets, such as the thymic hormone-like molecules thymosins.

      1.2 The use of thymosins in MS

      Evidence is emerging that the regulation of thymic peptide hormones, such as thymosins, have anti-inflammatory potential in inflammatory as well as autoimmune diseases (
      • Lunin S.M.
      • Novoselova E.G.
      Thymus hormones as prospective anti-inflammatory agents.
      ).
      In this context, in early studies conducted using thymosin fraction V to treat old mice (
      • Endoh M.
      • Tabira T.
      Effect of thymic hormones on induction of experimental allergic encephalomyelitis in old mice.
      ) or guinea pigs (
      • Woyciechowska J.
      • Goldstein A.L.
      • Driscoll B.
      Experimental allergic encephalomyelitis in guinea pigs. Influence of thymosin fraction V on the disease..
      ) subjected to the model of MS experimental autoimmune encephalomyelitis (EAE), the treatment showed no suppressive effect on incidence and severity of disease. In the first study (
      • Endoh M.
      • Tabira T.
      Effect of thymic hormones on induction of experimental allergic encephalomyelitis in old mice.
      ), however, EAE was induced in aged mice, in which susceptibility to disease may be significantly reduced. Indeed, in the same setting, also treatment with another thymic protein, the serum thymic factor, showed no effect, differently from that observed by other groups describing an intensive suppression of EAE symptoms (
      • Nagai Y.
      • Osanai T.
      • Sakakibara K.
      Intensive suppression of experimental allergic encephalomyelitis (EAE) by serum thymic factor and therapeutic implication for multiple sclerosis.
      ). Moreover, thymosin fraction V is a partially purified mixture of 10 major and at least other 30 polypeptides from the thymus gland with extremely varied and important biological properties that may act individually, sequentially, or in concert to influence the development of T cell subsets and that could also mediate inhibitory effects not observed in experiments conducted with single or a mixture of purified thymosins (
      • Hoch K.
      • Volk D.E.
      Structures of thymosin proteins.
      ,
      • Goldstein A.L.
      • Badamchian M.
      Thymosins: chemistry and biological properties in health and disease.
      ).
      Indeed, therapeutic benefits were observed in the damaged CNS of neurological disorders, including MS, when the synthetic form of thymosin β4 (Tβ4), a single peptide purified from thymosin fraction V able to pass the blood brain barrier (
      • Mora C.A.
      • et al.
      Biodistribution of synthetic thymosin beta 4 in the serum, urine, and major organs of mice.
      ), was exogenously administered. Using animal models of neurological injury, studies have demonstrated that Tβ4 can target multiple neural cells (including neurons, oligodendrocytes and microglia) and can also provide neuroprotection, immunosuppression, and neurorestoration, including remyelination, synaptogenesis, and axon growth (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ,
      • Zhang J.
      • et al.
      Function of thymosin beta-4 in ethanol-induced microglial activation.
      ,
      • Chopp M.
      • Zhang Z.G.
      Thymosin beta4 as a restorative/regenerative therapy for neurological injury and neurodegenerative diseases.
      ,
      • Santra M.
      • et al.
      Thymosin beta 4 mediates oligodendrocyte differentiation by upregulating p38 MAPK.
      ,
      • Santra M.
      • et al.
      Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.
      ,
      • Wang L.
      • et al.
      Therapeutic benefit of extended thymosin beta4 treatment is independent of blood glucose level in mice with diabetic peripheral neuropathy.
      ,
      • Wang L.
      • et al.
      Thymosin beta4 promotes the recovery of peripheral neuropathy in type II diabetic mice.
      ,
      • Cheng P.
      • et al.
      Beneficial effects of thymosin beta4 on spinal cord injury in the rat.
      ).
      Furthermore, studies showed that purified prothymosin α (ProTα), the precursor of thymosin α1 (Tα1), is able to regulate the defective phenotype of monocytes in MS impacting T cell activation (
      • Reclos G.J.
      • et al.
      Multiple sclerosis: II. Effects of prothymosin alpha on the autologous and allogeneic MLR in patients with multiple sclerosis..
      ,
      • Baxevanis C.N.
      • et al.
      Prothymosin-alpha enhances HLA-DR antigen expression on monocytes from patients with multiple sclerosis.
      ). More recently, the anti-inflammatory potential of Tα1 on the differentiation of regulatory subsets of lymphocytes was also studied in MS (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ).
      In this review we will describe in detail what is known on the role of Tβ4 and Tα1 in promotion of remyelination and anti-inflammatory responses in MS and the potential mechanisms of action.

      2. Methods

      2.1 Search strategy and selection criteria

      A literature search was carried out on PubMed/Medline database. The authors deemed important not to miss any potentially relevant study, therefore a comprehensive search strategy was set and no limits were fixed as to language or date of publication. Based on the review topic, the search strategy included controlled vocabulary and free-text words, synonyms and MeSH terms relating to thymosin “(thymosin/s", "thymus factor/s”, “thymus peptide/s”, “thymic peptide/s”, “thymosin fraction 5”, “thymosin beta 4”, “thymosin alpha 1”, “prothymosin”), combined with the main search filter (multiple sclerosis).
      The final reference list was generated considering titles relevant to the review topic based on the online searches updated to June 2018 as well as citations from other bibliographies or authors’ suggestions.

      3. Thymosins: the old and the new

      The thymus gland produces soluble hormone-like peptides that can mediate immune and non-immune physiological processes. Thymosin was originally prepared as a crude extract of mouse or rat thymus gland in 1966. In the next decades, the first biologically active thymic extract was purified and called thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, thus so named thymosins (
      • Goldstein A.L.
      History of the discovery of the thymosins.
      ). However, these molecules are genetically unrelated while being distributed widely throughout most tissues and play important, yet very different, roles in cells. The active peptides are typically short, highly charged with no or few aromatic amino acids and, therefore, are intrinsically unstructured proteins under natural conditions (
      • Hoch K.
      • Volk D.E.
      Structures of thymosin proteins.
      ).
      Starting with fraction V, several main peptides (ProTα, Tα1, polypeptide β1 and different Tβ) were isolated and tested for biological activity (Table 1).
      Table 1Thymosin proteins and their functions.
      NameSpeciesRoleReferences
      PROTHYMOSIN α (ProTα)mice, rat, humanchromatin remodeling, transcriptionally regulation, cell proliferation and survival(
      • Ueda H.
      • et al.
      Prothymosin alpha-deficiency enhances anxiety-like behaviors and impairs learning/memory functions and neurogenesis.
      ,
      • Samara P.
      • et al.
      Prothymosin alpha: an alarmin and more.
      ,
      • Samara P.
      • Ioannou K.
      • Tsitsilonis O.E.
      Prothymosin alpha and immune responses: are we close to potential clinical applications?.
      ,
      • Ueda H.
      • Matsunaga H.
      • Halder S.K.
      Prothymosin alpha plays multifunctional cell robustness roles in genomic, epigenetic, and nongenomic mechanisms.
      )
      THYMOSIN-α1 (Tα−1)rat, mice, humansimmunoregulation(
      • You J.
      • et al.
      Efficacy of thymosin alpha-1 and interferon alpha in treatment of chronic viral hepatitis B: a randomized controlled study.
      ,
      • Romani L.
      • et al.
      Thymosin alpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling.
      ,
      • King R.
      • Tuthill C.
      Immune Modulation with Thymosin Alpha 1 Treatment.
      ,
      • Liu F.
      • et al.
      The efficacy of thymosin alpha1 as immunomodulatory treatment for sepsis: a systematic review of randomized controlled trials.
      ,
      • Romani L.
      • et al.
      Thymosin alpha1 represents a potential potent single-molecule-based therapy for cystic fibrosis.
      ,
      • Garaci E.
      • et al.
      Thymosin alpha 1: from bench to bedside.
      )
      THYMOSIN-β4 (Tβ−4)mice, rats, humans, cattle, chimpazeesactin polymerization, angiogenesis, cell migration, collagen deposition, wound healing, fibrosis, neovasculogenesis, tissue repair and regeneration(
      • Chopp M.
      • Zhang Z.G.
      Thymosin beta4 as a restorative/regenerative therapy for neurological injury and neurodegenerative diseases.
      ,
      • Kuzan A.
      Thymosin beta as an actin-binding protein with a variety of functions.
      ,
      • Goldstein A.L.
      • Kleinman H.K.
      Advances in the basic and clinical applications of thymosin beta4.
      )
      THYMOSIN-β10 (Tβ−10)rat, mice, humans, cattle,cytoskeleton organization and morphology, proliferation, motility, anti-inflammatory effects, insulin secretion(
      • Sribenja S.
      • et al.
      Advances in thymosin beta10 research: differential expression, molecular mechanisms, and clinical implications in cancer and other conditions.
      ,
      • Zhang X.
      • et al.
      Thymosin beta 10 is a key regulator of tumorigenesis and metastasis and a novel serum marker in breast cancer.
      )
      THYMOSIN-β15 (Tβ−15)rat, mice, humanmotility, progression and metastatis of non-small cell lung cancer(
      • Banyard J.
      • Hutchinson L.M.
      • Zetter B.R.
      Thymosin beta-NB is the human isoform of rat thymosin beta15.
      )
      Thymosins are divided into 3 main groups based on the isoelectric focusing pattern of thymosin fraction V: α-thymosins below pH 5.0, β-thymosins between pH 5.0 and 7.0 and γ-thymosins above pH 7. The numerical subscript simply denotes the chronological order of isolation. The first two peptides isolated from fraction V were Tα1 and polypeptide β.
      In general, the peptides isolated from the β region of thymosin fraction V do not appear to be thymus-specific products. The most predominant band on the β region is polypeptide β1 that did not show any biological activity, though it was the most prominent component of thymosin fraction V. Later, it was identified as a 74-amino-acid residue fragment of ubiquitin, lacking two glycine residues at the C-terminus (
      • Hoch K.
      • Volk D.E.
      Structures of thymosin proteins.
      ). The next thymosin, which was isolated and sequenced, was termed Tβ4. Successively, β8, 9, 10 and 15 were isolated (
      • Hoch K.
      • Volk D.E.
      Structures of thymosin proteins.
      ).
      Many orthologs of human thymosin genes sharing chromosome location and/or sequence similarities have been characterized in different species. For a summary of the main thymosin genes’ characteristics and tissue distribution between human and mouse see Table 2.
      Table 2Thymosin genes: chromosome location and tissue distribution in human and mouse
      Name (gene)SpeciesChromosomeLocationTissues
      Prothymosin-α (PTMA)Homo sapiens2NC_000002.12Bone marrow, lymph node, appendix, endometrium, thyroid and urinary bladder
      Mus musculus1NC_000067.6CNS, limb, liver, thymus, adult ovary and placenta
      Thymosin beta 4 X-linked (TMSB4X)Homo sapiensXNC_000023.11spleen, lymph node, lung, appendix, bone marrow
      Mus musculusXNC_000086.7bladder adult, placenta adult and CNS
      Thymosin beta 4 Y-linked (TMSB4Y)Homo sapiens (homolog of TMSB4X)YNC_000024.10testis, prostate and colon
      No ortholog in Mus musculus
      Thymosin beta 10 (TMSB10)Homo sapiens2NC_000002.12appendix , colon, spleen, lymph node, lung, ovary
      Mus musculus6NC_000072.6CNS and placenta adult
      Thymosin beta 15a (TMSB15A)Homo sapiensXNC_000023.11prostate, endometrium, testis, thyroid
      Mus musculusXNC_000086.7Restricted expression toward testis adult
      Thymosin beta 15b (TMSB15B)Homo sapiensXNC_000023.11ovary, endometrium and prostate
      No ortholog in Mus musculus
      Several attempts were made in trying to characterize the cellular receptors involved in thymosins’ recognition; however, so far, none have been identified (
      • Rinaldi Garaci C.
      • et al.
      Receptors for thymosin alpha 1 on mouse thymocytes.
      ,
      • Brelinska R.
      • Warchol J.B.
      Receptors for thymosin fraction V on rat thymic lymphocytes.
      ).
      To-date, main members of the thymosin family are considered Tα1 and its precursor ProTα, as well as β-thymosins (
      • Mosoian A.
      Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies.
      ).
      ProTα is a 12.5-kDa, highly acidic protein, widely distributed in different cell types and expressed at both intracellular and extracellular levels. In humans, a family of 7 genes, 6 of which are considered pseudogenes, encode ProTα (
      • Mosoian A.
      Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies.
      ). The major intracellular functions of ProTα are linked to chromatin remodeling, cell proliferation, differentiation and apoptosis, and ProTα was also found overexpressed in different cancer types (
      • Mosoian A.
      Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies.
      ). The extracellular ProTα shares many features with interleukin (IL)-1α, thus representing an important endogenous stimulator of the innate immune system related to anti-viral, anti-cancer, anti-fungal and anti-ischemic activities, as well as an adjuvant for vaccines (
      • Mosoian A.
      Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies.
      ). Of note, ProTα signaling via toll-like receptor (TLR) 4 is required for its potent anti-human immunodeficiency virus (HIV) activity in macrophages via type I IFN induction (
      • Mosoian A.
      Intracellular and extracellular cytokine-like functions of prothymosin alpha: implications for the development of immunotherapies.
      ).
      Tα1 peptide, which is only 28 amino acids-long, is contained at the N-terminus of its precursor ProTα, nearly 100 bases-long. It was shown that a lysosomal asparaginyl endopeptidase (the so-called legumain) is able to proteolytically process the asparagynil-glycine residues Asn28-Gly29 of ProTα to generate Tα1 (
      • Sarandeses C.S.
      • et al.
      Prothymosin alpha is processed to thymosin alpha 1 and thymosin alpha 11 by a lysosomal asparaginyl endopeptidase.
      ).
      Since its discovery, investigations on Tα1 were mainly performed in the area of infectious diseases. Tα1 administration was studied in a wide variety of animal and human settings and its pharmacologic effects were shown to enhance cellular immunity inhibiting viral replication of different viruses. Based on these data, Tα1 was then used for the treatment of chronic hepatitis B and C (
      • Iino S.
      • et al.
      The efficacy and safety of thymosin alpha-1 in Japanese patients with chronic hepatitis B; results from a randomized clinical trial.
      ,
      • You J.
      • et al.
      Efficacy of thymosin alpha-1 and interferon alpha in treatment of chronic viral hepatitis B: a randomized controlled study.
      ), cytomegalovirus infection (
      • Bozza S.
      • et al.
      Thymosin alpha1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo.
      ) and invasive aspergillosis (
      • Romani L.
      • et al.
      Thymosin alpha 1 activates dendritic cells for antifungal Th1 resistance through toll-like receptor signaling.
      ). In addition, the post-marketing data on Zadaxin® (SciClone) clearly confirmed Tα1 immunomodulatory activities and related therapeutic potential also in cancer (such as Hepatocellular carcinoma, lung cancer, and melanoma), infectious diseases (sepsis, infections after bone marrow transplant, lung infections including chronic obstructive pulmonary disorder, Severe acute respiratory syndrome, and HIV) (
      • King R.
      • Tuthill C.
      Immune Modulation with Thymosin Alpha 1 Treatment.
      ,
      • Liu F.
      • et al.
      The efficacy of thymosin alpha1 as immunomodulatory treatment for sepsis: a systematic review of randomized controlled trials.
      ,
      • Matteucci C.
      • et al.
      Thymosin alpha 1 and HIV-1: recent advances and future perspectives.
      ,
      • Jia Z.
      • et al.
      Thymosin alpha1 plus routine treatment inhibit inflammatory reaction and improve the quality of life in AECOPD patients.
      ) and improvement in immune responses of elderly immunocompromised patients (for example for enhancement of response to vaccines) (
      • Tuthill C.
      • et al.
      Thymosin alpha1 continues to show promise as an enhancer for vaccine response.
      ).
      The Tβ family is composed of 20 short (40–44 amino acids) peptides; among these only three of them have been characterized in detail, Tβ4, Tβ10 and Tβ15. However, while Tβ15 was found only up-regulated in different malignancies, such as the human prostate cancer, representing a potential biomarker (
      • Bao L.
      • et al.
      Thymosin beta 15: a novel regulator of tumor cell motility upregulated in metastatic prostate cancer.
      ), in a healthy human body only Tβ4 and Tβ10 are expressed (
      • Huff T.
      • et al.
      Beta-Thymosins, small acidic peptides with multiple functions.
      ). These peptides play numerous different functions. Among others, they affect the processes of carcinogenesis, differentiation and angiogenesis, influence metalloproteinase activity and accelerate wound healing.
      Tβ4 is a highly conserved, 43-amino acidic peptide, which is widely distributed in mammalian tissues, including the CNS. Tβ4 is the major G-actin-sequestering molecule, and its primary physiological function is to regulate cell motility (
      • Bock-Marquette I.
      • et al.
      Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair.
      ). During development of CNS, Tβ4 regulates neurogenesis, tangential expansion, tissue growth and hemisphere folding (
      • Lever M.
      • et al.
      Thymosin beta4 overexpression regulates neuron production and spatial distribution in the developing avian optic tectum.
      ,
      • Wirsching H.G.
      • et al.
      Thymosin beta4 induces folding of the developing optic tectum in the chicken (Gallus domesticus).
      ,
      • Wirsching H.G.
      • et al.
      Thymosin beta 4 gene silencing decreases stemness and invasiveness in glioblastoma.
      ). Tβ4 was initially employed as an anti-inflammatory agent (
      • Badamchian M.
      • et al.
      Thymosin beta(4) reduces lethality and down-regulates inflammatory mediators in endotoxin-induced septic shock.
      ,
      • Girardi M.
      • et al.
      Anti-inflammatory effects in the skin of thymosin-beta4 splice-variants.
      ) and, subsequently, to inhibit proliferation and induce differentiation and apoptosis of leukemic cells (
      • Huang W.Q.
      • Wang B.H.
      • Wang Q.R.
      Thymosin beta4 and AcSDKP inhibit the proliferation of HL-60 cells and induce their differentiation and apoptosis.
      ). Recently, Tβ4 healing properties were described in skin, cornea and cardiac repair (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ,
      • Chopp M.
      • Zhang Z.G.
      Thymosin beta4 as a restorative/regenerative therapy for neurological injury and neurodegenerative diseases.
      ,
      • Bock-Marquette I.
      • et al.
      Thymosin beta4 activates integrin-linked kinase and promotes cardiac cell migration, survival and cardiac repair.
      ,
      • Badamchian M.
      • et al.
      Identification and quantification of thymosin beta4 in human saliva and tears.
      ,
      • Li Y.
      • et al.
      MiR-200a mediates protection of thymosin beta 4 in cardiac microvascular endothelial cells as a novel mechanism under hypoxia-reoxygenation injury.
      ,
      • Kim J.
      • et al.
      Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.
      ,
      • Goldstein A.L.
      • et al.
      Thymosin beta4: a multi-functional regenerative peptide. Basic properties and clinical applications..
      ).

      4. The established role of Tβ4 as a restorative therapy for MS

      4.1 The effect of Tβ4 on neuroprotection in the MS animal model

      Neuroprotection is a well-investigated effect of Tβ4 (
      • Santra M.
      • et al.
      Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.
      ,
      • Xiong Y.
      • et al.
      Neuroprotective and neurorestorative effects of thymosin beta4 treatment initiated 6 hours after traumatic brain injury in rats.
      ). In spinal cord injury and traumatic brain injury models, Tβ4 treatment significantly improved locomotor and sensorimotor functional recovery and spatial learning, as well as increased survival of neurons and OLs, and reduced cortical lesion volumes after neurological injuries (
      • Cheng P.
      • et al.
      Beneficial effects of thymosin beta4 on spinal cord injury in the rat.
      ,
      • Xiong Y.
      • et al.
      Neuroprotective and neurorestorative effects of thymosin beta4 treatment initiated 6 hours after traumatic brain injury in rats.
      ). In vitro, exogenous Tβ4 treatment significantly reduced apoptosis of the neural progenitor cells subjected to oxygen glucose deprivation (
      • Santra M.
      • et al.
      Thymosin beta 4 up-regulates miR-200a expression and induces differentiation and survival of rat brain progenitor cells.
      ). Moreover, Tβ4 may also drive neuroprotection in EAE. The EAE model is widely employed in investigation studies of MS since it exhibits significant neurological functional deficits as well as obvious pathological changes, including demyelination and inflammatory infiltration (
      • Procaccini C.
      • et al.
      Animal models of Multiple Sclerosis.
      ,
      • Eng L.F.
      • Ghirnikar R.S.
      • Lee Y.L.
      Inflammation in EAE: role of chemokine/cytokine expression by resident and infiltrating cells.
      ). When Tβ4 was administered as a prophylactic treatment on the day of proteolipid protein peptide (PLP139-151) immunization, Tβ4 treatment significantly delayed the EAE onset, and evoked a significantly improved neurological functional recovery. Since mature OLs and myelin support axonal integrity and function (
      • Nave K.A.
      Myelination and support of axonal integrity by glia.
      ), further investigation found that robust functional improvement accompanied increased numbers of mature OLs in the CNS of the EAE mice (
      • Zhang J.
      • et al.
      Neurological functional recovery after thymosin beta4 treatment in mice with experimental auto encephalomyelitis.
      ). Since the EAE model is induced by an autoimmune response, the anti-inflammatory and immunomodulatory properties of Tβ4 (
      • Badamchian M.
      • et al.
      Thymosin beta(4) reduces lethality and down-regulates inflammatory mediators in endotoxin-induced septic shock.
      ,
      • Girardi M.
      • et al.
      Anti-inflammatory effects in the skin of thymosin-beta4 splice-variants.
      ,
      • Sosne G.
      • et al.
      Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.
      ) via the suppression of nuclear factor-kappa B (NF-kB) activation (
      • Sosne G.
      • et al.
      Thymosin beta 4 suppression of corneal NFkappaB: a potential anti-inflammatory pathway.
      ) may protect OLs from damage and death. In addition to inflammatory cells (which infiltrate from peripheral circulation), microglia, the resident innate immune cells of the CNS, are activated by neuroinflammation and play a pivotal role in onset and pathological changes of disease. Thus, the effect of exogenous Tβ4 treatment on inhibition of microglial activation after damage may contribute to reduce the secretion of inflammatory mediators (
      • Zhang J.
      • et al.
      Function of thymosin beta-4 in ethanol-induced microglial activation.
      ,
      • Zhou T.
      • et al.
      Thymosin beta4 inhibits microglia activation through microRNA 146a in neonatal rats following hypoxia injury.
      ), and thereby prevent and/or reduce damage of OLs after EAE by attenuating immune onslaught (neuroprotection).
      The most important aspect of our study is in the novel evidence showing that prophylactic Tβ4 treatment may contribute to remyelination, since this treatment was able to promote an increase of new OLs generated from OPC proliferation and differentiation (
      • Zhang J.
      • et al.
      Neurological functional recovery after thymosin beta4 treatment in mice with experimental auto encephalomyelitis.
      ).

      4.2 The effects of Tβ4 on OPC differentiation and remyelination in the demyelination models

      To further investigate remyelination effects of Tβ4, the treatment window was delayed to permit the full development of demyelination damage in the CNS of EAE animals. When the therapeutic treatment of Tβ4 was administered after EAE symptoms’ onset instead of on the day of PLP immunization, functional outcomes revealed that this Tβ4 treatment approach evoked significant functional benefit (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ), and concurrently increased OLs in the demyelinating CNS. At the early stage of EAE (day 7 after onset), the protein level of myelin basic protein (MBP) and the numbers of OLs were strongly decreased. Conversely, OLs were significantly increased after Tβ4 treatment, suggesting that the newly generated OLs formed new myelin and contributed to improved functional recovery. This hypothesis was supported by results that also OPC differentiation was significantly induced, axons were re-wrapped, and axonal damage was reduced after Tβ4 treatment at the late stage of EAE (day 30 after onset), and that OPC differentiation significantly correlates with the neurological functional score (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ).
      This Tβ4 treatment approach increased myelin areas and improved functional outcome in the late disease stage, demonstrating the presence of a remyelination effect of Tβ4, in addition to its neuroprotective and anti-inflammatory effects. Complementing the EAE model, an additional demyelination model -induced by cuprizone diet- was employed to further confirm that the remyelination effect of Tβ4 derives from its direct action on OPCs. The cuprizone model is extensively used to study in vivo toxicity-induced demyelination (
      • Zhang J.
      • et al.
      MiR-146a promotes remyelination in a cuprizone model of demyelinating injury.
      ) with less infiltrated immune cells as compared to what found in EAE (
      • Procaccini C.
      • et al.
      Animal models of Multiple Sclerosis.
      ). Thus, this experimental model has utility for directly investigating the effects of a therapeutic agent on OPC differentiation and remyelination. Cuprizone-fed mice with demyelination treated by Tβ4 exhibited a significant increase of remyelination, accompanied with a robust increase of newly generated OLs, and elevation of MBP density in the demyelinating corpus callosum (
      • Zhang J.
      • et al.
      Function of thymosin beta-4 in ethanol-induced microglial activation.
      ). In concert, these results obtained from both the EAE and cuprizone models indicate that the in vivo effects of Tβ4 on OPC differentiation and remyelination are independent of its systemic anti-inflammatory effect.
      Data obtained from other models of neurological injury, which induce demyelination and white matter damage and result in neurological deficits, including stroke, peripheral neuropathy and traumatic brain injury, likewise show the benefits of Tβ4 on OPCs and myelin after delayed Tβ4 treatments. Rats with middle cerebral artery occlusion treated with Tβ4 demonstrated a significant overall improvement in functional outcome (
      • Morris D.C.
      • et al.
      Thymosin beta4 improves functional neurological outcome in a rat model of embolic stroke.
      ). Although lesion volumes were not reduced, Tβ4 treatment increased myelinated axons in the ischemic boundary, and augmented remyelination, which was associated with an increase of OPCs and myelinating OLs (
      • Morris D.C.
      • et al.
      Thymosin beta4 improves functional neurological outcome in a rat model of embolic stroke.
      ). In a model of diabetes-induced peripheral neuropathy, extended Tβ4 treatment of diabetic mice significantly improved neurological function, and was closely associated with increased axonal regeneration and remyelination in peripheral nerves (
      • Wang L.
      • et al.
      Therapeutic benefit of extended thymosin beta4 treatment is independent of blood glucose level in mice with diabetic peripheral neuropathy.
      ). Traumatic brain injury remains a leading cause of mortality and morbidity worldwide with no effective pharmacological treatments. Tβ4 treatment of traumatic brain injury in rats amplified endogenous remyelinating processes including oligodendrogenesis, neurogenesis and axonal remodeling, which appeared to drive functional recovery (
      • Xiong Y.
      • et al.
      Neuroprotective and neurorestorative effects of thymosin beta4 treatment initiated 6 hours after traumatic brain injury in rats.
      ). In vitro experiments provide evidence of the direct effects of Tβ4 on OPCs and neurons. After Tβ4 treatment of primary cultured OPCs, the differentiation of OPCs into mature OLs identified by the protein level of MBP significantly increased (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ,
      • Santra M.
      • et al.
      Thymosin beta4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.
      ). Thus, data generated from these in vivo and in vitro studies in concert, confirm the remyelination effect of Tβ4. Due to the combined benefits in neuroprotection and remyelination (summarized in Fig. 1), Tβ4 is an excellent candidate for the treatment of demyelinating diseases also based on data demonstrating safety, tolerability and efficacy of Tβ4 in clinical practice (
      • Crockford D.
      Development of thymosin beta4 for treatment of patients with ischemic heart disease.
      ,
      • Vasilopoulou E.
      • et al.
      The role of thymosin-beta4 in kidney disease.
      ,
      • Marks E.D.
      • Kumar A.
      Thymosin beta4: roles in development, repair, and engineering of the cardiovascular system.
      ).
      Fig. 1.
      Fig. 1Scheme of impact of Tβ4 on the promotion of OPC differentiation and remyelination via blocking the intrinsic and extrinsic inhibitors, thereby promoting MS recovery.
      In vivo and in vitro studies have also given mechanistic insights into the therapeutic effects of Tβ4. The epidermal growth factor receptor and the TLR signal transduction pathways may contribute to CNS remyelination and recovery of function induced by Tβ4 (
      • Zhang J.
      • et al.
      Thymosin beta4 promotes oligodendrogenesis in the demyelinating central nervous system.
      ,
      • Santra M.
      • et al.
      Thymosin beta4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.
      ). Hedgehog signaling pathway was shown to be involved in activation of stem cells after Tβ4 treatment, which may contribute to therapeutic benefit (
      • Kim J.
      • et al.
      Thymosin beta-4 regulates activation of hepatic stellate cells via hedgehog signaling.
      ).
      Furthermore, having in mind that microRNA (miRNA)−146a may directly induce OPC differentiation to OLs (
      • Zhang J.
      • et al.
      MiR-146a promotes remyelination in a cuprizone model of demyelinating injury.
      ,
      • Liu X.S.
      • et al.
      MicroRNA-146a promotes oligodendrogenesis in stroke.
      ), Tβ4 treatment may prominently stimulate the expression of this mRNA in primary cultured OPCs, thus promoting the proliferation and differentiation of OPCs and OLs (
      • Santra M.
      • et al.
      Thymosin beta4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.
      ), which may serve as a common remyelination therapeutic mechanism. In addition to increasing miR-146a expression in OPCs, Tβ4 remarkably also increased miR-146a expression in microglia and significantly inhibited secretion of pro-inflammatory mediators (
      • Zhou T.
      • et al.
      Thymosin beta4 inhibits microglia activation through microRNA 146a in neonatal rats following hypoxia injury.
      ), all of which may promote remyelination (
      • Crockford D.
      Development of thymosin beta4 for treatment of patients with ischemic heart disease.
      ).

      5. Tα1: a newcomer in MS field

      5.1 Tα1 as a pleiotropic immunoregulator

      Tα1 has been shown to have beneficial effects on numerous immune system parameters, related to both innate and adaptive immune cells, including macrophages, neutrophils, natural killer cells and DC in addition to the well-characterized effects on the differentiation and maturation of T cells (
      • Serafino A.
      • et al.
      Thymosin alpha1 as a stimulatory agent of innate cell-mediated immune response.
      ). For these features, Tα1 was used as an adjuvant or immunotherapeutic agent to treat disparate human diseases, including viral infections, immunodeficiencies and malignancies (
      • Romani L.
      • et al.
      Jack of all trades: thymosin alpha1 and its pleiotropy.
      ). Tα1, however, demonstrated to be a powerful pleiotropic molecule able not only to induce anti-viral and pro-inflammatory responses, but also to promote a regulatory milieu depending on the context, as others’ and our data showed (
      • Romani L.
      • et al.
      Thymosin alpha1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance.
      ,
      • Giacomini E.
      • et al.
      Dual effect of Thymosin alpha 1 on human monocyte-derived dendritic cell in vitro stimulated with viral and bacterial toll-like receptor agonists.
      ). These observations are consistent with the fact that Tα1 can negatively control inflammation during immunopathological pro-inflammatory infections and diseases, exerting an interesting, and until now not-completely understood, role as a homeostasis regulator (
      • Romani L.
      • et al.
      Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance.
      ).
      In this context it is important to underline another interesting property of Tα1 that relies on the modulation of regulatory T cell (Treg) function by acting on signals delivered through TLR in response to pathogen associated molecular patterns (
      • Romani L.
      • et al.
      Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance.
      ,
      • Montagnoli C.
      • et al.
      Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism.
      ). This characteristic was also recently tested in cystic fibrosis where Tα1 increases the altered maturation of the cystic fibrosis transmembrane conductance regulator and reduces the chronic inflammation caused by the excessive activation of the innate immune response (
      • Romani L.
      • et al.
      Thymosin alpha1 represents a potential potent single-molecule-based therapy for cystic fibrosis.
      ). Indeed, owing to its ability to activate the tolerogenic pathway of tryptophan catabolism via the immunoregulatory enzyme indoleamine 2,3-dioxygenase 1 (
      • Puccetti P.
      • Grohmann U.
      IDO and regulatory T cells: a role for reverse signalling and non-canonical NF-kappaB activation.
      ), Tα1 specifically potentiates immune tolerance in the lung, breaking the vicious circle that perpetuates chronic lung inflammation in response to a variety of infectious noxae (
      • Romani L.
      • et al.
      Thymosin alpha1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance.
      ).
      Collectively, these data suggest that Tα1 represents a promising molecule to control inflammation, immunity and tolerance in a variety of clinical settings, including organ transplantation, tumors as well as autoimmune diseases, such as MS (
      • Serafino A.
      • et al.
      Thymosin alpha1 as a stimulatory agent of innate cell-mediated immune response.
      ).

      5.2 Tα1 and the anti-inflammatory effect in MS: focus on B cells

      Due to the Tα1 ability to establish a regulatory environment for balance of inflammation and tolerance, we recently hypothesized a possible role for this molecule in novel therapeutic applications towards autoimmune diseases, and in particular for MS (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ).
      Concentration of Tα1 in human serum is very high in fetuses and newborns, when the immune system is first developing, but rapidly drops in early childhood coincident with the maturation of T cells in the body remaining to a steady-state level throughout adulthood. However, deregulation in Tα1 serum concentrations was found in different types of cancers or infectious diseases and, more recently, in patients affected with chronic inflammatory autoimmune diseases such as psoriatic arthritis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), that have a much lower level of serum Tα1 as compared to healthy controls (
      • Pica F.
      • et al.
      Serum thymosin alpha 1 levels in patients with chronic inflammatory autoimmune diseases.
      ). Similarly, we also recently found that serum Tα1 is significantly lower in RRMS patients than in matched controls (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ) and we hypothesized that the deficient endogenous Tα1 level found in patients’ sera could be related to MS-associated altered inflammatory status.
      As for Tα1 always studied as a modulator of T cell responses, MS was considered by far a T cell-mediated autoimmune disease. Nonetheless, over the past few years B cells have been increasingly recognized as disease-relevant in MS and recent evidence on their immunopathogenic support on MS development has been collected, highlighting their central role (
      • Franciotta D.
      • et al.
      B cells and multiple sclerosis.
      ), historically overshadowed by the emphasis on T cell research.
      Traditionally, B cells have been implicated in MS for their ability to produce pathogenic antibodies (Abs) or auto-Abs, found to be present in CSF and brain tissue of MS patients. Accordingly, characteristic oligoclonal IgG bands in their CSF are thought to be a quite specific marker for MS (
      • Walsh M.J.
      • et al.
      Immunoglobulin G, A, and M–clonal restriction in multiple sclerosis cerebrospinal fluid and serum–analysis by two-dimensional electrophoresis.
      ). Numerous B lymphocytes were also described together with T lymphocytes, DC and plasma cells in white matter lesions, with a very high frequency in acute lesions (
      • Nyland H.
      • Mork S.
      • Matre R.
      In-situ characterization of mononuclear cell infiltrates in lesions of multiple sclerosis.
      ). However, new impetus on the central role of B cells in MS pathogenesis was received from the identification in the meninges of SPMS patients of ectopic lymphoid follicles enriched with B and plasma cells, whose establishment in the brain of these individuals could provide a microenvironment in which B cell expansion and maturation, and hence local Ig production, may occur (
      • Serafini B.
      • et al.
      Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.
      ). Nowadays, however, important Ab-independent pathogenic roles for B cells are emerging, also in light of successful results of B cell-depleting therapies in MS (
      • Barun B.
      • Bar-Or A.
      Treatment of multiple sclerosis with anti-CD20 antibodies.
      ). In particular, selective depletion of B cells via monoclonal Abs against the B cell lineage specific surface marker CD20 (i.e. rituximab, ocrelizumab, and ofatumumab) proved to be remarkably effective in the induction of long-lasting suppression of lesion activity and clinical relapses but showed no effect on plasma cell differentiation and Ab production (
      • Barun B.
      • Bar-Or A.
      Treatment of multiple sclerosis with anti-CD20 antibodies.
      ).
      Importantly, B cells can efficiently present antigens to T cells and modulate local immune responses by secreting soluble factors. In humans, different B cell subsets were described producing distinct effector cytokines. In particular, CD19 + CD27- naïve B cells release mainly the anti-inflammatory IL-10; while CD19 + CD27 + memory B cells largely express pro-inflammatory factors, such as lymphotoxin (LT), TNF-α and IL-6. In human B cells the effector cytokine profile is stringently context-dependent with a reciprocal regulation of pro- and anti-inflammatory response. In MS, this cytokine network is dysregulated, with a much lower production of the anti-inflammatory factor IL-10 (
      • Duddy M.
      • et al.
      Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis.
      ). Furthermore, B cells of MS patients exhibit aberrant pro-inflammatory responses, with increased LT:IL-10 ratio and exaggerated LT and TNF-α secretion, that may mediate ‘bystander activation’ of disease-relevant pro-inflammatory T cells, resulting in new relapsing MS disease activity (
      • Bar-Or A.
      • et al.
      Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?.
      ). The term “regulatory B cells” was first introduced by Mizoguchi and Bhan to indicate a subset of B cells with the ability to produce IL-10 and to suppress inflammatory cellular immune responses (
      • Mizoguchi A.
      • Bhan A.K.
      A case for regulatory B cells.
      ). Since then the family of B reg is expanding with many subsets identified and shown to arise at different stages of B cell differentiation in a context-dependent manner (
      • Mauri C.
      • Menon M.
      The expanding family of regulatory B cells.
      ). In particular, among many others, two main B reg populations have been deeply characterized: CD24 + CD38hi transitional-immature B reg arising from CD27- B cell compartment (
      • Blair P.A.
      • et al.
      CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.
      ) and CD24low/negCD38hi plasmablast-like B reg cells differentiating from CD27 + memory B cells (
      • Matsumoto M.
      • et al.
      Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation.
      ). While much evidence indicate that B cell-derived IL-10 production is strongly down-regulated in MS patients (
      • Duddy M.
      • et al.
      Distinct effector cytokine profiles of memory and naive human B cell subsets and implication in multiple sclerosis.
      ,
      • Bar-Or A.
      • et al.
      Abnormal B-cell cytokine responses a trigger of T-cell-mediated disease in MS?.
      ), only few studies have characterized so far B reg populations in MS by using different Ab cocktails leading to contrasting results (
      • Knippenberg S.
      • et al.
      Reduction in IL-10 producing B cells (Breg) in multiple sclerosis is accompanied by a reduced naive/memory Breg ratio during a relapse but not in remission.
      ,
      • Michel L.
      • et al.
      Unaltered regulatory B-cell frequency and function in patients with multiple sclerosis.
      ,
      • Li R.
      • et al.
      Proinflammatory GM-CSF-producing B cells in multiple sclerosis and B cell depletion therapy.
      ,
      • Habib J.
      • et al.
      Blood B cell and regulatory subset content in multiple sclerosis patients.
      ).
      Based on this background and having in mind the strong immunomodulatory and pleiotropic activity of Tα1 molecule as well as our previous data showing a TLR7-driven dysregulation of B cell response in MS patients (
      • Giacomini E.
      • et al.
      IFN-beta therapy modulates B-cell and monocyte crosstalk via TLR7 in multiple sclerosis patients.
      ,
      • Rizzo F.
      • et al.
      Interferon-beta therapy specifically reduces pathogenic memory B cells in Multiple Sclerosis patients by inducing a FAS-mediated apoptosis.
      ), we set up an in vitro PBMC-based experimental procedure to study differentiation of B reg subsets and the impact of Tα1 in this context (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ). In particular, differently from studies on purified B cells, the mixed cell population of PBMC resemble the in vivo scenario and, upon stimulation with a specific TLR7 agonist (the so-called Imiquimod), could account for cytokine production as well as triggering of CD40 signaling generating an in vitro B cell proliferating milieu unique for either healthy individuals or patients. By using this setting, our study demonstrated a striking difference in the ability of B cells from RRMS patients to differentiate into both CD24 + CD38hi transitional-immature and CD24low/negCD38hi plasmablast-like B reg subsets, as compared to matched controls (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ). Very interestingly, in vitro exposure to Tα1 drastically reduced the TLR7-induced production of the pro-inflammatory cytokines IL-1β, IL-8 and IL-6, while increasing expression of the anti-inflammatory mediators IL-10 and IL-35. Accordingly, Tα1 treatment restored the ability of RRMS B cells to differentiate into IL-10-producing transitional-immature B reg and regulatory plasmablasts to the level found in healthy controls (Fig. 2). Furthermore, the B reg subsets expanded by Tα1 treatment display a suppressive activity reducing both IFN-γ and IL-17 production found in TLR7-treated MS patients-derived PBMC cultures (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ). Unfortunately, there is no evidence on the role of Tα1 in EAE. However, Janeway and colleagues first observed that B10.PL mice lacking B cells suffered an unusually severe and chronic form of EAE, suggesting that anti-inflammatory B cells in charge of negatively regulating inflammatory reactions may be depleted (
      • Wolf S.D.
      • et al.
      Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice.
      ). Therefore, we envisage that treatment with Tα1, impacting induction of both B and T regulatory cell subsets, may exert protective effects during EAE by reducing disease severity.
      Fig. 2.
      Fig. 2Scheme of impact of synthetic Tα1 treatment on B cell differentiation in MS.
      In concert, these findings highlight the therapeutic potential of Tα1 in MS as well as in other autoimmune conditions that show reduced differentiation of B reg subsets and chronic immune activation.

      6. Conclusive remarks

      Ongoing research in MS therapeutics seeks strategies to target or modulate the pro-inflammatory responses into a more anti-inflammatory scenario, in which antigen-specific immune tolerance may be induced and, in this regard, manipulating B reg subsets may be successful. However, since approved treatments for MS work by reducing immune system activity or blocking entry of immune cells into the CNS, thus reducing relapse rate and severity of attacks, but they do not repair immune-mediated damage to the myelin sheaths surrounding axons, drug repurposing or development of new treatments to promote myelin repair and neuronal protection is desirable.
      In this scenario, we reviewed the state-of-the-art on thymosins and MS, with particular attention to the neuroprotective and remyelinating action of Tβ4 and to the more recently characterized anti-inflammatory activity of Tα1.
      The clinical usage of Tα1 in the past in cancer or chronic infections or as adjuvant in vaccine formulations (
      • Garaci E.
      • et al.
      Thymosin alpha 1: from bench to bedside.
      ), in which an immune potentiation is needed, may seem difficult to reconcile with its use in autoimmune conditions where an anti-inflammatory action is desirable. However, our results on Tα1 potentiation of B reg differentiation in MS (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ), together with its well-characterized pleiotropic activity able to shift inflammation toward a more anti-inflammatory milieu depending on the stimuli or pathological conditions (
      • Romani L.
      • et al.
      Jack of all trades: thymosin alpha1 and its pleiotropy.
      ,
      • Giacomini E.
      • et al.
      Dual effect of Thymosin alpha 1 on human monocyte-derived dendritic cell in vitro stimulated with viral and bacterial toll-like receptor agonists.
      ) and its capacity to induce Treg generation and IL-10 production (
      • Romani L.
      • et al.
      Thymosin alpha1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance.
      ,
      • Romani L.
      • et al.
      Thymosin alpha1 represents a potential potent single-molecule-based therapy for cystic fibrosis.
      ), indicate beneficial and advisable functions for a drug to be used in autoimmune diseases. These data may pave the way for a repurposing potential of Tα1, alone or in combination with other approved drugs, for treatment of MS or other autoimmune conditions that display reduced differentiation of B reg subsets and chronic immune activation.
      In addition, Tβ4 has a broad net of protective and restorative effects on neurological degeneration and injury in the CNS. Recent studies investigated the remarkable capacity of Tβ4 not only on promotion of OPC proliferation, but also on OPC differentiation and remyelination, and demonstrated significant improvement of functional and behavioral outcomes in animal models of MS. The ability of Tβ4 to target many diverse processes via multiple molecular pathways that drive oligodendrogenesis and axonal remodeling may also be mediated by miRNAs, particularly, miR-146a (
      • Santra M.
      • et al.
      Thymosin beta4 up-regulation of microRNA-146a promotes oligodendrocyte differentiation and suppression of the Toll-like proinflammatory pathway.
      ).
      Thus, Tβ4 has substantial potential for clinical translation as a multiple-target therapy for MS/EAE or other neurological demyelinating diseases, with the remyelination effect supplementing its anti-inflammatory and neuroprotective role, as previously found in studies involving Tβ4 prophylactic treatment (
      • Zhang J.
      • et al.
      Neurological functional recovery after thymosin beta4 treatment in mice with experimental auto encephalomyelitis.
      ).
      Since low level of endogenous Tα1 is present in sera of patients affected with chronic inflammatory autoimmune diseases such as psoriatic arthritis, RA and SLE (
      • Pica F.
      • et al.
      Serum thymosin alpha 1 levels in patients with chronic inflammatory autoimmune diseases.
      ) as well as in RRMS patients (
      • Giacomini E.
      • et al.
      Thymosin-alpha1 expands deficient IL-10-producing regulatory B cell subsets in relapsing-remitting multiple sclerosis patients.
      ), an interesting approach might also be to quantify the levels of serum Tβ4 in the same categories of patients to investigate whether a deregulation of both these thymic peptides may be related to MS-associated autoimmune responses. Tβ4 level in the MS population has been reported to be down-regulated in CSF (
      • Liguori M.
      • et al.
      Proteomic profiling in multiple sclerosis clinical courses reveals potential biomarkers of neurodegeneration.
      ) and this down-regulation is a potential biomarker of MS.
      Furthermore, one may also envisage that treatment with currently approved disease modifying therapies would affect a dysregulated endogenous thymosin system in MS.
      Thus, it may be interesting to investigate whether endogenous Tα1 and/or Tβ4 expression level in cells or sera are modified in patients undergoing treatment with disease-modifying therapies. In particular, immune-modulatory therapies, such as IFN-β, glatiramer acetate or teriflunomide, could impact the low circulating level of Tα1 and/or Tβ4 in MS patients. Or possibly, the combination of synthetic Tα1 with B cell-directed therapeutics, as the specific anti-CD20 mAbs ocrelizumab and ofatunumab or those mAbs, including natalizumab and alemtuzumab, affecting both T and B lymphocytes, may complement their ability to affect the B cell compartment. The profound and rapid depletion of B cells that arises after treatment with these mAbs is then followed by repletion of lymphocytes with a more regulatory phenotype. However, in some cases (as for example following CD52 depletion) there is a hyperpopulation of immature B cells that may be responsible for secondary autoimmunity, a major drawback of alemtuzumab therapy (
      • Baker D.
      • et al.
      Interpreting lymphocyte reconstitution data from the pivotal phase 3 trials of Alemtuzumab.
      ). As in the combination regimens of chemotherapeutics plus Tα1 adopted for treatment of many cancer types (
      • Garaci E.
      • et al.
      Historical review on thymosin alpha1 in oncology: preclinical and clinical experiences.
      ), in MS a combination of disease modifying therapies with synthetic Tα1 may help in addressing and correctly modulating the repopulation of B cell compartment towards a more regulatory and anti-inflammatory phenotype.
      Furthermore, the induction of miR-146a mediated by Tβ4 can modulate the innate and adaptive immune response (
      • Wu T.
      • Chen G.
      miRNAs Participate in MS pathological processes and its therapeutic response.
      ). For example, the increased level of miR-146a can suppress IFN-γ-dependent Th1 responses and reduce Th1-mediated lesions by Tregs inhibition of signal transducer and activator of transcription 1 (
      • Lu L.F.
      • et al.
      Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses.
      ), and diminish adhesion of T cells to endothelial cells (
      • Wu D.
      • et al.
      Brain endothelial miR-146a negatively modulates T-cell adhesion through repressing multiple targets to inhibit NF-kappaB activation.
      ). Currently, FDA-approved disease modifying therapies for MS mainly focus on immunomodulation, and there is lack of remyelination treatments. In this review, Tβ4 has been demonstrated to promote remyelination, synaptogenesis and axon growth, in addition to its effect on immunosuppression. In future clinical treatment, Tβ4 may be employed as a monotherapy or combined with other disease modifying therapies to treat MS patients. Thus, therapeutic approaches should attempt to decrease severity of disease by immunosupression in order to reduce the damage to myelin and axons as well as to promote repair of damaged myelin and axons and, thereby, enhance functional recovery of MS patients.
      We believe that further studies will be needed to address this very interesting aspect of thymosin biology, such as the effects of Tβ4 on MS patients, and that of Tα1 on the EAE model, having in mind that treating MS patients with Tα1 and Tβ4 alone or together with other approved drugs may meet the requirements for a desirable but-still-unknown therapy for MS acting on both immune dysregulation and CNS damage.

      Funding

      Our work was in part supported by grant GR-2016-02363749 from Italian Ministry of Health (to MS).

      Conflict of interest

      The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
      Enrico Garaci is a patent holder.
      Henry Ford Hospital has a Material Transfer Agreement with RegeneRx Biopharmaceuticals, Inc., Rockville, MD. A US Provisional Patent 61/163,556 has been filed for use of Tβ4 in neurological injury.
      The other authors have no conflict to declare.

      Acknowledgments

      The authors acknowledge Professor Marco Salvetti and his team (Centre for Experimental Neurological Therapies -CENTERS, Sapienza University of Rome, Rome, Italy) for the helpful discussion on the Thymosin results collected during the long-lasting collaboration in MS study with E.M. Coccia's team; Eugenio Morassi (Division Service for Data Management, Documentation, Library, and Publishing Activities, Istituto Superiore di Sanità, Rome, Italy) for preparing drawings and Letizia Sampaolo (Division Service for knowledge and scientific communication, Istituto Superiore di Sanità, Rome, Italy) for helping with literature search strategy in PubMed/Medline database.

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