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ANNALS OF THE NEW YORK ACADEMY OF SCIENCES
Issue: Thymosins in Health and Disease

Thymosin a1: the regulator of regulators?

Bonifazi Pierluigi,1 Carmen D’Angelo,1 Francesca Fallarino,1 Silvia Moretti,1 Teresa Zelante,1 Silvia Bozza,1 Antonella De Luca,1 Francesco Bistoni,1 Enrico Garaci,2 and Luigina Romani1
1Department of Experimental Medicine and Biochemical Science, University of Perugia, Perugia, Italy. 2Department of

Experimental Medicine and Biochemical Science, University of Tor Vergata, Rome, Italy Address for correspondence: Luigina Romani, M.D., Ph.D., Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. lromani@unipg.it

The peripheral immune system can promote either immunity or tolerance when presented with newantigens. Current knowledgewithholds thatpopulations of suppressor orregulatory Tcells(Treg cells)constituteapivotal mechanism of immunological tolerance. The potential role of malfunctioning Treg cells in chronic inflammatory immune and autoimmune diseases is well-documented. Learning how to successfully manipulate Treg responses could result in more effective vaccines and immunomodulators. We have already shown that Thymosin α1(Tα1), a naturally occurring thymic peptide first described and characterized by Allan Goldstein in 1972, by modulating signals delivered through innate immune receptors on dendritic cells, affects adaptive immune responses via modulation of Th cell effector and regulatory functions. We will discuss recent molecular mechanisms underlying the ability of Tα1 to activate or inhibit immune responses.

Introduction

The peripheral immune system can promote either immunity or tolerance when presented with new antigens. Dendritic cells (DCs) have a crucial role in determining immune outcomes by acquiring antigens, collating environmental cues, and then becoming cells that are either potent stimulators or suppressors of T-cell responses.1 Current knowledge withholds that populations of suppressor or regulatory T cells (Treg cells) constitute a pivotal mechanism of immunological tolerance.2 Regulatory T cells are characterized by high-level surface CD25 and intracellular forkhead family transcription factor (FoxP3) expression. Diverse types of Treg cells, with disparate and multiple functions have been discovered. Natural Treg cells are selected by high-avidity interactions in the thymus, whereas inducible, adaptive Foxp3+CD4+ Treg cells develop outside the thymus during chronic inflammation. Induced Treg cells are essential in mucosal immune tolerance and in the control of severe chronic allergic inflammation, and most likely are one of the main barriers to the eradication of tumors, whereas natural Treg cells are thought to prevent autoimmunity and raise the activation threshold for all immune responses. The potential role of malfunctioning Treg cells in chronic inflammatory immune and auto immune diseases is well-documented.3

Given the recognized importance of Treg cells in regulating immune responses, understanding their mechanism of action and interplay with other means of immunological tolerance, and their therapeutic exploitation is one major challenge in immunology. Increased comprehension of how Treg cells exert their function holds the promise for therapeutic intervention by manipulating one of the most sophisticated features of immunity to either boost responses in cancer and microbial diseases or suppress those unwanted in autoimmunity and transplantation. In addition, learning how to successfully manipulate Treg cell responses could result in more effective vaccines and immunomodulators.

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential amino acid tryptophan.4 The concept that cells expressing IDO can suppress T-cell responses and promote tolerance is a relatively new paradigm in immunology. IDO is a haeme-containing enzyme that catabolizes compounds containing indole rings, such as the essential amino acid tryptophan. IDO protein is encoded by a tightly regulated gene that is responsive to inflammatory mediators in a limited range of cell types. Enzymatic activity of IDO correlates with reduced T-cell-mediated responses in several experimental (mouse) systems, including models of autoimmune diseases, cancer, organ and tissue transplant rejection, and pregnancy.5 Mature DCs that express functional IDO enzyme activity can be potent suppressors of T-cell responses in vivo and in vitro. The synthetic immunomodulatory reagent cytotoxic T lymphocyte antigen 4 (CTLA4)– immunoglobulin fusion protein is a potent inducer of IDO expression by DCs.6 Regulatory T cells that express CTLA4 induce IDO-competent DCs to express IDO, indicating that CTLA4+ Treg cells use the IDO mechanism to suppress T-cell responses and promote tolerance. More speculatively, IDO-expressing DCs might promote the development of Treg cells; if so, Treg cells and IDO-competent DCs might cooperate to form a self-amplifying immunoregulatory network.7

Thymosin a1(Ta1) is a naturally occurring thymic peptide first described and characterized by Goldstein et al.8 Although the peptide is produced in small amounts in several peripheral lymphoid and nonlymphoid tissues, the highest concentrations of Ta1 are found in the thymus. Prepared as a 28mer synthetic amino-terminal acylated peptide, Ta1 is in clinical trials worldwide for the treatment of some viral infections, either as a monotherapy or in combination with IFN-

a. Additional indications are some immunodeficiencies, malignancies, and HIV/AIDS.9 The mechanism of action of the synthetic polypeptide is thought to be related to its immunomodulating activities, centered primarily on the augmentation of T-cell function. However, mechanistically, Ta1has shown an action beyond its effect on T lymphocytes to include an ability to act as an endogenous regulator of both the innate and adaptive immune systems.

In this review, we discuss current knowledge of molecula mechanisms by which Ta1may affect immunity and tolerance in various experimental settings.

Thymosin α1 activates dendritic cells for antimicrobial immunity

Ta1 strongly upregulated the expression of Toll-like receptors (TLR2)and TLR9 by murine DCs and activated NF-kB and JNK/p38/AP-1 pathways.10,11 Studies with human DC confirmed this finding and further showed that Ta1 upregulated the expression of TLR2 by myeloid or conventional DC (cDC) and of TLR9 by plasmacytoid dendritic cells (pDC). Induction of TLR2 or TLR9 expression by DC by Ta1 was associated with a distinct activation program in both murine and human DC subsets. Ta1 induced the production of IL-12p70 in cDC and IL-10 in pDC. Production of IL-12p70 was reduced in TLR2−/− mice, whereas IL-10 was reduced in TLR9−/− mice. The production of both cytokines was severely reduced in DC lacking the myeloid differentiation factor 88 (MyD88), an adaptor protein essential for the production of cytokines upon signaling by all the TLR family members. Therefore, consistent with the finding showing that Ta1 directly signaled through TLR9 and potentiated ligand-induced TLR2 signaling by TLR-transfected HEK293 cells,10 these data indicate that Ta1activates DC subsets through distinct TLR and involves MyD88.

The immunomodulatory effects of Ta1onDCs correlated with a therapeutic effect of the peptide in experimental fungal or viral infections.12 Administration of Ta1 to mice with Aspergillus fumigatus resulted in a state of full protection to the fungus as revealed by the increased survival after the infection that paralleled the reduced fungal growth, the promotion of IFN-"-producing Th1 cells, and the inhibition of IL-4-producing Th2 cells. Despite the fact that Ta1 increased the recovery and functional activity of effector macrophages and neutrophils, the myeloid functional recovery could not per se account for the efficacy of Ta1. In fact, recovery from neutropenia alone, as by treatment with granulocyte colony stimulating factor, was not sufficient to mediate a degree of antifungal resistance comparable to that obtained with Ta1.10 Therefore, the achievement of a state of full protection to the fungus required the coordinated action of innate effector cells and protective Th1 responses, an activity successfully promoted by Ta1.10,12 Consistent with the in vitro data on TLR expression, the therapeutic efficacy of Ta1 in vivo was dependent on My D88 signaling activated by TLR2 and partially by TLR9. Therefore, despite a degree of redundancy in the TLR usage, the My D88-dependent signaling pathway is essential in the antifungal activity of Ta1 in vitro and in vivo.

The ability of Ta1 to modulate DC functioning through TLR9 correlated wit han effect on Cytomegalo virus sensing by DCs in vitro and in vivo.13 The antiviral effect of Ta1 led to the activation of IRF7 and the promotion of the IFN-a/IFN-"dependent effector pathway in pDC via the TLR9 dependent viral recognition sensing. In infection, Ta1 decreased the viral load in both susceptible and resistant mice with primary MCMV infection and induced the expansion of cytolytic NK1.1+ cells, IFN-"-producing CD8+ or CD4+ T cells, and the productionofIL-12p70, IFN-a,IFN-", and IL-10.13 Together, these data indicate that Ta1 affects both the innate and adaptive antimicrobial immune responses in vivo.

Thymosin α1 activates dendritic cells for IDO-mediated immune tolerance

The induction of tolerance is critical for the maintenance of immune homeostasis. DCs not only play a key role in the induction of immune responses but also in the induction and maintenance of immune tolerance.1 Tryptophan catabolism and kynurenines play a crucial role in the induction of peripheral tolerance.4,5 IDO is widely expressed in a variety of human tissues as well as in macrophages and in DC, and is induced in inflammatory states by IFN-" and other proinflammatory cytokines. TLR9 stimulation may lead to the activation of the tryptophan catabolism pathway in DC,14 and IDO is a molecular signature of tolerogenic pDC.15 Ta1 was found to induce IDO activity in DCs through TLR9 and type I Interferon receptor signaling.16 IDO blockade prevented the activation of IL-10– producing CD4+ T cells, while sparing that of IFN-"-producing cells, a finding confirming the causal link of IDO with priming for IL-10– producing T cells. Consistent with the finding that TLR9 stimulation can promote pDC mediated generation of CD4+CD25+ Treg cells,17 CD4+CD25+ T cells induced by Ta1-treated DC expressed FoxP3. Therefore, Ta1 induced CD4+CD25+Treg cells through the activation of a TLR9-dependent immunosuppressive pathway of tryptophan catabolism. Studies on experimental hematopoietic stem cell transplantation (HSCT) and respiratory allergy established a proof-of concept principle that the induction of immune tolerance via IDO-induced Treg cells could be exploited for immunoregulatory therapy with Ta1or Ta1-conditioned DCs. Treatment with Ta1 or the infusion of Ta1-treated DC resulted in pathogen clearance, prompt resolution of inflammatory pathology, and prevention of graft-versus host disease in HSCT.16 Similarly, Ta1given eitherprophylactically, during the sensitization phase or therapeutically, at the elicitation phase, attenuated signs of inflammatory allergy in a murine model of fungal allergy by promoting the tolerogenic program of lung DC and priming for CD25+ Treg cells inhibiting allergic Th2 responses.12

Balancing immunity and tolerance by Tα1: the regulator of regulators

Figure 1. Balancing immunity and tolerance by Ta1. A view of the possible actions of Ta1 on dendritic cells through TLR exploitation eventually leading to the induction of antimicrobial Th priming and tolerance via the IDO/Treg axis. The ability of Ta1 to affect the skewing of Th1/Th17 cell activation through a direct action on polyclonal-stimulated T cells in vitro (unpublished observations) is also shown. IDO, indoleamine 2,3-dioxygenase; Treg, regulatory T cells, TLR, Toll-like receptors.

Learning how to successfully manipulate Treg cell responses could result in more effective vaccines by boosting responses in cancer and microbial diseases and immunomodulatory strategies to suppress unwanted responses in autoimmunity and transplantation.2,3 By affecting the balance between immunogenic and tolerogenic DCs through TLR exploitation, Ta1 fulfills the requirement of a promising adjuvant candidate for strategies aimed at the control of inflammation, immunity, and tolerance in a variety of clinical settings (Fig. 1). The real challenge will be to discover molecular pathways underlying these apparently opposite activities of Ta1 on the immune system. Our own unpublished studies would suggest an ability of Ta1 to affect the balance between inflammatory/antiinflammatory NFKB pathways in DCs. NF-KB is a family of seven structurally related transcription factors that play a central role in the stress response and inflammation by controlling gene network expression.18,19 Although the NF-KB subunits are ubiquitously expressed, their actions are regulated in a cell type-and stimulus-specific manner, allowing for a diverse spectrum of effects. Recent molecular dissection of its mechanisms for activation has shown that NFKB can be induced by the so-called “canonical” and “noncanonical” pathways, leading to distinct patterns in the individual subunits activated and downstream genetic responses produced. Although much attention hasbeen focusedon thepro-inflammatory signaling of canonical NF-KB, recent data indicate that noncanonical NF-KB could have opposing roles, limiting canonical NF-KB activity, inducing IDO, and controlling inflammation.7 Thus, the intersection between canonical and noncanonical signaling pathways may be crucial in promoting an optimally protective response balanced between inflammation and tolerance by Ta1. This unanticipated finding could have significant implications in immune regulation by Ta1, including the development of Treg cell responses.

Acknowledgments

We thank Dr. Cristina Massi Benedetti for digital art and editing. The original studies conducted in the authors’ laboratory were supported by the Italian Projects PRIN 2007KLCKP8˙004 (to LR), 2007XYB9T9˙001 (to SB), and 2007KLCKP8˙005 (to FB), and by the Project 2008.021.338 from Fondazione Cassa di Risparmio di Perugia.

Conflicts of interest

The authors declare no conflicts of interest.

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Immunoregulation by Thymosin α1

Human plasmacytoid dendritic cells activated by CpG oligodeoxynucleotides induce the generation of CD4+CD25+ regulatory T cells. J. Immunol. 173: 44334442.

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© 2010 New York Academy of Sciences.


   

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