COVID-19 and beyond: Reassessing the role of thymosin alpha1 in lung infections.

The recent COVID-19 pandemic has catalyzed the attention of the scientific community to the long-standing issue of lower respiratory tract infections. The myriad of airborne bacterial, viral and fungal agents to which humans are constantly exposed represents a constant threat to susceptible individuals and bears the potential to reach a catastrophic scale when the ease of inter-individual transmission couples with a severe pathogenicity. While we might be past the threat of COVID-19, the risk of future outbreaks of respiratory infections is tangible and argues for a comprehensive assessment of the pathogenic mechanisms shared by airborne pathogens. On this regard, it is clear that the immune system play a major role in dictating the clinical course of the infection. A balanced immune response is required not only to disarm the pathogens, but also to prevent collateral tissue damage, thus moving at the interface between resistance to infection and tolerance. Thymosin alpha1 (Tα1), an endogenous thymic peptide, is increasingly being recognized for its ability to work as an immunoregulatory molecule able to balance a derailed immune response, working as immune stimulatory or immune suppressive in a context-dependent manner. In this review, we will take advantage from the recent work on the COVID-19 pandemic to reassess the role of Tα1 as a potential therapeutic molecule in lung infections caused by either defective or exaggerated immune responses. The elucidation of the immune regulatory mechanisms of Tα1 might open a new window of opportunity for the clinical translation of this enigmatic molecule and a potential new weapon in our arsenal against lung infections.

Thymosin ß4 and the anti-fibrotic switch

Wound healing involves a rapid response to the injury by circulating cells, followed by inflammation with an influx of inflammatory cells that release various factors. Soon after, cellular proliferation begins to replace the damaged cells and extracellular matrix, and then tissue remodeling restores normal tissue function. Various factors can lead to pathological wound healing when excessive and irreversible connective tissue/extracellular matrix deposition occurs, resulting in fibrosis. The process is initiated when immune cells, such as macrophages, release soluble factors that stimulate fibroblasts. TGFß is the most well-characterized macrophage derived pro-fibrotic mediator. Other soluble mediators of fibrosis include connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), and interleukin 10 (IL-10). Thymosin ß4 (Tß4) has shown therapeutic benefit in preventing fibrosis/scarring in various animal models of fibrosis/scarring. The mechanism of action of Tß4 appears related, in part, to a reduction in the inflammatory response, including a reduction in macrophage infiltration, decreased levels of TGFß and IL-10, and reduced CTGF activation, resulting in both prevention of fibroblast conversion to myofibroblasts and production of normally aligned collagen fibers. The amino N-terminal end of Tß4, SDKP (serine-aspartate-lysine-proline), appears to contain the majority of anti-fibrotic activity and has shown excellent efficacy in many animal models of fibrosis, including liver, lung, heart, and kidney fibrosis. Ac-SDKP not only prevents fibrosis but can reverse fibrosis. Unanswered questions and future directions will be presented with regard to therapeutic uses alone and in combination with already approved drugs for fibrosis.

Thymosin α1 protects from CTLA-4 intestinal immunopathology.

The advent of immune checkpoint inhibitors has represented a major boost in cancer therapy, but safety concerns are increasingly being recognized. Indeed, although beneficial at the tumor site, unlocking a safeguard mechanism of the immune response may trigger autoimmune-like effects at the periphery, thus making the safety of immune checkpoint inhibitors a research priority. Herein, we demonstrate that thymosin α1 (Tα1), an endogenous peptide with immunomodulatory activities, can protect mice from intestinal toxicity in a murine model of immune checkpoint inhibitor-induced colitis. Specifically, Tα1 efficiently prevented immune adverse pathology in the gut by promoting the indoleamine 2,3-dioxygenase (IDO) 1-dependent tolerogenic immune pathway. Notably, Tα1 did not induce IDO1 in the tumor microenvironment, but rather modulated the infiltration of T-cell subsets by inverting the ratio between CD8+ and Treg cells, an effect that may depend on Tα1 ability to regulate the differentiation and chemokine expression profile of DCs. Thus, through distinct mechanisms that are contingent upon the context, Tα1 represents a plausible candidate to improve the safety/efficacy profile of immune checkpoint inhibitors.

Thymosin ß4 promotes autophagy and repair via HIF-1α stabilization in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a genetic disorder of the NADPH oxidase characterized by increased susceptibility to infections and hyperinflammation associated with defective autophagy and increased inflammasome activation. Herein, we demonstrate that thymosin ß4 (Tß4), a g-actin sequestering peptide with multiple and diverse intracellular and extracellular activities affecting inflammation, wound healing, fibrosis, and tissue regeneration, promoted in human and murine cells noncanonical autophagy, a form of autophagy associated with phagocytosis and limited inflammation via the death-associated protein kinase 1. We further show that the hypoxia inducible factor-1 (HIF-1)α was underexpressed in CGD but normalized by Tß4 to promote autophagy and up-regulate genes involved in mucosal barrier protection. Accordingly, inflammation and granuloma formation were impaired and survival increased in CGD mice with colitis or aspergillosis upon Tß4 treatment or HIF-1α stabilization. Thus, the promotion of endogenous pathways of inflammation resolution through HIF-1α stabilization is druggable in CGD by Tß4.

A Reappraisal of Thymosin Alpha1 in Cancer Therapy.

Thymosin alpha1 (Tα1), an endogenous peptide first isolated from the thymic tissue in the mid-sixties, has gained considerable attention for its immunostimulatory activity that led to its application to diverse pathological conditions, including cancer. Studies in animal models and human patients have shown promising results in different types of malignancies, especially when Tα1 was used in combination with other chemo- and immune therapies. For this reason, the advancements in our knowledge on the adjuvant role of Tα1 have moved in parallel with the development of novel cancer therapies in a way that Tα1 was integrated to changing paradigms and protocols, and tested for increased efficacy and safety. Cancer immunotherapy has recently experienced a tremendous boost following the development and clinical application of immune checkpoint inhibitors. By unleashing the full potential of the adaptive immune response, checkpoint inhibitors were expected to be very effective against tumors, but it soon became clear that a widespread and successful application was not straightforward and shortcomings in efficacy and safety clearly emerged. This scenario led to the development of novel concepts in immunotherapy and the design of combination protocols to overcome these limitations, thus opening up novel opportunities for Tα1 application. Herein, we summarize in a historical perspective the use of Tα1 in cancer, with particular reference to melanoma, hepatocellular carcinoma and lung cancer. We will discuss the current limitations of checkpoint inhibitors in clinical practice and the mechanisms at the basis of a potential application of Tα1 in combination protocols.

Thymosin ß4 limits inflammation through autophagy.

INTRODUCTION: Thymosin ß4 (Tß4) is a thymic hormone with multiple and different intracellular and extracellular activities affecting wound healing, inflammation, fibrosis and tissue regeneration. As the failure to resolve inflammation leads to uncontrolled inflammatory pathology which underlies many chronic diseases, the endogenous pathway through which Tß4 may promote inflammation resolution becomes of great interest. In this review, we discuss data highlighting the efficacy of Tß4 in resolving inflammation by restoring autophagy. AREAS COVERED: The authors provide an overview of the Tß4's anti-inflammatory properties in several pathologies and provide preliminary evidence on the ability of Tß4 to resolve inflammation via the promotion of non-canonical autophagy associated with the activation of the DAP kinase anti-inflammatory function. EXPERT OPINION: Based on its multitasking activity in various animal studies, including tissue repair and prevention of chronic inflammation, Tß4 may represent a potential, novel treatment for inflammatory diseases associated with defective autophagy.

Cellular proteostasis: a new twist in the action of thymosin α1.

INTRODUCTION: Thymosin alpha 1 (Tα1) is a naturally occurring polypeptide of 28 amino acids, whose mechanism of action is thought to be related to its ability to signal through innate immune receptors. Tα1 (ZADAXIN®) is used worldwide for treating viral infections, immunodeficiencies, and malignancies. Owing to its ability to activate the tolerogenic pathway of tryptophan catabolism - via the immunoregulatory enzyme indoleamine 2,3-dioxygenase - Tα1 potentiates immune tolerance mechanisms, breaking the vicious circle that perpetuates chronic inflammation in response to a variety of infectious noxae. AREAS COVERED: Tα1 has never been studied in Cystic fibrosis (CF) in which the hyperinflammatory state is associated with early and nonresolving activation of innate immunity, which impairs microbial clearance and promotes a self-sustaining condition of progressive lung damage. Optimal CF treatments should, indeed, not only rescue CF transmembrane conductance regulator protein localization and functionality but also alleviate the associated hyperinflammatory pathology. Because of the inherent complexity of the pathogenetic mechanisms, a multidrug approach is required. EXPERT OPINION: By providing a multipronged attack against CF, i.e. restraining inflammation and correcting the basic defect, Tα1 favorably opposed CF symptomatology in preclinical relevant disease settings, thus suggesting its possible exploitation for 'real-life' clinical efficacy in CF. This could represent a major conceptual advance in the CF field, namely the proposal of a drug with the unique activity to correct CFTR defects through regulation of inflammation.

Author Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, some labels in Fig. 1f are incorrect. The "ß-actin" labels on the second and fourth rows of blots should instead be "ß-tubulin". The error has been corrected in the HTML and PDF versions of this article.

Publisher Correction: Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

In the version of this article originally published, the amino acid sequence for Tα1 described in the Online Methods is incorrect. The sequence is described as "Ac-SDAAVDTSSEITTJDLKEKKEVVEEAEN-OH". It should be "Ac-SDAAVDTSSEITTKDLKEKKEVVEEAEN-OH". The error has been corrected in the HTML and PDF versions of this article.

Thymosin α1 represents a potential potent single-molecule-based therapy for cystic fibrosis.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) that compromise its chloride channel activity. The most common mutation, p.Phe508del, results in the production of a misfolded CFTR protein, which has residual channel activity but is prematurely degraded. Because of the inherent complexity of the pathogenetic mechanisms involved in CF, which include impaired chloride permeability and persistent lung inflammation, a multidrug approach is required for efficacious CF therapy. To date, no individual drug with pleiotropic beneficial effects is available for CF. Here we report on the ability of thymosin alpha 1 (Tα1)-a naturally occurring polypeptide with an excellent safety profile in the clinic when used as an adjuvant or an immunotherapeutic agent-to rectify the multiple tissue defects in mice with CF as well as in cells from subjects with the p.Phe508del mutation. Tα1 displayed two combined properties that favorably opposed CF symptomatology: it reduced inflammation and increased CFTR maturation, stability and activity. By virtue of this two-pronged action, Tα1 has strong potential to be an efficacious single-molecule-based therapeutic agent for CF.

Introduction for the Fourth International Symposium on Thymosins in Health and Disease.

The Fourth International Symposium on Thymosins in Health and Disease brought together many of the leading scientists, clinicians and thought-leaders from the United States, Israel, Europe, China and Japan to discuss the latest advances and clinical applications of the thymosins in both basic and clinical areas. The symposium, held in Rome, Italy, on October 23 - 25, 2014, was sponsored by The George Washington University and the University of Rome 'Tor Vergata.'

Advances in the basic and clinical applications of thymosin ß4.

INTRODUCTION: Thymosin ß4 (Tß4), a multifunctional peptide, has been used successfully in several clinical trials involving tissue repair and regeneration. The review will first update the current information on the common underlying cellular cascades and pathways that are basic to Tß4's regenerative activity and second, on the current and potential uses of this protein in the clinic. AREAS COVERED: Significant advances in our understanding of the actions of Tß4 have occurred in directing stem cell maturation and in regeneration and repair of injuries. Many of its activities directly affect the repair cascade following injury. Using PubMed, we summarize the discovery and isolation of Tß4 as well as the studies on tissue repair, which have provided the scientific foundation for ongoing and projected trials in the treatment of eye injuries, dermal wounds, repair of the heart following myocardial infarction and healing of the brain following stroke, trauma or neurological diseases. EXPERT OPINION: Based on its multifunctional activities during tissue regeneration in various animal studies, Tß4 has the potential for new clinical applications such kidney and liver disease, as well as repair of spinal cord, bone and ligament damage. In addition, it may be useful in the treatment of a wide range of other applications, including the consequences of aging and viral infections.

The regenerative peptide thymosin ß4 accelerates the rate of dermal healing in preclinical animal models and in patients.

Chronic nonhealing cutaneous wounds are a worldwide problem with no agent able to promote healing. A naturally occurring, endogenous repair molecule, thymosin beta 4 (Tß4), has many biological activities that promote dermal repair. It is released by platelets at the site of injury and initiates the repair cascade. Tß4 accelerated dermal healing of full-thickness punch wounds in various animal models, including normal rats and mice, steroid-treated rats, diabetic mice, and aged mice. Furthermore, in two phase 2 clinical trials of stasis and pressure ulcers, it was found to accelerate healing by almost a month in those patients that did heal. Tß4 likely acts to repair and regenerate wounds by promoting cell migration and stem cell mobilization and differentiation, and by inhibiting inflammation, apoptosis, and infection. We conclude that Tß4 is a multifunctional regenerative peptide important in dermal repair.

Development of an analytical HPLC methodology to study the effects of thymosin ß4 on actin in sputum of cystic fibrosis patients.

A high-performance liquid chromatography (HPLC) methodology is presented. Using bovine and rabbit F- and G-actin, this methodology results in both fractions as being well-resolved peaks, which were confirmed by dot blot immunoassay and fluorescence microscopy. F- and G-actin were incubated with thymosin ß4 (Tß4) and DNase and then analyzed by HPLC, which indicated that Tß4 and DNase inhibit G-actin polymerization and that Tß4 depolymerizes F-actin in a dose- and time-dependent manner. The F- and G-actin content in sputum from healthy controls and cystic fibrosis (CF) patients were measured by HPLC before and after incubation with Tß4, DNase, and gelsolin. These data demonstrate higher quantities of F-actin in the sputum of CF patients compared to healthy individuals, and also demonstrate a significantly increased F/G-actin ratio in CF sputum. Further, Tß4, DNase, and gelsolin each increase the depolymerization of F-actin in CF sputum in a dose-dependent fashion that is additive when these agents are combined.

Thymosin ß4: a multi-functional regenerative peptide. Basic properties and clinical applications.

INTRODUCTION: Thymosin ß(4), a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin ß(4), is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin ß(4) binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue. Thymosin ß(4) also decreases the number of myofibroblasts in wounds, resulting in decreased scar formation and fibrosis. AREAS COVERED: This article will cover the many thymosin ß(4) activities that directly affect the repair and regeneration cascade with emphasis on its therapeutic uses and potential. Our approach has been to evaluate the basic biology of the molecule as well as its potential for clinical applications in the skin, eye, heart and brain. EXPERT OPINION: The considerable advances in our understanding of the functional biology and mechanisms of action of thymosin ß(4) have provided the scientific foundation for ongoing and projected clinical trials in the treatment of dermal wounds, corneal injuries and in the regeneration and repair of heart and CNS tissue following ischemic insults and trauma.